HK1154264B - Ts23 alpha-amylase variants with altered properties - Google Patents

Description

TS23 alpha-amylase variants with altered properties

Priority

This application claims priority from U.S. provisional patent application serial No. 61/026056, filed on day 2/4 of 2008, and 61/059,403, filed on day 6/2008, which are incorporated herein by reference in their entirety.

Technical Field

The present invention discloses compositions and methods relating to variants of TS-23 alpha-amylase (alpha-amylase) having altered biochemical properties and advantageous performance characteristics relative to a parent amylase. The variants disclosed herein are suitable for use in, for example, starch conversion, ethanol production, laundry and dish washing, hard surface cleaning, textile desizing, and/or sweetener production.

Background

The starch is a mixture of amylose (15-30% w/w) and amylopectin (70-85% w/w). Amylose is a linear chain of alpha-1, 4 linked glucose units having a Molecular Weight (MW) of from about 60,000 to about 800,000. Amylopectin is a branched polymer which contains alpha-1, 6 branch points per 24-30 glucose units. The molecular weight can be as high as 1 hundred million.

Sugars in the form of concentrated glucose syrups are currently produced from starch by an enzymatic process involving: (1) liquefying (or thinning) solid starch with an alpha-amylase to dextrins having an average degree of polymerization of about 7-10, and (2) saccharifying the liquefied starch produced (i.e., starch hydrolysate) with an amyloglucosidase (also known as glucoamylase or GA). The resulting syrup has a high glucose content. Most of the glucose syrups produced commercially are subsequently enzymatically isomerized to a dextrose/fructose mixture, known as isomerous syrup (isosyrup).

Alpha-amylases (alpha-1, 4-glucan-4-glucanohydrolases, e.c.3.2.1.1) are a group of enzymes that hydrolyze starch, glycogen and related polysaccharides by randomly cleaving the intrinsic alpha-1, 4-glycosidic bonds. This group of enzymes has a number of important commercial applications, for example as detergents in the initial stages of starch processing (liquefaction), in desizing of textiles, in deinking of recycled paper, starch modification in the paper and pulp industry, in wet corn milling, in ethanol production, in the production of sweeteners (e.g. sugars), in the beverage industry, in brewing, in oil mines, in animal feed and in detergent matrices. For example, these enzymes can be used to remove starch stains in dishware and laundry processes.

Alpha-amylases are isolated from a variety of bacterial, fungal, plant and animal sources. Industrially, many important alpha-amylases are isolated from bacilli. One characterized alpha-amylase is that of the alkalophilic Bacillus species TS-23 strain, which produces at least five classes of enzymes exhibiting starch hydrolyzing activity. (Lin et al, 1998, "Production and properties of a raw-stable-mapping amyloasefrom the therophilic and alcaliphilic Bacillus sp.TS-23," Biotechnol.appl.biochem.28: 61-68). Although it is stable over a wide range of pH (i.e., pH4.7 to 10.8), the optimum pH for the alpha-amylase of Bacillus species TS-23 strain is 9. Although the enzyme is active at lower temperatures, e.g.15-20 ℃ the optimum temperature is 45 ℃.

There is also a need for alpha-amylase variants that have altered biochemical characteristics and provide improved performance in industrial applications.

Disclosure of Invention

Variants (mutants) of TS-23 alpha-amylase are disclosed which exhibit altered properties, which are advantageous for a variety of industrial processes, such as starch processing (e.g., starch liquefaction, saccharification, etc.), textile processing (e.g., desizing), and as an additive to detergents (e.g., for cleaning starchy stains).

Such changes include, but are not limited to, changes in specific activity, substrate specificity, substrate binding, substrate cleavage pattern, thermostability, oxidative stability, Ca²⁺Dependence, pH/activity profile, pH/stability profile, and other properties of interest. An exemplary altered pH/stability profile is increased stability at low pH (e.g., pH < 6 or even pH < 5) and/or high pH (e.g., pH > 9).

In one aspect of the invention, variants of a parent AmyTS23 a-amylase are provided that have an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% identical to the parent a-amylase and that comprise at least two of the following features: (a) a truncation of the C-terminus, (b) a substitution of amino acid 201, or (C) a deletion of residues R180 and S181, and wherein the variant has alpha-amylase activity (numbering of amino acids using SEQ ID NO: 1). In some embodiments, the parent alpha-amylase is SEQ ID NO: 1. in some embodiments, the parent alpha-amylase hybridizes to SEQ ID NO: 1 has the indicated homology.

Another aspect of the invention relates to a hand or automatic dishwashing composition comprising a Bacillus species TS-23 strain alpha-amylase or variant thereof. The composition may further comprise one or more of surfactants, detergent builders, complexing agents, polymers, bleaching systems, stabilizers, foaming agents, suds suppressors, anti-corrosion agents, soil suspending agents, anti-soil redeposition agents, dyes, bactericides, hydrotropes, tarnish inhibitors, and perfumes. The dishwashing composition may be a composition for hand washing or automatic dishwashing.

A related aspect of the invention relates to a laundry detergent additive comprising a Bacillus species TS-23 strain alpha-amylase or variant thereof. As noted above, the composition may further comprise one or more of a surfactant, detergent builder, complexing agent, polymer, bleaching system, stabilizer, foaming agent, suds suppressor, anti-corrosion agent, soil suspending agent, anti-soil redeposition agent, dye, germicide, hydrotrope, tarnish inhibitor, and fragrance. The composition may also comprise one or more of surfactants, detergent builders, complexing agents, polymers, bleaching systems, stabilizers, foaming agents, suds suppressors, anti-corrosion agents, soil suspending agents, anti-soil redeposition agents, dyes, bactericides, hydrotropes, optical brighteners, fabric conditioning agents and perfumes.

Further aspects relate to nucleic acids encoding the above variants and vectors comprising such nucleic acids. It also relates to cells into which the nucleic acid is inserted, for example by vector, phage or virus insertion. Such an isolated host cell may be a microorganism, such as a bacterium or a fungus. The bacteria may be gram positive bacteria selected from the group consisting of: bacillus subtilis (Bacillus subtilis), Bacillus licheniformis (b. licheniformis), Bacillus lentus (b. lentus), Bacillus brevis (b. brevis), Bacillus stearothermophilus (g. stearothermophilus, formerly known as b. stearothermophilus), Bacillus alkalophilus (b. alkalophilus), Bacillus amyloliquefaciens (b. amyloliquefaciens), Bacillus coagulans (b. coemulsifens), Bacillus circulans (b. circulans), Bacillus lautus (b. lautus), Bacillus thuringiensis (b. thuringiensis), Streptomyces lividans (Streptomyces lividans), and Streptomyces murinus (s. murinus); or a gram-negative bacterium, wherein the gram-negative bacterium is an Escherichia coli (Escherichia coli) and Pseudomonas species (Pseudomonas).

Other aspects relate to methods of making the variant polypeptides and the use of the variant polypeptides in various industrial processes (e.g., in starch liquefaction) alone or in combination with other enzymes, including alpha-lytic amylases. Some aspects relate to the use of a plurality of polypeptides for laundry or dish washing. Also relates to methods of cleaning fabrics and/or other hard surfaces with the various polypeptides described above. Another aspect relates to the use of an alpha-amylase or any of the alpha-amylase variants described herein in a fabric desizing composition, e.g., wherein the composition is an aqueous solution. Also relates to a method for desizing fabrics with the composition.

The variant polypeptide may optionally be present as a non-dusting granulate, a particulate, a stabilized liquid, or a protected enzyme. Another aspect relates to a detergent additive or detergent composition further comprising an enzyme selected from the group consisting of: cellulases, proteases, acyltransferases, aminopeptidases, amylases, carbohydrases, carboxypeptidases, catalases, chitinases, cutinases (cutinases), cyclodextrin glucanotransferases (cyclodextrincransferases), deoxyribonucleases, esterases, alpha-galactosidases, beta-galactosidases, glucoamylases, alpha-glucosidases, beta-glucosidases, haloperoxidases, invertases, laccases, lipases, mannosidases, oxidases, pectinolytic enzymes, peptidoglutamidases, peroxidases, phytases, polyphenoloxidases, proteolytic enzymes, ribonucleases, transglutaminase, xylanases, pullulanases, isoamylases, carrageenases (carrageenases) and any combination thereof. Other amylases useful in the composition include two or more other alpha-amylases, beta-amylases, isoamylases, or glucoamylases.

Some aspects relate to compositions for starch processing in aqueous solution, comprising a bacillus species TS-23 strain alpha-amylase or variant thereof. Also relates to a method for processing starch using such a composition. The methods and compositions may further include a glucoamylase, an isoamylase, a pullulanase, a phytase, or a combination thereof. Yet other aspects relate to a biofilm degrading (e.g., hydrolyzing) composition in a solution or gel comprising a bacillus species TS-23 strain alpha-amylase or variant thereof, optionally further comprising a cellulase, hemicellulase, xylanase, lipase, protease, pectinase, antimicrobial agent, or any combination thereof. Also relates to a method for hydrolyzing a biofilm using the composition.

Another aspect relates to a composition of saccharified starch in solution, comprising a Bacillus species TS-23 strain alpha-amylase or variant thereof. Therefore, it also relates to a method of saccharifying starch comprising applying a composition comprising the amylase for a period of time sufficient to saccharify starch.

Another aspect relates to a composition of liquefied starch in solution comprising bacillus species TS-23 strain alpha-amylase or variant thereof. Also relates to a method of liquefying starch comprising applying the composition for a period of time sufficient to liquefy the starch.

Some specific aspects of the compositions and methods are described below.

In one aspect, a variant of a parent AmyTS23 a-amylase is provided, wherein the variant has an amino acid sequence that is at least 80% identical to the parent a-amylase and comprises at least two of the following characteristics:

(a) the truncation of the C-terminal end,

(b) substitution of residue 201, or

(c) The deletion of residues R180 and S181,

wherein the amino acid residue refers to SEQ ID NO: 1. In some embodiments, the variant has alpha-amylase activity.

In some embodiments, the variant has at least 90% identity to the parent alpha-amylase. In some embodiments, the variant has at least 95% identity to the parent alpha-amylase. In a specific embodiment, the parent alpha-amylase has the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the variant further comprises a substitution at one or more residues selected from the group consisting of: residue 87, residue 225, residue 272 and residue 282.

In another aspect, a variant of a parent AmyTS23 a-amylase is provided, wherein the variant has an amino acid sequence at least 85% identical to the parent a-amylase and comprises a truncation at the C-terminus. In some embodiments, the variant has the amino acid sequence of SEQ ID NO: 2. The variant may have increased cleaning activity on starch stains in cold water compared to the parent amylase.

In some embodiments, the variant further comprises deletions of residues at positions R180 and S181, wherein the amino acid residue positions are referenced to SEQ ID NO: 1. The variant may have improved detergent stability compared to the parent amylase.

In some embodiments, the variant further comprises a substitution at residue 201, wherein the amino acid residue position is referenced to SEQ ID NO: 1. The variant may have improved oxidative stability compared to the parent amylase. The variant may have the substitution M201L.

Any of the variants described above may further comprise substitutions at one or more residues selected from the group consisting of: residue 87, residue 225, residue 272 and residue 282, wherein the amino acid residue positions are referenced to SEQ ID NO: 1.

In a related aspect, there is provided a nucleic acid encoding a variant described herein. In some embodiments, an expression vector comprising the nucleic acid under the control of a suitable promoter is provided. In some embodiments, a host cell comprising the expression vector is provided.

In a related aspect, there is provided a hand or automatic dishwashing composition comprising a variant as described herein and one or more of the following: surfactants, detergent builders, complexing agents, polymers, bleaching systems, stabilizers, foaming agents, foam inhibitors, anti-corrosion agents, soil suspending agents, anti-soil redeposition agents, dyes, bactericides, hydrotropes, tarnish inhibitors and perfumes.

In a related aspect, there is provided a laundry detergent additive comprising a variant as described herein and one or more of the following: surfactants, detergent builders, complexing agents, polymers, bleaching systems, stabilizers, foaming agents, foam inhibitors, anti-corrosion agents, soil suspending agents, anti-soil redeposition agents, dyes, bactericides, hydrotropes, optical brighteners, fabric conditioners, and perfumes.

In another aspect, there is provided a method of removing starch from a fabric, comprising: incubating a fabric in the presence of a variant of a parent AmyTS23 alpha-amylase, wherein the variant has an amino acid sequence that is at least 80% identical to the parent alpha-amylase and comprises at least two of the following characteristics:

(a) the truncation of the C-terminal end,

(b) substitution of residue 201, or

(c) The deletion of residues R180 and S181,

wherein the amino acid residues are referenced to SEQ ID NO: 1, and wherein the incubating removes starch from the fabric.

In a related aspect, there is provided a method of processing starch, the method comprising incubating a fabric in the presence of a variant of a parent AmyTS23 a-amylase, wherein the variant has an amino acid sequence at least 80% identical to the parent a-amylase and comprises at least two of the following characteristics:

(a) the truncation of the C-terminal end,

(b) substitution of residue 201, or

(c) The deletion of residues R180 and S181,

wherein the amino acid residues are referenced to SEQ ID NO: 1, and wherein the incubating hydrolyzes the starch.

These and other aspects and embodiments of the present compositions and methods will be apparent from the disclosure herein and the accompanying drawings.

Drawings

FIG. 1 shows the amino acid sequence of the parent AmyTS23 alpha-amylase (full length, mature; SEQ ID NO: 1).

FIG. 2 shows the amino acid sequence of the AmyTS23t truncated polypeptide (mature; SEQ ID NO: 2). Residues indicated in bold and underlined represent SEQ ID NO: 2 at amino acid residues R180, S181 and M201.

FIG. 3 shows the DNA sequence of the optimized amyTS23 gene (SEQ ID NO: 3).

FIG. 4 shows the DNA sequence of the optimized amyTS23t gene (SEQ ID NO: 4).

Fig. 5 illustrates the expression cassettes of AmyTS23 and AmyTS23 t.

Fig. 6 is a graph depicting the results of a sample cleaning assay using full-length AmyTS23 amylase (AmyTS23fl) and the OxAm control.

FIG. 7 is a graph depicting the results of sample cleaning assays performed using amylase Amy TS23fl and the Oxam control.

FIG. 8 is a graph depicting the results of sample cleaning assays performed with the amylase AmyTS23t and the Oxam control.

Fig. 9 is a graph depicting the results of sample cleaning assays performed using AmyTS23t and the OxAm control.

Fig. 10 is a graph depicting increased stability studies of AmyTS23t and AmyTS23t Δ RS in two different laundry detergents.

Fig. 11 is a graph depicting the oxidative stability of AmyTS23t, AmyTS23t Δ RS, and AmyTS23t (M201L + Δ RS).

FIG. 12 is a graph depicting the performance of AmyTS23t Δ RS on rice starch samples in liquid detergent.

FIG. 13 is a graph depicting residual activity as a function of charge change.

FIG. 14: other amino acid and nucleotide sequences referenced in this disclosure are described.

Detailed Description

Disclosed herein are compositions and methods relating to alpha-amylases of bacillus species TS-23 strain and variants thereof. Variants of TS-23 have altered biochemical properties and exhibit high performance in, for example, laundry and dishwashing detergent applications. These and other features of these variations and applications in which these variations are used are described in detail below.

1. Definitions and abbreviations

In this detailed description, the following abbreviations and definitions apply. The singular forms "a", "an" and "the" encompass references to the plural forms as well, unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such enzymes, and reference to "the preparation" includes reference to one or more preparations and equivalents thereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Singleton et al, DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY, second edition, John Wiley AND Sons, New York (1994) AND Hale & Markham, THE HARPER COLLINSDITIONARY OF BIOLOGY, Harper Perennial, New York (1991) provide the artisan with a number OF comprehensive dictionaries for which the terms are used herein.

In certain aspects, the compositions and methods described herein rely on conventional techniques and methods used in the fields of genetic engineering and molecular biology. The following resources comprise a description of the general methodology used in the compositions and methods of the invention: sambrook et al, Molecula CLONING: ALABORATORY MANUAL (second edition, 1989); kreigler, GENE TRANSFERAND EXPRESSION; edited by Ausubel et al, Current promoters IN MOLECULAR BIOLOGY (1994). These general references provide definitions and methods well known in the art. However, the compositions and methods of this invention should not be limited to any of the specific techniques, protocols, and reagents described, as these may vary. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the compositions and methods of use disclosed herein, the preferred methods and materials are described herein.

When describing proteins and their encoding genes, it is common for the gene names to be italicized and not capitalized, whereas the protein names are generally not italicized and are capitalized on the first letter.

All patents and publications, including all sequences disclosed within such patents and publications, cited herein are expressly incorporated herein by reference.

1.1. Definition of

The term "starch" as used herein refers to a complex polysaccharide carbohydrate comprising plants, comprising a polysaccharide having the general formula (C)₆H₁₀O₅)_x(wherein X may be any number) of amylose and amylopectin. The term especially relates to any plant-based material including, but not limited to, cereals, grasses, tubers and roots, more specifically to wheat, barley, maize, rye, rice, sorghum, bran, cassava, millet, potato, sweet potato and tapioca starch.

As used herein, an "amylase" is an enzyme capable of catalyzing the degradation of starch. Amylases are hydrolases which cleave the alpha-D- (1 → 4) O-glycosidic bond in starch. In general, an alpha-amylase (EC 3.2.1.1; alpha-D- (1 → 4) -glucan glucohydrolase) is defined as an endonuclease that cleaves alpha-D- (1 → 4) O-glycosidic linkages within a starch molecule in a random fashion. In contrast, exo-amylases, such as beta-amylase (EC 3.2.1.2; α -D- (1 → 4) -glucanohydrolase), and some product-specific amylases, such as maltogenic alpha-amylase (EC 3.2.1.133), cleave starch molecules from the non-reducing ends of the substrate. Beta-amylases, alpha-glucosidases (EC 3.2.1.20; alpha-D-glucoside glucohydrolases), glucoamylases (EC 3.2.1.3; alpha-D- (1 → 4) -glucan glucohydrolases), and product-specific amylases can produce malto-oligosaccharides of specific length from starch. As used herein, "amylase" includes any/all amylases, including glucoamylases, alpha-amylases, beta-amylases, and wild-type alpha-amylases, e.g., of Bacillus species (e.g., B.licheniformis and B.subtilis).

As used herein, "Bacillus species TS-23 strain alpha-amylase" and similar phrases refer to alpha-amylase derived from Bacillus species TS-23 strain. The gene encoding the alpha-amylase can be a wild-type gene or a codon-optimized polynucleotide encoding the alpha-amylase. The mature alpha-amylase of Bacillus species TS-23 strain is (amino to carboxyl orientation) (SEQ ID NO: 1; FIG. 1):

ntapinetmm qyfewdlpnd gtlwtkvkne aanlsslgit alwlppaykg 50

tsqsdvgygv ydlydlgefn qkgtirtkyg tktqyiqaiq aakaagmqvy 100

advvfnhkag adgtefvdav evdpsnrnqe tsgtyqiqaw tkfdfpgrgn 150

tyssfkwrwy hfdgtdwdes rklnriykfr stgkawdwev dtengnydyl 200

mfadldmdhp evvtelknwg twyvnttnid gfrldavkhi kysffpdwlt 250

yvrnqtgknl favgefwsyd vnklhnyitk tngsmslfda plhnnfytas 300

kssgyfdmry llnntlmkdq pslavtlvdn hdtqpgqslq swvepwfkpl 350

ayafiltrqe gypcvfygdy ygipkynipg lkskidplli arrdyaygtq 400

rdyidhqdii gwtregidtk pnsglaalit dgpggskwmy vgkkhagkvf 450

ydltgnrsdt vtinadgwge fkvnggsvsi wvaktsnvtf tvnnatttsg 500

qnvyvvanip elgnwntana ikmnpssypt wkatialpqg kaiefkfikk 550

dqagnviwes tsnrtytvpf sstgsytasw nvp 583

as used herein, "Bacillus species TS-23 strain alpha-amylase variant" and similar phrases refer to variants/mutants of the wild-type Bacillus species TS-23 strain alpha-amylase, including sequence substitutions, insertions, and/or deletions with respect to the amino acid sequence of the parent (wild-type, reference) amylase of the Bacillus species TS-23 strain. The term "variant" may be used interchangeably with the term "mutant". The Bacillus species TS-23 strain alpha-amylase variant can include a mutation in the signal sequence compared to the parent signal sequence. In addition, the alpha-amylase variant of Bacillus species TS-23 strain can be in the form of a fusion protein comprising a heterologous alpha-amylase signal sequence, such as the signal sequence from Bacillus Licheniformis (LAT).

As used herein, the phrases "Bacillus species TS-23 strain alpha-amylase parent", "wild-type Bacillus species TS-23 strain alpha-amylase", "Bacillus species TS-23 strain alpha-amylase reference", and similar phrases, refer to polypeptides of the Bacillus species TS-23 strain. For convenience, the term may be abbreviated as "parent enzyme", "wild-type enzyme", "parent polypeptide", "reference polypeptide", and the like. The Bacillus species TS-23 strain alpha-amylase parent can include a mutation in a signal sequence of a parent polypeptide. Also, the alpha-amylase parent of bacillus species TS-23 strain can be a fusion protein comprising a heterologous alpha-amylase signal sequence (e.g., a signal sequence from bacillus licheniformis, LAT).

"parent nucleic acid/polynucleotide", "wild-type nucleic acid/polynucleotide" or "reference nucleic acid/polynucleotide" refers to the nucleic acid sequence encoding the parent polypeptide and the nucleic acid complementary thereto.

"variant nucleic acid/polynucleotide" refers to a nucleic acid sequence encoding a variant polypeptide or a nucleic acid complementary thereto, or a polynucleotide sequence having a substitution, insertion or deletion of at least one base with respect to a parent polynucleotide sequence or a nucleic acid complementary thereto. Specifically, the nucleic acid may include those having a specified degree of homology with the reference sequence, or capable of hybridizing with the reference sequence, for example, capable of hybridizing under stringent conditions (e.g., 50 ℃ and 0.2 × SSC {1 × SSC ═ 0.15M NaCl, 0.015M trisodium citrate, ph7.0}) or high stringent conditions (e.g., 65 ℃ and 0.1 × SSC {1 × SSC ═ 0.15M NaCl, 0.015M trisodium citrate, ph7.0 }). Variant nucleic acids may be optimized to reflect the use of codons preferred for a particular host organism, such as methylotrophic yeasts (e.g., Pichia (Pichia), Hansenula (Hansenula), etc.) or filamentous fungi (e.g., Trichoderma (Trichoderma) such as Trichoderma reesei (t. reesei)) or other expression hosts (e.g., bacillus, Streptomyces (Streptomyces), etc.

The term "recombinant" as used in reference to a cell, nucleic acid, protein or vector, refers to a subject that has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in the native (non-recombinant) form of the cell, or express native genes that would otherwise be abnormally expressed, down-regulated, or not expressed at all.

The terms "recovered", "separated" and "isolated" are used herein to refer to a compound, protein, cell, nucleic acid or amino acid that is separated from at least one other component with which it is naturally associated and naturally occurring.

As used herein, the term "purified" refers to a substance (e.g., an isolated polypeptide or polynucleotide) that is in a relatively pure state, e.g., at least about 90% pure, at least about 95% pure, at least about 98% pure, or even at least about 99% pure.

The terms "thermostable" and "thermostability" refer to the ability of the enzyme to retain activity after exposure to elevated temperatures. Thermostability of enzymes (e.g. alpha-amylase) by their half-life (t)_1/2) Measurement, half-life (t)_1/2) Is measured in minutesHours or days, during which half of the enzyme activity is lost under defined conditions. The half-life is calculated by measuring the residual alpha-amylase activity after exposure to elevated temperature (i.e. stimulation with elevated temperature).

"pH range" refers to the range of pH values at which the enzyme exhibits the ability to exhibit catalytic activity.

As used herein, "pH stable" and "pH stability" refer to the ability of an enzyme to maintain activity over a wide pH range for a predetermined period of time (e.g., 15 minutes, 30 minutes, 1 hour, etc.).

As used herein, "amino acid sequence" is synonymous with the terms "polypeptide," "protein," and "peptide," and is used interchangeably. When such an amino acid sequence exhibits activity, it may be referred to as an "enzyme". The amino acid residues are referred to herein using the conventional single or three letter code.

The term "nucleic acid" includes DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. The nucleic acid may be single-stranded or double-stranded, and may be a chemically modified form thereof. The terms "nucleic acid" and "polynucleotide" are used interchangeably herein. Because the genetic code is degenerate, more than one codon can be used to encode a particular amino acid, and the compositions and methods described include nucleotide sequences that encode a particular amino acid sequence.

Unless otherwise indicated, nucleic acids are written from left to right in the 5 'to 3' direction, and amino acid sequences are written from left to right in the amino to carboxyl direction, respectively.

"homolog" shall mean an entity that has a degree of identity to the subject amino acid sequence and the subject nucleotide sequence. Homologous sequences include amino acid sequences that are at least about 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% identical to the subject sequence, as aligned using conventional sequence alignment tools (e.g., Clustal, BLAST, etc.). Unless otherwise indicated, typically, homologues comprise the same active site residues as the subject amino acid sequence.

As used herein, "hybridization" refers to the process of base pairing one strand of a nucleic acid with a complementary strand, as occurs in dot hybridization and PCR techniques.

As used herein, a "synthetic" molecule is one that is prepared by in vitro chemical or enzymatic synthesis, rather than by biological preparation.

As used herein, the terms "transformed," "stably transformed," and "transgenic" when referring to a cell mean that the cell has a non-native (e.g., heterologous) nucleic acid sequence that is integrated into the genome of the cell or carried as an episomal plasmid that is maintained over multiple passages.

The term "introduced" in the context of inserting a nucleic acid sequence into a cell means "transfection" or "transformation" or "transduction" as is known in the art.

A "host strain" or "host cell" is an organism into which has been introduced an expression vector, phage, virus or other DNA construct comprising a polynucleotide encoding a polypeptide of interest (e.g., an alpha-amylase variant). Exemplary host strains are bacterial cells. The term host cell includes protoplasts prepared from cells (e.g., cells of a Bacillus species).

The term "heterologous" in reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell.

The term "endogenous" in reference to a polynucleotide or protein refers to a polynucleotide or protein that naturally occurs in a host cell.

The term "expression" as used herein refers to the process of producing a polypeptide based on the nucleic acid sequence of a gene. The process includes transcription and translation.

The term "selectable marker" or "selectable marker" refers to a gene that is capable of being expressed in a host, allowing for easy selection by the host carrying the gene. Examples of selectable markers include, but are not limited to, antibiotics (e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage on the host cell, such as a nutritional advantage.

"culturing" refers to growing a population of microbial cells in a liquid or solid medium under suitable conditions. The cultivation involves fermentative bioconversion of a starch substrate comprising granular starch to the final product (usually in a vessel or reactor).

"fermentation" is the enzymatic and anaerobic breakdown of organic substrates by microorganisms to produce simpler organic compounds. Although fermentation occurs under anaerobic conditions, this does not mean that the term is limited to strictly anaerobic conditions only, since fermentation also occurs in the presence of oxygen.

"Gene" refers to a DNA segment involved in the production of a polypeptide, including the coding region, regions preceding and following the coding region, and intervening sequences (introns) between individual coding segments (exons).

"vector" refers to a polynucleotide sequence designed to introduce a nucleic acid into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, expression cassettes and the like.

An "expression vector" refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest operably linked to appropriate control sequences capable of effecting its expression in an appropriate host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding a suitable ribosome binding site on the mRNA, enhancers and sequences which control termination of transcription and translation.

A "promoter" is a regulatory sequence involved in binding RNA polymerase to initiate transcription of a gene. The promoter may be an inducible promoter or a constitutive promoter. An exemplary promoter is the Bacillus licheniformis alpha-amylase (AmyL) promoter.

"operably linked" means that the components so described are in a relationship (including, but not limited to, juxtaposition) that allows them to function in their intended manner. For example, a regulatory sequence operably linked to a coding sequence is operably linked to such that expression of the coding sequence is under the control of a control sequence.

The term "under transcriptional control" refers to the transcription of a polynucleotide sequence (usually a DNA sequence), depending on the elements to which it is operably linked that contribute to transcription initiation or promote transcription.

The term "under translational control" refers to the translation of a polynucleotide sequence (typically an RNA sequence) into a polypeptide, depending on the elements to which it is operably linked that aid in translation initiation or promote translation.

"Signal sequence" refers to an amino acid sequence that binds to the N-terminal portion of a protein, which facilitates secretion of the protein to the outside of a cell. The mature form of the extracellular protein lacks the signal sequence that is cleaved off during secretion.

As used herein, "biological activity" is a sequence having a particular biological activity, e.g., an enzymatic activity. In the case of the amylase of the invention, the activity is an alpha-amylase activity.

"Water hardness" is a measure of the minerals (e.g., calcium and magnesium) present in water.

"saccharification" refers to the enzymatic conversion of starch to glucose.

"gelatinization" refers to the dissolution of starch molecules by cooking to form a viscous suspension.

"liquefaction" refers to the starch conversion stage in which gelatinized starch is hydrolyzed to low molecular weight soluble dextrins.

The term "Degree of Polymerization (DP)" refers to the number of anhydroglucopyranose units of a known saccharide. Examples of DP1 are monosaccharides such as glucose and fructose. Examples of DP2 are disaccharides such as maltose and sucrose. DP > 3 represents polymers having a degree of polymerization of greater than 3.

For starch conversion, the term "end product" or "end product of interest" refers to a molecule derived from a specific carbon source from the enzymatic conversion of a starch substrate.

The term "dry solids content (ds)" as used herein refers to the percentage of total solids in the slurry in% dry weight.

The term "slurry" refers to an aqueous mixture containing undissolved solids.

The term "residual starch" refers to the starch (soluble or insoluble) remaining in the composition after fermentation or enzymatic hydrolysis of the starch-containing substrate.

As used herein, "recirculation step" refers to the recirculation of constituents of the mash, which may include residual starch, enzymes, and/or microorganisms that ferment the starch-containing substrate.

The term "mash" refers to an aqueous mixture comprising a fermentable carbon source (e.g., a carbohydrate) that may be used to produce a fermentation product (e.g., ethanol). The terms "beer" and "mash" are used interchangeably.

The term "stillage" refers to a mixture of unfermented solids and water, which is the residue after removal of ethanol from the fermented mash.

The terms "Distillers Dried Grains (DDG)" and "distillers dried grains with Solubles (DDGs)" refer to useful byproducts of grain fermentation.

As used herein, "ethanologenic microorganism" refers to a microorganism capable of converting a saccharide or oligosaccharide to ethanol. Ethanologenic microorganisms are capable of producing ethanol as a result of their ability to express one or more enzymes, which enzymes, alone or together, can convert sugars into ethanol.

The term "ethanol producer" or "ethanologenic microorganism" as used herein refers to any organism or cell capable of producing ethanol from hexose or pentose sugars. Generally, ethanologenic cells contain alcohol dehydrogenase and pyruvate decarboxylase. Examples of ethanologenic microorganisms include fungal microorganisms, such as yeast. Preferred yeasts include saccharomyces, in particular saccharomyces cerevisiae (s.

For an amylase and its substrate, the term "contacting" refers to placing the enzyme close enough to its substrate to enable the enzyme to convert the substrate to the final product. The contacting may include mixing.

The term "derived from" means, depending on the context, that the terms "originate from", "based on", "derived from" or "obtainable from" and "isolated from".

The term "enzymatic conversion" generally refers to the modification of a substrate (e.g., starch) by the action of an enzyme (e.g., amylase).

The term "specific activity" as used herein refers to the number of moles of substrate converted to product by an enzyme preparation per unit time under specified conditions. The specific activity is expressed in units (U)/mg of protein.

The term "yield" refers to the amount of end product produced by a process, e.g., expressed as a concentration, volume, amount, or percentage of starting material.

"ATCC" refers to the American Standard culture Collection, located in Mr. 20108(ATCC) of Venturia, Virginia.

"NRRL" refers to the American type culture Collection for agricultural research, the national center for agricultural applications research (formerly called USDA, research laboratory in the northern region of the United states department of agriculture), in Pioriya, Illinois.

Numerical ranges include the numbers defining the range.

In general, the headings are descriptive and should not be construed as limiting.

1.2. Abbreviations

Unless otherwise indicated, the following abbreviations apply:

AE alcohol ethoxylates

AEO alcohol ethoxylates

AEOS alcohol ethoxy sulfate

AES alcohol ethoxy sulfate

AFAU acid fungal alpha-amylase units

AGU glucoamylase Activity Unit

AOS alpha-olefin sulfonates

AS alcohol sulfate salt

BAA Bacillus amyloliquefaciens alpha-amylase

BLA Bacillus licheniformis (or LAT)

BSA bovine serum albumin

cDNA complementary DNA

CMC carboxymethyl cellulose

DNA deoxyribonucleic acid

Degree of polymerization of DP3 trisubunit

Degree of polymerization of DPn subunit

DTMPA Diethylenetriamine pentaacetic acid

Enzyme Committee for enzyme Classification of EC

EDTA ethylene diamine tetraacetic acid

EO Oxirane

F & HC fabrics and home care

FAU fungal amylase units

GA glucoamylase

gpg granule per gallon

HFCS high fructose corn syrup

HFSS high fructose starch-based syrup

IPTG isopropyl-beta-D-1-thiogalactopyranoside

LAS linear alkyl benzene sulfonate

LOM Launder fastness tester (launcher-O-meter)

LU Liquiphon Unit

MW molecular weight

MWU modified Wohlgemuth units

NOBS nonanoyloxy benzene sulfonate

NTA nitrilotriacetic acid

PCR polymerase chain reaction

PEG polyethylene glycol

PVA poly (vinyl alcohol)

PVP poly (vinylpyrrolidone)

RNA ribonucleic acid

SAS secondary alkane sulfonate

TAED tetraacylethylenediamine

TCA trichloroacetic acid

TSB trypsin digestion soy peptone culture medium

UFC ultrafiltration concentration

w/v weight/volume

w/w weight/weight

wt wild type

1.3 nomenclature

In the description and claims of the present invention, the conventional single and three letter codes for amino acid residues are used. For ease of reference, the following nomenclature is used to describe the α -amylase variants of the compositions and methods of the invention:

original amino acids: position: substitution of amino acids.

According to this nomenclature, for example, the substitution of alanine for serine at position 242 is represented as:

ser242Ala or S242A

The alanine deletion at position 30 is represented as:

Ala30^*or A30^*Or Δ A30

The insertion of additional amino acid residues, such as lysine, is expressed as:

ala30AlaLys or A30 AK.

Deletions of contiguous stretches of amino acid residues, e.g. amino acid residues 30-33, are indicated as (30-33)^*Or Δ (A30-N33) or Δ 30-33. Deletions of two consecutive amino acids, e.g.amino acid residues R180-S181, are indicated as Δ RS or Δ 180-.

When a particular alpha-amylase comprises a "deletion" when compared to other alpha-amylases, and an insertion is made at this location, it is expressed as:

by using^*36Asp or^*36D indicates the insertion of aspartic acid at position 36.

Multiple mutations are separated by plus signs, i.e.:

with Ala30Asp + Glu34Ser or A30N + E34S

Indicating the replacement of asparagine and serine at positions 30 and 34 with alanine and glutamic acid, respectively.

When one or more alternative amino acid residues can be inserted at a given position, it is expressed as:

a30N, E; or

A30N or a 30E.

Also, when a site suitable for modification is identified herein but does not specify any particular modification, it is understood that any amino acid residue may be substituted for the amino acid residue at that position. Thus, for example, when referring to a modification of alanine at position 30 but not indicated, it is to be understood that alanine may be deleted or substituted for any other amino acid, i.e. any one of the following amino acids:

R、N、D、A、C、Q、E、G、H、I、L、K、M、F、P、S、T、W、Y、V。

additionally, "a 30X" refers to any of the following alternatives:

a30R, a30N, a30D, a30C, a30Q, a30E, a30G, a30H, a30I, a30L, a30K, a30M, a30F, a30P, a30S, a30T, a30W, a30Y, or a 30V;

or simply: a30R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V.

If the parent enzyme used for numbering already contains the amino acid residue in question at which a substitution is proposed, in the case when, for example, one of N or V is present in the wild type, the following nomenclature is used:

"X30N" or "X30N, V". This therefore means that the other relevant parent enzyme is replaced by "Asn" or "Val" at position 30.

1.4 amino acid residue characteristics

Charged amino acids:

Asp，Glu，Arg，Lys，His

negatively charged amino acids (aligned from the most negatively charged residue):

Asp，Glu

positively charged amino acids (arranged from the most positively charged residue):

Arg，Lys，His

neutral amino acids:

Gly，Ala，Val，Leu，Ile，Phe，Tyr，Trp，Met，Cys，Asn，Gln，Ser，Thr，Pro

hydrophobic amino acid residues (most hydrophobic residues are ranked last):

Gly，Ala，Val，Pro，Met，Leu，Ile，Tyr，Phe，Trp，

hydrophilic amino acids (most hydrophilic residues are listed last):

Thr，Ser，Cys，Gln，Asn

1.5 homology (identity)

A polynucleotide or polypeptide that has a certain percentage (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99%) sequence identity to another sequence refers to the percentage of bases or amino acid residues that are identical in the two sequences compared when the two sequences are compared. Such alignments and percent homologies or identities can be determined using any suitable software program known IN the art, such as those described IN CURRENT promoters IN MOLECULARBIOLOGY (edited by f.m. ausubel et al, 1987, supplement 30, section 7.7.18). Preferred programs include Vector NTI Advance^TM9.0(Invitrogen Corp. Carlsbad, Calif.), GCG Pileup program, FASTA (Pearson et al (1988) Proc. Natl Acad. Sci USA 85: 2444-. Another preferred alignment program is ALIGN Plus (Scientific and economic Software, PA), which preferably uses default parameters. Another available sequence software program is the TFASTA data search program, which can be derived from the sequence software package version 6.0 (university of wisconsin, madison).

Homology can be determined as the degree of identity between two sequences, indicating that the first sequence is derived from the second. Homology is suitably determined using computer programs known in the art, for example GAPs provided in the GCG package (as described above). Thus, GAP GCG v8 can be used with the default scoring matrix identification and the following default parameters: a gap creation penalty of 5.0 and a gap extension penalty of 0.3, for nucleic acid sequence comparisons, respectively; a gap creation penalty of 3.0 and a gap extension penalty of 0.1 are used for protein sequence comparisons, respectively. GAP was measured using Needleman and Wunsch, (1970), j.mol.biol.48: 443-.

A structural alignment between AmyTS23(SEQ ID NO: 1) and, for example, another alpha-amylase can be used to identify equivalent/corresponding sites in other alpha-amylases that have a high degree of homology, e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% with AmyTS 23. One way to achieve this structural alignment is to use the default values for the gap penalty, i.e., gap creation penalty of 3.0 and gap extension penalty of 0.1, using the Pile Up program from the GCG package. Other methods of structural alignment include hydrophobic cluster analysis (Gaboriaud et al, FEBS letters.224: pp.149-155, 1987) and back-cues (Huber, T. and Torda, A.E., Protein science.7 (1): pp.142-149 (1998)).

1.6 hybridization

Oligonucleotide probes for characterizing AmyTS23 described above can be prepared based on, as appropriate, the full-length or partial nucleotide or amino acid sequence of the alpha-amylase under study.

Suitable conditions for detecting hybridization include pre-soaking in 5 XSSC and pre-hybridization at 40 ℃ for 1 hour in: 20% formamide solution, 5 XDenhardt's solution, 50mM sodium phosphate, pH6.8 and 50mg denatured sonicated calf thymus DNA. Then hybridized in the same solution supplemented with 100mM ATP at 40 ℃ for 18 hours. The filters (filters) were then washed three times with the following conditions: 2 XSSC, 0.2% SDS, 40 ℃,30 minutes of washing (low stringency conditions); washing at 50 ℃ (medium stringency conditions) is preferred; more preferably at 65 ℃ (high stringency conditions); further preferably at 75 deg.C (very high stringency conditions). For more details on the hybridization method see: sambrook et al, Molecular Cloning: a Laboratory Manual, second edition, Cold Spring Harbor, 1989.

In this context, "derived from" refers not only to the strain from which the alpha-amylase is or can be produced from the organism in question, but also to the DNA sequence encoding the alpha-amylase isolated from this strain and produced by a host organism transformed with this DNA sequence. Finally, the term is intended to refer to alpha-amylases encoded by synthetic and/or cDNA-derived DNA sequences and having properties identical to those of the alpha-amylase in question. The term is also intended to mean that the parent alpha-amylase may be a variant of a naturally occurring alpha-amylase, i.e., a variant, which is the result of one or more amino acid residue modifications (insertions, substitutions, deletions) of the naturally occurring alpha-amylase.

One skilled in the art will appreciate that the sequences encompassed by the compositions and methods of the invention are also defined by the ability to hybridize under stringent hybridization conditions to an exemplary amyTS23 sequence (e.g., SEQ ID NO: 4 as shown in FIG. 4). A nucleic acid is capable of hybridizing to another nucleic acid sequence when in a single stranded form capable of renaturing the nucleic acid under suitable conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known in the art (see, e.g., Sambrook (1989), supra, particularly chapters 9 and 11). In some embodiments, stringent conditions correspond to a Tm of 65 ℃ and 0.1 x SSC, 0.1% SDS.

1.7 parent alpha-amylases

As described above, any AmyTS23 a-amylase may be used as a parent (i.e., background) a-amylase according to the present invention. In a preferred embodiment, the parent alpha-amylase is derived from a Bacillus species TS-23 strain, such as one of those referred to above, e.g., having the amino acid sequence of SEQ ID NO: 1 (see fig. 1) of the amino acid sequence shown in TS-23 a-amylase.

1.8 altered Properties

The following section illustrates the relationship between the mutants present in the variant amylases described herein, and the changes in desirable properties that may result therefrom (as compared to the parent TS-23. alpha. -amylase). Variations encompassed by the compositions and methods of the present invention are described in detail throughout the specification and are summarized only in the following paragraphs.

As described above, the compositions and methods of the invention relate in one aspect to alpha-amylases derived or derivable from an alpha-amylase of bacillus species TS-23, including variants/mutants having altered properties relative to a parent amylase. The parent amylase is a hybrid or chimeric amylase of the parent TS-23 alpha-amylase described above and comprising at least a portion of the TS-23 alpha-amylase (e.g., the amino acid sequence of the mature polypeptide).

While Bacillus species TS-23 alpha-amylase (SEQ ID NO: 1) is used as a starting point for discussing variant amylases, it will be understood that other Bacillus species alpha-amylases having high homology to Bacillus species TS-23 alpha-amylase may be used as parent amylases without departing from the scope of the compositions and methods of the invention. This is particularly true for other naturally occurring Bacillus species alpha-amylases which differ only slightly from the Bacillus species TS-23 alpha-amylase and do not include the substitutions, deletions or insertions that are the subject of the present invention.

In a first aspect of the compositions and methods of the invention, there is provided a variant of a parent bacillus species strain alpha-amylase, wherein the variant comprises at least two of the following alterations:

(a) c terminal truncation;

(b) substitution of amino acid 201 (i.e., M201) with SEQ ID NO: 1 counting the amino acids; or

(c) Deletion of at least two residues selected from the group consisting of R180, S181, T182 and G183. Note that the numbering of the amino acid residues refers to SEQ ID NO: 1. in some embodiments, such changes include (a) and (b). In other embodiments, the changes comprise (a) and (c). In some embodiments, the variant may further comprise a substitution at one or more residues selected from the group consisting of residue 87, residue 225, residue 272, and residue 282. The variant amylase preferably has alpha-amylase activity. Other remaining amino acid sequences of the variant amylase may have amino acid sequences identical to SEQ ID NO: 1, is at least 85% amino acid sequence identity.

In a related aspect, a variant of a parent AmyTS23 a-amylase is provided, wherein the variant has an amino acid sequence at least 85% identical to the parent a-amylase and comprises a truncation of the C-terminus. The variant may have the sequence of SEQ ID NO: 2. The variant may have improved cleaning ability of starch stains in cold water compared to the parent amylase.

In some embodiments, variants comprising a C-terminal truncation may further comprise deletions of residues at positions R180 and S181 (see amino acid sequence of SEQ ID NO: 1). The resulting variants may have higher detergent stability than the parent amylase.

In some embodiments, a variant comprising a C-terminal truncation may further comprise a substitution of the residue at position 201 (see also the amino acid sequence of SEQ ID NO: 1). The resulting variants may have a higher oxidative stability than the parent amylase.

In some embodiments, any of the above variants may further comprise substitutions at one or more residues selected from the group consisting of residue 87, residue 225, residue 272, and residue 282.

1.8.1 stability

In the context of the variants described herein, mutations (including amino acid substitutions and deletions) important to obtain an altered stability (i.e., higher or lower) including any mutation listed in the section "altered properties", particularly an improved stability, at a particular elevated temperature (i.e., 70-120 ℃) and/or extreme pH (i.e., low or high pH, i.e., pH4-6 or pH8-11, respectively), particularly a free (i.e., unbound, and therefore in solution) calcium concentration of less than 60ppm, are included. Stability can be determined as described in the "methods" section below.

1.8.2Ca²⁺Stability of

Altered Ca²⁺Stability means that the enzyme is in Ca²⁺The stability under reduced conditions is improved, i.e. higher or lower stability. In the context of the presently described variants, for Ca altered²⁺Mutations important for stability (including amino acid substitutions and deletions) include any of the mutations listed in the section "altered Properties", the altered Ca²⁺Stability especially of increased Ca²⁺Stability, higher or lower stability at a particular high pH (i.e., pH 8-10.5).

1.8.3 specific activity

In a further aspect, important mutations, including amino acid substitutions and deletions, for obtaining variants exhibiting an altered specific activity, which means, in particular, an increased or decreased specific activity, in particular at 10-60 ℃, preferably 20-50 ℃, in particular 30-40 ℃, include any mutation listed in the "altered properties" section. Specific activity can be determined as described in the "methods" section below.

1.8.4 oxidative stability

The variant may also have an altered oxidative stability, in particular a higher oxidative stability, compared to the parent alpha-amylase. For example, increased oxidative stability is advantageous in detergent compositions, while decreased oxidative stability is advantageous in compositions for starch liquefaction. Oxidative stability can be measured as described in the "methods" section below.

Modified pH Spectrum

Important positions and mutations for the resulting variants with altered pH profiles, which refer to improved activity, particularly at exceptionally high pH (i.e., pH8-10.5) or low pH (i.e., pH4-6), include mutations of amino acid residues located near the active site residue.

Preferred specific mutations/substitutions are those listed above in the section "altered properties" for the position in question. Suitable assays are described in the "methods" section below.

1.8.6 Wash Performance

Important positions and mutations for the resulting variants with improved wash performance at particularly high pH (i.e., pH8.5-11) include the specific mutations/substitutions listed above in the "altered properties" section for the positions in question. Wash performance can be measured as described in the "methods" section below.

2. Method for preparing alpha-amylase variants

Thus, in one aspect, the invention provides for the production of recombinant forms of the alpha-amylase sequence of Bacillus species TS-23 strain, which include other previously identified amino acid substitutions, deletions, transversions, insertions, and combinations thereof, to produce variants of the alpha-amylase of Bacillus species TS-23 strain. These variants may have additional yield increases, increased pH stability, increased temperature stability, reduced Ca pair²⁺Increased specific activity, increased dishwashing or washing performance, increased solubility, increased storage stability, or a combination thereof. Methods of recombinantly producing variants can be performed using the sequences and vectors provided, or in other ways known in the art.

Several methods for introducing mutations into genes are known in the art. After a brief discussion of the cloning of the alpha-amylase encoding DNA sequence, the method of generating mutations at specific sites within the alpha-amylase coding sequence will be discussed.

2.1 cloning of DNA sequences encoding alpha-Amylase

The DNA sequence encoding the parent alpha-amylase may be isolated from any cell or microorganism producing the alpha-amylase by a variety of methods known in the art. First, a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA of the organism producing the alpha-amylase to be studied. Then, if the amino acid sequence of the alpha-amylase is known, homologous, labeled oligonucleotide probes can be synthesized for use in identifying clones encoding alpha-amylase from genomic libraries prepared from the organism in question. Alternatively, labeled oligonucleotide probes containing sequences homologous to known α -amylase genes can be used to identify probes encoding α -amylase clones by hybridization and washing under low stringency conditions.

Another method for identifying clones encoding alpha-amylase involves inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming alpha-amylase negative bacteria with the resulting pool of genomic DNA, and plating the transformed bacteria on agar containing a substrate for alpha-amylase, thereby identifying clones expressing alpha-amylase.

Alternatively, the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, for example the phosphoramidite method described in S.L. Beaucage and M.H. Caruthers, (1981), or the method described in Matthes et al (1984). In the phosphoramidite method, oligonucleotides are synthesized, for example on an automated DNA synthesizer, purified, annealed, ligated and cloned into appropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, and prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, fragments corresponding to various parts of the entire DNA sequence) by standard techniques. The DNA sequence may also be prepared by Polymerase Chain Reaction (PCR) using specific primers, as described, for example, in U.S. Pat. No.4,683,202 or R.K. Saiki et al (1988).

2.2 site-directed mutagenesis

Once the DNA sequence encoding the alpha-amylase has been isolated, the site of the desired mutation is determined, and the mutation can be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; the mutant nucleotide is inserted during oligonucleotide synthesis. In one particular method, a single-stranded DNA gap bridging the alpha-amylase coding sequence is formed in a vector carrying the alpha-amylase gene. The synthetic nucleotide with the desired mutation then anneals to the homologous site of the single-stranded DNA. The remaining gaps were then filled in with DNA polymerase I (Klenow fragment) and the construct was ligated using T4 ligase. Specific examples of such methods are described in Morinaga et al (1984). U.S. Pat. No.4,760,025 discloses a method of introducing oligonucleotides encoding multiple mutations by making minor changes in the expression cassette. However, a greater number of mutations can be introduced at any one time using the Morinaga method, as multiple oligonucleotides of various lengths can be introduced.

Another method for introducing mutations into DNA sequences encoding alpha-amylase is described in Nelson and Long (1989). It involves a three-step method for generating a PCR fragment containing a mutation to be introduced using a chemically synthesized DNA strand as a primer in a PCR reaction. From the PCR-generated fragment, a DNA fragment carrying the mutation may be isolated by cleavage with a restriction enzyme and then inserted into an expression plasmid.

Other methods of providing variants include gene shuffling, e.g., as described in WO95/22625(Affymax Technologies N.V.) or WO 96/00343(Novo Nordisk A/S), or other related techniques to obtain hybrid enzymes comprising the mutations in question, e.g., substitutions and/or deletions.

2.3 expression of alpha-amylase variants

DNA sequences encoding alpha-amylase variants produced by the methods described above, or other methods known in the art, can be used to express the variant amylases (i.e., enzymes) using expression vectors that typically include regulatory sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and optionally a repressor gene or various activator genes.

The recombinant expression vector carrying the DNA sequence encoding the alpha-amylase variant may be any vector which may conveniently be subjected to recombinant DNA procedures, the choice of vector often being dependent on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, which replicates the replication of an independent hand chromosome, e.g., a plasmid, a phage or an extrachromosomal element, a minichromosome, or an artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

In the vector, the DNA sequence should be operably linked to an appropriate promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing transcription of a DNA sequence encoding an alpha-amylase variant of the compositions and methods of the invention, particularly in a bacterial host, are: the promoter of the lac operon of Escherichia coli, the dagA promoter of the agarose gene of Streptomyces coelicolor, the promoter of the alpha-amylase gene (amyL) of Bacillus licheniformis, the promoter of the maltogenic amylase gene (amyM) of Bacillus stearothermophilus, the promoter of alpha-amylase (amyQ) of Bacillus amyloliquefaciens, and the promoters of the xylA and xylB genes of Bacillus subtilis, and the like. Examples of useful promoters for transcription in a fungal host are promoters derived from the genes encoding Aspergillus oryzae (A.oryzae) TAKA amylase, Rhizomucor miehei (Rhizomucor miehei) aspartate proteolytic enzyme, Aspergillus niger (Aspergillus niger) neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger glucoamylase, Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, or Aspergillus nidulans (A.dunalans) acetamidase.

The expression vector may also include a suitable transcription terminator operably linked to the DNA sequence encoding the alpha-amylase variant of the compositions and methods of the invention and, in eukaryotes, polyadenylation sequences. The termination sequence and polyadenylation sequence may suitably be derived from the same source as the promoter.

The vector may also comprise a DNA sequence permitting the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ 702.

The vector may also include a selectable marker, e.g., a gene the product of which complements a defect in the host cell, e.g., the dal genes from B.subtilis or B.licheniformis, or a gene which confers antibiotic resistance (e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance). Furthermore, the vector may comprise Aspergillus selection markers, such as amdS, argB, niaD and sC (markers giving rise to hygromycin resistance), or selection may be achieved by co-transformation, see, for example, WO 91/17243.

While intracellular expression may be beneficial in some respects, for example when using certain bacteria as host cells, extracellular expression is generally preferred. In general, the Bacillus alpha-amylases referred to herein include a proregion (preregion) that allows secretion of the expressed protease into the culture medium. If desired, the preregions may be replaced by different preregions or signal sequences, which may be conveniently effected by substitution of the DNA sequences encoding the respective preregions.

The procedures used to ligate the DNA construct encoding the alpha-amylase variant, the promoter, terminator and other elements, respectively, and to insert them into a suitable vector containing the information necessary for replication are well known in the art (see, e.g., Sambrook et al, Molecularclong: A LABORATORY MANL, 2 nd Ed., Cold Spring Harbor, 1989).

Advantageously, cells comprising the DNA construct or the expression vector are used as host cells for the recombinant production of the alpha-amylase variant. Cells can be conveniently transformed with the DNA constructs of the compositions and methods of the invention encoding the variants by integrating (in one or more copies) the DNA construct into the host chromosome. This integration is generally considered to be advantageous because the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA construct into the host chromosome may be achieved according to conventional methods, e.g.by homologous or heterologous recombination. Alternatively, the cells may be transformed with the above-described expression vectors in connection with different types of host cells. The cell may be a cell of a higher organism, such as a mammalian or insect cell, but is preferably a microbial cell, such as a bacterial or fungal (including yeast) cell.

Examples of suitable bacteria are: gram-positive bacteria, such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus (Geobacillus stearothermophilus), Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, Streptomyces lividans or Streptomyces murinus; or gram-negative bacteria, such as E.coli. Transformation of the bacteria can be carried out, for example, by protoplast transformation or in a known manner using competent cells.

Suitable yeast organisms may be selected from: saccharomyces species (Saccharomyces) or Schizosaccharomyces species (Schizosaccharomyces), such as Saccharomyces cerevisiae. The filamentous fungus may advantageously belong to the species Aspergillus (Aspergillus), such as Aspergillus oryzae (Aspergillus oryzae) or Aspergillus niger (Aspergillus niger). Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a known manner. Suitable methods for transformation of Aspergillus host cells are described in EP 238023.

In yet another aspect, a method of producing an alpha-amylase variant is provided, the method comprising culturing the host cell described above under conditions conducive to production of the variant, and recovering the variant from the cell and/or culture medium.

The medium used to culture the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the alpha-amylase variant. Suitable media are available from commercial suppliers or may be prepared according to published preparations, for example as described in catalogues of the American Type Culture Collection (ATCC).

The alpha-amylase variant secreted from the host cells may be conveniently recovered from the culture medium by well-known methods, including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by a salt such as ammonium sulphate, followed by the use of chromatographic methods such as ion exchange chromatography, affinity chromatography, and the like.

3. Industrial applications

The alpha-amylase variants of the invention have valuable properties for use in a variety of industrial applications. In particular, the enzyme variants are useful as ingredients in laundry, dishwashing and hard surface cleaning detergent compositions.

One or more variants with altered properties may be used in starch processing, particularly starch conversion, especially liquefaction of starch (see, e.g., U.S. Pat. No. 3,912,590, european patent applications 252730 and 63909, WO 99/19467 and WO 96/28567, all references herein incorporated by reference). Also relates to compositions of starch conversion interest which, in addition to variants of the compositions and methods of the invention, include glucoamylases, pullulanases and other alpha-amylases.

Additionally, one or more variants may also be used in the production of sweeteners and ethanol (see, e.g., U.S. Pat. No. 5,231,017, incorporated herein by reference), such as the production of fuel ethanol, potable ethanol, and industrial ethanol from starch or whole grain.

The variants of the invention may also be used for the desizing of textiles, fabrics and garments (see for example WO 95/21247, U.S. Pat. No.4,643,736 and EP 119,920, incorporated herein by reference); producing or brewing fermented mash; and pulp and paper production.

3.1 starch conversion

Conventional starch conversion processes, such as liquefaction and saccharification processes, are described, for example, in U.S. Pat. No. 3,912,590 and European patent publications 252,730 and 63,909, which are incorporated herein by reference.

In one embodiment, the starch conversion process to degrade starch to lower molecular weight carbohydrate components (e.g., sugar or fat substitutes) includes a debranching step.

3.2 conversion of starch to sugar

In the case of the conversion of starch to sugar, the starch is depolymerized. Such depolymerization process may consist of a pretreatment step and two or three consecutive steps, such as a liquefaction step, a saccharification step, and optionally an isomerization process depending on the desired end product.

3.3 pretreatment of native starch

Native starch consists of microscopic particles, which are insoluble in water at room temperature. When the aqueous starch slurry is heated, the particles swell and eventually break, releasing starch molecules into solution. During this "gelatinization" process, the viscosity increases dramatically. Since in typical industrial processes the solids level is 30-40%, the starch has to be thinned or "liquefied" to enable it to be handled. This reduction in viscosity is currently obtained in large part by enzymatic degradation.

3.4 liquefaction

In the liquefaction step, the long-chain starch is degraded by alpha-amylase into branched and linear shorter units (maltodextrins). The liquefaction process is typically carried out at 105 ℃ and 110 ℃ for 5-10 minutes and then at 95 ℃ for 1-2 hours at a pH between 5.5 and 6.2. To ensure optimal enzyme stability under these conditions, 1mM calcium (40ppm free calcium ion) was added. After this treatment, the liquefied starch will have a "dextrose equivalent" (DE) of 10-15.

3.5 saccharification

After the liquefaction step, by adding a glucoamylase (e.g., OPTIDEX)L-400) and debranching enzymes (e.g., isoamylase (u.s.pat. No.4,335,208) or pullulanase) to convert maltodextrins to glucose. Prior to this step, the pH is lowered to a value below 4.5, and a high temperature (above 95 ℃) is maintained to inactivate the liquefying α -amylase, in order to reduce the formation of short chain oligosaccharides called "panose precursors", which cannot be correctly hydrolyzed by the branching enzymes.

Glucoamylase and debranching enzyme may be added to bring the temperature down to 60 ℃. The saccharification step is typically carried out for 24-72 hours.

Typically, when the alpha-amylase is denatured after the liquefaction step, about 0.2-0.5% of the saccharification product is a branched trisaccharide, Glc p α 1-6Glc p α 1-4Glc (panose), which cannot be degraded by pullulanase. If the active amylase from the liquefaction step is present in the saccharification step, i.e.not denatured, the level of this sugar can be as high as 1-2%, which is a highly undesirable result as it greatly reduces the yield of the saccharification.

3.6 isomerization

When the desired final sugar product is, for example, high fructose syrup, the glucose syrup can be converted to fructose. After the saccharification process, the pH is raised to a value in the range of 6-8, preferably pH7.5, and calcium is removed by ion exchange. The glucose syrup is then treated, for example, with an immobilized glucose isomerase (e.g., GENSWEET)IGI-HF) into high fructose syrup.

3.7 ethanol production

The production of ethanol from whole grain can generally be divided into four main steps: grinding; liquefying; saccharification and fermentation.

3.7.1 grinding

Milling the grain opens up its structure, allowing further processing. Two milling methods that can be used are wet milling or dry milling. In the dry milling process, the entire core is milled and used in the remainder of the process. The wet milling process provides good separation of germ and flour (starch granules and protein) and, with few exceptions, is used in parallel syrup production.

3.7.2 liquefaction

During this liquefaction, the starch granules are solubilized by hydrolysis into mostly maltodextrins with a DP higher than 4. The hydrolysis may be carried out by acid treatment or by enzymatic treatment with alpha-amylase. Acid hydrolysis is used on a limited matrix. The feedstock may be milled whole grain or a side stream (by-product) of starch processing.

Enzymatic liquefaction is typically carried out as a three-step hot slurry process. The slurry is heated to 60-95 deg.C, preferably 80-85 deg.C, and enzyme is added. The slurry is then jet cooked at 95-140 deg.C, preferably 105-125 deg.C, cooled to 60-95 deg.C, and more enzyme is added to obtain the final hydrolysis. The liquefaction process is carried out at a pH of 4.5-6.5, typically at a pH between 5 and 6. The milled and liquefied grain is also known as mash.

3.7.3 saccharification

For producing low-molecular sugar DP which can be metabolized by yeasts_1-3The maltodextrin produced in the liquefaction must be further hydrolyzed. Typically, hydrolysis is carried out enzymatically, by glucoamylase, alternative alpha-glucosidases or acid alpha-amylases may also be used. The complete saccharification step may last up to 72 hours, but usually only a pre-saccharification of typically 40-90 minutes is performed, followed by a complete saccharification (SSF) in the fermentation. The saccharification is usually carried out at 30-65 ℃ and generally at about 60 ℃ and pH 4.5.

3.7.4 fermentation

Yeasts from Saccharomyces species are typically added to the mash and fermented for 24-96 hours, for example typically 35-60 hours. The temperature is between 26-34 deg.C, usually about 32 deg.C, and the pH is 3-6, preferably about 4-5.

It is noted that a widely used process is a Simultaneous Saccharification and Fermentation (SSF) process, where there is no holding period for saccharification, meaning that yeast and enzyme are added together. When SSF is performed, a pre-saccharification step is typically introduced at a temperature above 50 ℃ just prior to fermentation.

3.8 distillation

After fermentation, the mash is distilled to extract ethanol. The ethanol obtained according to the method can be used, for example, as fuel ethanol; the neutral alcoholic beverage can be drunk by drinking ethanol; or industrial ethanol.

3.9 by-products

The remaining grain of the fermentation is usually used as animal feed in liquid or dry form.

Further details of how liquefaction, saccharification, fermentation, distillation and ethanol recovery are known to those skilled in the art.

According to the methods described herein, saccharification and fermentation can be performed simultaneously or separately.

3.10 pulp and paper production

The alpha-amylase of the invention may also be used for the production of lignocellulosic materials, such as pulp, paper and cardboard, from starch-enhanced waste paper and cardboard, especially when repulping is performed at a pH above 7, the amylase promotes disintegration of the waste material by degradation of the enhanced starch. Alpha-amylases are particularly useful in processes for making papermaking pulp from starch coated printing paper. The process may be carried out as described in WO95/14807, comprising the steps of:

a) decomposing paper to produce paper pulp;

b) treating with a starch degrading enzyme before, during or after step a); and

C) separating the ink particles from the pulp after steps a) and b).

The alpha-amylases described herein may also be used to modify starch, enzymatically modified starch with alkaline fillers such as calcium carbonate, kaolin and clay for use in papermaking. With the alpha-amylase of the compositions and methods of the invention, it is possible to modify starch in the presence of fillers, thus allowing for a simpler integrated process.

3.11 desizing of textiles, fabrics and garments

The alpha-amylases of the invention may also be used for desizing of textiles, fabrics or garments. In the textile processing industry, alpha-amylases are commonly used as an adjuvant in desizing processes to facilitate the removal of starch-containing size, which size functions as a protective coating on yarns during weaving. Complete removal of the size coating after weaving is important to ensure optimal results for subsequent processes in which the fabric is washed, bleached and dyed. Enzymatic starch breakdown is preferred as this does not have any detrimental effect on the textile material. In order to reduce processing costs and increase grinding throughput, desizing processes are sometimes performed in conjunction with washing and bleaching steps. In this case, non-enzymatic auxiliaries, such as bases or oxidizing agents, are usually used to break down the starch, since classical α -amylases are very unsuitable for high pH and bleaching agents. Non-enzymatic breakdown of starch slurry may result in some fiber damage because of the more aggressive chemicals used. Therefore, alpha-amylase variants using the compositions and methods of the invention would be desirable because of their improved performance in alkaline solutions. The alpha-amylase may be used alone or in combination with cellulase when desizing cellulose containing fabrics or textiles.

Desizing and bleaching processes are well known in the art. This process is described, for example, in WO 95/21247, u.s.pat. No.4,643,736 and EP 119,920, which are incorporated herein by reference. Commercial products for desizing include OPTISIZE from GenencorFLEX。

The invention also relates to compositions and methods for treating fabrics (e.g., textile desizing) using one or more bacillus species TS-23 strain alpha-amylases or variants thereof. It is well known in the art that the enzyme can be used in any fabric treatment process, see for example U.S. Pat. No. 6,077,316. For example, in one aspect, the hand and appearance of a fabric is improved by a method comprising contacting the fabric with a solution of bacillus species TS-23 strain alpha-amylase or variant thereof. In one aspect, the fabric is treated with the solution under pressure.

In one aspect, the enzyme is used during or after textile weaving, or in a desizing stage, or in one or more additional fabric processing steps. During textile weaving, the yarns are exposed to considerable mechanical tension. Prior to weaving on a mechanical loom, the warp yarns are typically coated with a starch or starch derivative size to increase their tensile strength and prevent breakage. These enzymes may be applied to remove these starch or starch derivative slurries. After the textile is woven, the fabric may enter a desizing stage. This may be followed by one or more further fabric processing steps. Desizing is the act of removing size from a textile. After weaving the size coat must be removed before further processing of the fabric to ensure a uniform and wash-durable effect. Also provided herein are desizing methods comprising enzymatic hydrolysis of the above-described slurry by the action of a bacillus species TS-23 strain alpha-amylase or variant thereof.

The enzyme may be used as a detergent additive, alone or in combination with other desizing chemicals and/or desizing enzymes, to desize fabrics, including cotton-containing fabrics, in, for example, aqueous compositions. The bacillus species TS-23 strain alpha-amylase or variants thereof can also be used in compositions and methods for producing a stonewashed appearance on indigo dyed denim fabric and clothing. To produce a garment, the fabric may be cut and sewn into a garment or garment, followed by finishing. In particular, for the production of denim jeans (denim jeans), different enzymatic finishing processes have been developed. The finishing of denim garments usually starts with an enzymatic desizing step during which amylolytic enzymes act on the garment to soften the fabric and make the cotton more susceptible to a subsequent enzymatic finishing step. The enzymes can be used in processes for finishing denim garments (e.g. "bio-polishing"), enzymatic desizing and providing softness to fabrics, and/or finishing. The dosage of amylase varies with the type of process. Smaller doses will require more time than larger doses of the same enzyme. However, there is no upper limit to the amount of desizing amylase present, other than controlled by the physical constraints of the solution. Thus, the limit of the enzyme may be an amount that can be dissolved in a solution. Typically, a desizing enzyme, such as an alpha-amylase, is added to the treatment composition in an amount from about 0.00001% to about 2% enzyme protein, from about 0.0001% to about 1% enzyme protein, from about 0.001% to about 0.5% enzyme protein, by weight of the fabric, and in another embodiment, may be in an amount from about 0.01% to about 0.2% enzyme protein, by weight of the fabric.

3.12 production of beer

The variant alpha-amylases provided herein can also be used in a beer production process; alpha-amylase is typically added during starch saccharification.

3.13 detergent compositions

The alpha-amylase variants described herein may be incorporated into and thus become an ingredient of a detergent composition.

For example, the detergent compositions provided herein can be formulated as hand or machine laundry detergent compositions, including laundry additive compositions suitable for pretreating soiled fabrics and rinse added fabric softener compositions, or as detergent compositions for general household hard surface cleaning operations, or as dishwashing operations for hand or machine washing.

In a particular aspect, there is provided a detergent additive comprising a variant enzyme as described herein. The detergent additive and detergent composition may comprise one or more other enzymes, such as a protease, a lipase, a peroxidase, another amylolytic enzyme (e.g. another alpha-amylase), a glucoamylase, a maltogenic amylase, a cgtase and/or a cellulase, a mannanase (e.g. MANNASTAR ex Danisco u.s.a.inc., Genencor Division)^TM) Pectinase, pectin lyase, cutinase and/or laccase.

Generally, the properties of the enzyme selected should be compatible with the detergent selected (i.e., pH optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme should be present in an effective amount.

Protease: suitable proteases include those of animal, vegetable or microbial origin. Preferably of microbial origin. Chemically modified or protein engineered mutants are included. The protease may be a serine protease or a metalloprotease, preferably an alkaline microbial protease or a trypsin-like protease or a chymotrypsin-like protease. Examples of alkaline proteases are subtilisins, in particular those derived from Bacillus, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described, for example, in WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g.of porcine or bovine origin), and the Fusarium proteases described in WO 89/06270 and WO 94/25583.

Examples of useful proteases also include, but are not limited to, the variants described in WO98/23732, WO99/20770, WO 92/19729, WO 98/20115, WO 98/20116 and WO 98/34946, in particular variants having substitutions in one or more of the following positions: 27. 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, and 274.

Exemplary commercially available proteases include ALCALASESAVINASEPRIMASEDURALASEESPERASEAnd KANNASE(purchased from Novozymes A/S), MAXATASEMAXACAL、MAXAPEMPROPERASEPURAFECTPURAFECT OXPFN2FN3And FN4(Genencor)。

Lipase: suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include, but are not limited to, lipases from the genus Humicola (Humicola), synonymous with Thermomyces (Thermomyces), e.g. from Humicola lanuginosa (h.lanuginosa) (t.lanuginosus) (see EP258068 and EP 305216) or from Humicola insolens (h.insolens) (as described in WO 96/13580); lipases from the genera Pseudomonas (Pseudomonas) such as from Pseudomonas alcaligenes (P.alcaligenes) or Pseudomonas pseudoalcaligenes (EP 218272), Pseudomonas cepacia (P.cepacia) (EP 331376), Pseudomonas stutzeri (P.stutzeri) (GB 1,372,034), Pseudomonas fluorescens (P.fluoroscens), Pseudomonas sp. SD 705(WO 95/06720 and WO 96/27002), P.wisconsinensis (WO 96/12012), lipases from the genera Bacillus such as Bacillus subtilis (Dartois et al, (1993), Biochemica et Biophysica Acta, 1131: 253 and 360, Bacillus stearothermophilus (B.stearothermophilus) (JP 64/744992) or Bacillus pumilus (B.puuus) (WO 91/4022) other lipases which can be considered for use in formulations include, for example, variants such as those from Pseudomonas aeruginosa (P.alcaligenes) or Pseudomonas pseudoalcaligenes (EP 397264, EP 727264, EP 72387) which can be used in formulations such as Bacillus subtilis (Dartosis, Pseudomonas fluorescens, Pseudomonas aeruginosa (WO 3911/4022), and Bacillus stearothermophilus (Bacillus stearothermophilus) are described in this disclosure, Those described in WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.

Some commercially available lipases include LIPOLASE^TMAnd LIPOLASE ULTRA^TM(Novozymes，A/S)

A polyesterase: suitable polyesterases may be included in the composition. Suitable polyesterases include, for example, those described in WO 01/34899 and WO 01/14629.

Amylase: one or more additional amylases (in addition to the amylase variants described herein) may also be included. Suitable amylases (. alpha.and/or. beta.) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases from Bacillus, such as the particular strain of Bacillus licheniformis more particularly described in GB 1,296,839. Examples of useful alpha-amylases are the variants described in WO 94/18314, WO 96/39528, WO94/02597, WO 94/18314, WO 96/23873 and WO 97/43424, in particular variants having substitutions at one or more of the following positions: 15. 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.

The commercially available alpha-amylase is DURAMYL^TM、LIQUEZYME^TMTERMAMYL^TM、NATALASE^TM、STAINZYME^TM PLUS、STAINZYME^TMULTRA、FUNGAMYL^TMAnd BAN^TM(Novozymes A/S)、RAPIDASE^TMAnd PURASTAR^TM(available from Genencor).

Cellulase: cellulase may be added to the composition. Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include, but are not limited to, cellulases from the genera bacillus, pseudomonas, trichoderma, humicola, fusarium, Thielavia (Thielavia), Acremonium (Acremonium), e.g., as disclosed in U.S. Pat. nos. 4,435,307; 5,648,263; 5,691,178; 5,776,757 and WO89/09259 disclose fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum. Examples of trichoderma reesei cellulases are disclosed in us patents 4,689,297, 5,814,501 and 5,324,649, and WO 92/06221 and WO 92/06165. Exemplary bacillus cellulases are disclosed in us patent 6,562,612.

Commercially available cellulases include CELLUZYMEAnd CAREZYME(Novozymes A/S)，CLAZINASEAnd PURADAX HA(Genencor International Inc.) and KAC-500(B)(Kao Corporation)。

Peroxidase/oxidase: suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus (e.g.Coprinus cinereus (C.cinereus)) and variants thereof as described in WO 93/24618, WO 95/10602 and WO 98/15257.

Commercially available peroxidases include GUARDZYME(Novozymes A/S)。

Detergent enzymes may be included in detergent compositions by the addition of separate additives containing one or more enzymes, or by the addition of additives including combinations of all of these enzymes. The compositions of the invention and the detergent additives of the invention, i.e. separate additives or combined additives, may be formulated, for example, as granules, liquids, slurries and the like. Preferred detergent additive formulations are: of the granular type, in particular dust-free granules; liquids, in particular stabilized liquids; or a slurry.

Non-dusting granules can be produced, for example, as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 and can optionally be coated by methods known in the art. Examples of waxy coating materials are polyethylene oxide products (polyethylene glycol, PEG) having an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenols having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are from 15 to 80 ethylene oxide units; a fatty alcohol; a fatty acid; and monoacylglycerols and diacylglycerols and triacylglycerols of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given, for example, in GB 1483591. The liquid enzyme preparation may be stabilized in accordance with established methods, for example by adding polyols such as propylene glycol, sugars or sugar alcohols, lactic acid or boric acid. The protected enzymes may be prepared according to the methods disclosed in EP 238,216, for example.

Generally, the detergent composition may be in any convenient form, such as a bar, tablet, powder, granule, paste or liquid. Liquid detergents may be aqueous, typically containing up to about 70% water, and 0% to about 30% organic solvent. The concentrated detergent gel contains, for example, about 30% or less water.

The detergent composition may comprise one or more surfactants which may be non-ionic, including semi-polar and/or anionic and/or cationic and/or zwitterionic. The surfactant is typically present at a level of 0.1% to 60% by weight.

When included in a detergent, the detergent will typically contain from about 1% to about 40% anionic surfactant, such as linear alkylbenzene sulfonates, alpha-olefin sulfonates, alkyl sulfates (fatty alcohol sulfates), alcohol ethoxy sulfates, secondary alkyl sulfonates, alpha-sulfo fatty acid methyl esters, alkyl or alkenyl succinic acids, or soaps.

When included in a detergent, the detergent will typically contain from about 0.2% to about 40% of a nonionic surfactant, such as an alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or an N-acyl N-alkyl derivative of glucosamine ("glucamide").

The detergent may contain 0% to 65% of a detergent builder or complexing agent (complexing agent), such as a zeolite, diphosphate, triphosphate, phosphate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl-or alkenylsuccinic acid, soluble silicate or layered silicate (e.g. SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples are carboxymethylcellulose, polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, poly (vinylpyridine-N-oxide), polyvinylimidazole, polycarboxylates (e.g.polyacrylates, maleic/acrylic acid copolymers) and lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system, which may include H₂O₂Sources (e.g. perborate or percarbonate) which can be formed withPeracid bleach activators such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate are used in combination. Alternatively, the bleaching system may comprise peroxyacids (e.g. of the amide, imide or sulfone type). The bleaching system may also be an enzymatic bleaching system. See, for example, WO 05/056782.

Conventional stabilizers may be used to stabilize the enzymes in the detergent compositions of the present compositions and methods, such as: a polyol (such as propylene glycol or glycerol), a sugar or sugar alcohol, lactic acid, boric acid or a boric acid derivative (e.g., an aromatic ester of boric acid), or a phenyl boronic acid derivative (e.g., 4-formylphenyl boronic acid). The compositions may be formulated as described in WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredients such as fabric conditioners including clays, foaming agents, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors or perfumes.

It is presently believed that in detergent compositions, especially the Bacillus species TS-23 strain alpha-amylase or variant thereof may be added in an amount corresponding to from about 0.01 to about 100mg enzyme protein per liter of wash liquor (e.g., from about 0.05 to about 5.0mg enzyme protein per liter of wash liquor, or from about 0.1 to about 1.0mg enzyme protein per liter of wash liquor).

One or more of the variant enzymes described herein may additionally be added to the detergent formulations disclosed in WO 97/07202, which is incorporated herein by reference.

4. Composition and use

The variant enzyme(s) described herein may also be used in detergents (especially laundry and dishwashing detergent compositions), hard surface cleaning compositions, in processes using alpha-amylase, in fabric, textile or laundry desizing compositions, in the production of pulp and paper, beer production, ethanol production and starch conversion processes as described above.

4.1 laundry detergent compositions and uses

According to this embodiment, typically one or more bacillus species TS-23 strain alpha-amylases, or variants thereof, may be a component of a laundry detergent composition. As such, it may be included in the detergent composition in the form of a non-dusting granulate, a stable liquid, or a protected enzyme. The dried formulation may be in the form of granules or microparticles. Non-dusting granules may be produced, for example, as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452, and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly (ethylene oxide) products (polyethylene glycol, PEG) having an average molar mass of 1,000 to 20,000; ethoxylated nonylphenols having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are from 15 to 80 ethylene oxide units; a fatty alcohol; a fatty acid; and monoacylglycerols and diacylglycerols and triacylglycerols of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given, for example, in UK patent 1483591. The liquid enzyme preparation may be stabilized in accordance with established methods, for example by adding polyols such as propylene glycol, sugars or sugar alcohols, lactic acid or boric acid. Other enzyme stabilizers are well known in the art. The protected enzymes may be prepared, for example, according to the methods disclosed in European application No.238,216. Polyols have long been recognized as stabilizers for proteins, and for improving the solubility of proteins. See, e.g., J.K.Kaushik et al, "where a trehalose an empirical protein stabilization of the thermal stability of proteins in the presence of the compatible acidic trehalose," J.biol.chem.278: 26458-65(2003) and the references cited therein; and Monica Conti et al, "Capillary isoelectric focusing: the recipe of protein solubility, "J.chromatography 757: 237-245(1997).

The composition may comprise as a major enzyme component the Bacillus species TS-23 strain alpha-amylase or variant thereof, e.g.a one-component composition. Alternatively, the composition can include a variety of enzymatic activities, such as aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase, as well as other enzymes described below. Other enzymes can be produced by microorganisms belonging to the genera: aspergillus, trichoderma, huminase (e.g., humicola insolens) and Fusarium (Fusarium). Exemplary members of the genus Aspergillus include Aspergillus aculeatus (Aspergillus aculeatus), Aspergillus awamori (Aspergillus awamori), Aspergillus niger (Aspergillus niger) or Aspergillus oryzae (Aspergillus oryzae). Exemplary members of the genus Fusarium include Fusarium bactridioides (Fusarium bactridioides), Fusarium cerealis, Fusarium crookwellense (Fusarium culmorum), Fusarium graminearum (Fusarium graminearum), Fusarium graminum (Fusarium graminum), Fusarium heterosporum (Fusarium heterosporum), Fusarium negundi (Fusarium negundim), Fusarium oxysporum (Fusarium oxysporum), Fusarium reticulatum (Fusarium reticulatum), Fusarium roseum (Fusarium roseum), Fusarium sambucinum (Fusarium sambucinum), Fusarium sarcochroum (Fusarium sarcinatum), Fusarium suzurum (Fusarium suum), Fusarium sapphium, Fusarium trichothecioides (Fusarium) or Fusarium trichothecioides.

The detergent composition may be in any useful form, such as a powder, granule, paste or liquid. Liquid detergents may be aqueous, typically containing up to about 70% water, and 0% to about 30% organic solvent. It is also possible to have a detergent composition in the form of a concentrated gel comprising only about 30% water. The enzyme may be used in any detergent composition that is compatible with the stability of the enzyme. The enzymes can generally be protected from the harmful components in known encapsulated forms, for example by granulation or isolation in hydrogels. Enzymes and in particular alpha-amylases are not limited to laundry and dishwashing applications but may also be used in surface cleaners and in the production of ethanol from starch or biomass.

The detergent composition may comprise one or more surfactants, each of which may be anionic, nonionic, cationic or zwitterionic. The detergent will typically comprise from 0% to about 50% of an anionic surfactant, such as linear alkyl benzene sulphonate (LAS); alpha-olefin sulfonates (AOS); alkyl sulfates (fatty Alcohol Sulfates) (AS); alcohol ethoxy sulfate (AEOS or AES); secondary Alkyl Sulfonates (SAS); alpha-sulfo fatty acid methyl ester; alkyl or alkenyl succinic acids or soaps. The composition may also comprise from 0% to about 40% of a nonionic surfactant, such as an alcohol ethoxylate (AEO or AE), a carboxylated alcohol ethoxylate, a nonylphenol ethoxylate, an alkylpolyglycoside, an alkyldimethylamine oxide, an ethoxylated fatty acid monoethanolamide, a fatty acid monoethanolamide, or a polyhydroxyalkyl fatty acid amide (as described, for example, in WO 92/06154).

The detergent composition may additionally comprise any combination of one or more other enzymes, such as lipases, cutinases, proteases, cellulases, peroxidases, and/or laccases. As described above.

The detergent may optionally contain from about 1% to about 65% of a detergent builder or complexing agent, such as a zeolite, diphosphate, triphosphate, phosphate, citrate, nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTMPA), alkyl-or alkenylsuccinic acid, soluble silicate or layered silicate (e.g., SKS-6 from Hoechst). The detergent may also be builder-free, i.e. essentially free of detergent builder.

The detergent may optionally include one or more polymers. Examples include carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.

The detergent may optionally containBleaching system, which can comprise H₂O₂A source (e.g. perborate or percarbonate) which can be used in conjunction with a peracid-forming bleach activator such as Tetraacetylethylenediamine (TAED) or Nonanoyloxybenzenesulfonate (NOBS). Alternatively, the bleaching system may comprise peroxyacids (e.g. of the amide, imide or sulfone type). The bleaching system may also be an enzymatic bleaching system, wherein a perhydrolase enzyme activates peroxides, such as those described in WO 2005/056783.

Conventional stabilizers may be used to stabilize enzymes in detergent compositions, for example: a polyol (such as propylene glycol or glycerol), a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative (e.g., an aromatic ester of boric acid). The compositions may be formulated as described in WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredients such as fabric conditioners including clays, foaming agents, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners or perfumes.

The pH (measured in aqueous solution at the use concentration) is typically neutral or basic, e.g., a pH of about 7.0 to about 11.0.

Specific forms in which detergent compositions comprising the bacillus species TS-23 strain alpha-amylase or variant thereof may be formulated include:

1) a detergent composition in the form of granules having a bulk density (bulk density) of at least 600g/L comprising from about 7% to about 12% linear alkylbenzene sulphonate calculated as acid; about 1% to about 4% alcohol ethoxy sulfate (e.g., C)_12-18Alcohols, 1-2 Ethylene Oxide (EO)) or alkyl sulfates (e.g. C)_16-18) (ii) a About 5% to about 9% alcohol ethoxylate (e.g. C)_14-15Alcohol, 7 EO); about 14% to about 20% sodium carbonate (e.g., Na)₂CO₃) (ii) a About 2% to about 6% soluble silicate (e.g., Na)₂O，2SiO₂) (ii) a About 15% to about 22% of a zeolite (e.g., NaAlSiO @)₄) (ii) a 0% to about 6% sodium sulfate (e.g., Na)₂SO₄) (ii) a About 0% to about 15% sodium citrate/citric acid (e.g., C)₆H₅Na₃O₇/C₆H₈O₇) (ii) a Sodium perborate (e.g., NaBO) from about 11% to about 18%₃H₂O); about 2% to about 6% TAED; 0% to about 2% carboxymethylcellulose (CMC); 0-3% of a polymer (e.g. maleic/acrylic acid copolymer, PVP, PEG); 0.0001-0.1% protein enzyme (calculated as pure enzyme); and 0 to 5% of minor ingredients (e.g. suds suppressors, perfumes, optical brighteners, photobleaches).

2) A detergent composition in the form of granules having a bulk density of at least 600g/L comprising from about 6% to about 11% linear alkylbenzene sulphonate (calculated as acid); about 1% to about 3% alcohol ethoxy sulfate (e.g., C)_12-18Alcohols, 1-2EO) or alkyl sulfates (e.g. C)_16-18) (ii) a About 5% to about 9% alcohol ethoxylate (e.g. C)_14-15Alcohol, 7 EO); about 15% to about 21% sodium carbonate (e.g., Na)₂CO₃) (ii) a About 1% to about 4% soluble silicate (e.g., Na)₂O，2SiO₂) (ii) a About 24% to about 34% of a zeolite (e.g., NaAlSiO @)₄) (ii) a About 4% to about 10% sodium sulfate (e.g., Na)₂SO₄) (ii) a 0% to about 15% sodium citrate/citric acid (e.g. C)₆H₅Na₃O₇/C₆H₈O₇) (ii) a 0% to about 2% carboxymethylcellulose (CMC); 1-6% of a polymer (e.g. maleic/acrylic acid copolymer, PVP, PEG); 0.0001-0.1% enzyme (calculated as pure enzyme protein); 0 to 5% of minor ingredients (e.g. suds suppressors, perfume).

3) A detergent composition in the form of granules having a bulk density of at least 600g/L comprising from about 5% to about 9% linear alkylbenzene sulphonate (calculated as acid); about 7% to about 14% alcohol ethoxylate (e.g. C)_12-15Alcohol, 7 EO); about 1% to about 3% of soap such as fatty acid (e.g. C)_16-22Fatty acids); about 10% to about 17% sodium carbonate (e.g., Na)₂CO₃) (ii) a About 3% to about 9% of a soluble silicate (e.g., Na)₂O，2SiO₂) (ii) a About23% to about 33% of a zeolite (e.g., NaAlSiO @)₄) (ii) a 0% to about 4% sodium sulfate (e.g., Na)₂SO₄) (ii) a Sodium perborate (e.g., NaBO) from about 8% to about 16%₃H₂O); about 2% to about 8% TAED; 0% to about 1% phosphate (e.g., EDTMPA); 0% to about 2% carboxymethylcellulose (CMC); 0-3% of a polymer (e.g. maleic/acrylic acid copolymer, PVP, PEG); 0.0001-0.1% enzyme (calculated as pure enzyme protein); 0 to 5% of minor ingredients (e.g. suds suppressors, perfume, optical brighteners).

4) A detergent composition in the form of granules having a bulk density of at least 600g/L comprising from about 8% to about 12% linear alkylbenzene sulphonate (calculated as acid); about 10% to about 25% alcohol ethoxylate (e.g. C)_12-15Alcohol, 7 EO); about 14% to about 22% sodium carbonate (e.g., Na)₂CO₃) (ii) a About 1% to about 5% soluble silicate (e.g., Na)₂O，2SiO₂) (ii) a About 25% to about 35% of a zeolite (e.g., NaAlSiO @)₄) (ii) a 0% to about 10% sodium sulfate (e.g., Na)₂SO₄) (ii) a 0% to about 2% carboxymethylcellulose (CMC); 1-3% of a polymer (e.g. maleic/acrylic acid copolymer, PVP, PEG); 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0 to 5% minor ingredients (e.g. suds suppressors, perfume).

5) An aqueous liquid detergent composition comprising from about 15% to about 21% linear alkylbenzene sulfonate (calculated as acid); about 12% to about 18% alcohol ethoxylate (e.g. C)_12-15Alcohol, 7EO or C_12-15Alcohol, 5 EO); from about 3% to about 13% soap such as fatty acids (e.g., oleic acid); 0% to about 13% of alkenyl succinic acid (C)_12-14) (ii) a About 8% to about 18% aminoethanol; about 2% to about 8% citric acid; 0% to about 3% phosphate ester; 0% to about 3% of a polymer (e.g., PVP, PEG); 0% to about 2% of a borate (e.g. B)₄O₇) (ii) a 0% to about 3% ethanol; about 8% to about 14% propylene glycol; 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0 to 5% of minor ingredients (e.g. dispersants, suds suppressors, perfumes, optical brighteners).

6) An aqueous structured liquid detergent composition comprising from about 15% to about 21% linear alkylbenzene sulfonate (calculated as acid); 3-9% alcohol ethoxylate (e.g. C)_12-15Alcohol, 7EO or C_12-15Alcohol, 5 EO); from about 3% to about 10% soap such as fatty acids (e.g., oleic acid); about 14% to about 22% of a zeolite (e.g., NaAlSiO @)₄) (ii) a From about 9% to about 18% potassium citrate; 0% to about 2% of a borate (e.g. B)₄O₇) (ii) a 0% to about 2% carboxymethylcellulose (CMC); 0% to about 3% of a polymer (e.g., PEG, PVP); 0% to about 3% of an anchoring polymer (e.g., lauryl methacrylate/acrylic acid copolymer; molar ratio 25: 1, MW 3800); 0% to about 5% glycerin; 0.0001-0.1% enzyme (calculated as pure enzyme protein); and from 0% to 5% of minor ingredients (e.g. dispersants, suds suppressors, perfumes, optical brighteners).

7) A detergent composition in the form of granules having a bulk density of at least 600g/L comprising from about 5% to about 10% fatty alcohol sulfate; from about 3% to about 9% ethoxylated fatty acid monoethanolamide; 0-3% soap such as fatty acid; about 5% to about 10% sodium carbonate (e.g., Na)₂CO₃) (ii) a About 1% to about 4% soluble silicate (e.g., Na)₂O，2SiO₂) (ii) a About 20% to about 40% of a zeolite (e.g., NaAlSiO @)₄) (ii) a About 2% to about 8% sodium sulfate (e.g., Na)₂SO₄) (ii) a Sodium perborate (e.g., NaBO) from about 12% to about 18%₃H₂O); about 2% to about 7% TAED; about 1% to about 5% of a polymer (e.g., maleic/acrylic acid copolymer, PEG); 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0 to 5% of minor ingredients (e.g. optical brighteners, suds suppressors, perfumes).

8) A detergent composition in the form of granules comprising from about 8% to about 14% linear alkylbenzene sulfonate (calculated as acid); from about 5% to about 11% ethoxylated fatty acid monoethanolamide; from 0% to about 3% soap such as fatty acids; about 4% to about 10% sodium carbonate (e.g., Na)₂CO₃) (ii) a About 1% to about 4% soluble silicic acidSalt (Na)₂O，2SiO₂) (ii) a About 30% to about 50% of a zeolite (e.g., NaAlSiO @)₄) (ii) a About 3% to about 11% sodium sulfate (e.g., Na)₂SO₄) (ii) a About 5% to about 12% sodium citrate (e.g., C)₆H₅Na₃O₇) (ii) a About 1% to about 5% of a polymer (e.g., PVP, maleic/acrylic acid copolymer, PEG); 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0 to 5% minor ingredients (e.g. suds suppressors, perfume).

9) A detergent composition in the form of granules comprising from about 6% to about 12% linear alkylbenzene sulfonate (calculated as acid); from about 1% to about 4% of a nonionic surfactant; from about 2% to about 6% soap such as fatty acid; about 14% to about 22% sodium carbonate (e.g., Na)₂CO₃) (ii) a About 18% to about 32% of a zeolite (e.g., NaAlSiO @₄) (ii) a About 5% to about 20% sodium sulfate (e.g., Na)₂SO₄) (ii) a About 3% to about 8% sodium citrate (e.g., C)₆H₅Na₃O₇) (ii) a About 4% to about 9% sodium perborate (e.g., NaBO)₃H₂O); from about 1% to about 5% of a bleach activator (e.g., NOBS or TAED); 0% to about 2% carboxymethylcellulose (CMC); about 1% to about 5% of a polymer (e.g., polycarboxylate or PEG); 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0 to 5% of minor ingredients (e.g. optical brighteners, perfumes).

10) An aqueous liquid detergent composition comprising from about 15% to about 23% linear alkylbenzene sulfonate (calculated as acid); from about 8% to about 15% alcohol ethoxy sulfate (e.g., C)_12-15Alcohol, 2-3 EO); about 3% to about 9% alcohol ethoxylate (e.g. C)_12-15Alcohol, 7EO or C_12-15Alcohol, 5 EO); from 0% to about 3% soap such as fatty acids (e.g., lauric acid); about 1% to about 5% aminoethanol; from about 5% to about 10% sodium citrate; about 2% to about 6% of a hydrotrope (e.g., sodium toluene sulfonate); 0% to about 2% of a borate (e.g. B)₄O₇) (ii) a 0% to about 1% carboxymethylcellulose; about 1% to about 3% ethanol; about 2% to about 5% propylene glycol; 0.0001-0.1% ofEnzyme (calculated as pure enzyme protein); and 0 to 5% of minor ingredients (e.g. polymers, dispersants, perfumes, optical brighteners).

11) An aqueous liquid detergent composition comprising from about 20% to about 32% linear alkylbenzene sulfonate (calculated as acid); 6-12% alcohol ethoxylate (e.g. C)_12-15Alcohol, 7EO or C_12-15Alcohol, 5 EO); about 2% to about 6% aminoethanol; about 8% to about 14% citric acid; about 1% to about 3% of a borate (e.g., B)₄O₇) (ii) a From 0% to about 3% of a polymer (e.g., maleic/acrylic acid copolymer, an anchoring polymer such as lauryl methacrylate/acrylic acid copolymer); about 3% to about 8% glycerin; 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0 to 5% of minor ingredients (e.g. hydrotropes, dispersants, perfumes, optical brighteners).

12) A detergent composition in the form of granules having a bulk density of at least 600g/L comprising from about 25% to about 40% of an anionic surfactant (linear alkylbenzene sulphonate, alkyl sulphate, alpha-olefin sulphonate, alpha-sulphonic fatty acid methyl ester, alkyl sulphonate, soap); from about 1% to about 10% of a nonionic surfactant (e.g., alcohol ethoxylate); about 8% to about 25% sodium carbonate (e.g., Na)₂CO₃) (ii) a About 5% to about 15% soluble silicate (e.g., Na)₂O，2SiO₂) (ii) a 0% to about 5% sodium sulfate (e.g., Na)₂SO₄) (ii) a About 15% to about 28% of zeolite (NaAlSiO)₄) (ii) a Sodium perborate (e.g., NaBO) 0% to about 20%₃·4H₂O); from about 0% to about 5% of a bleach activator (TAED or NOBS); 0.0001-0.1% enzyme (calculated as pure enzyme protein); 0 to 3% of minor ingredients (e.g. perfumes, optical brighteners).

13) The detergent composition as described in the above compositions 1) to 12), wherein all or part of the linear alkylbenzene sulfonate is substituted by (C)₁₂-C₁₈) Alkyl sulfates.

14) Detergent composition in the form of granules having a bulk density of at least 600g/L, packageComprising about 9% to about 15% (C)₁₂-C₁₈) An alkyl sulfate; from about 3% to about 6% alcohol ethoxylate; from about 1% to about 5% of a polyhydroxy alkyl fatty acid amide; about 10% to about 20% of a zeolite (e.g., NaAlSiO @)₄) (ii) a From about 10% to about 20% of a layered disilicate (e.g., SK56 from Hoechst); about 3% to about 12% sodium carbonate (e.g., Na)₂CO₃) (ii) a 0% to about 6% soluble silicate (e.g., Na)₂O，2SiO₂) (ii) a From about 4% to about 8% sodium citrate; about 13% to about 22% sodium percarbonate; about 3% to about 8% TAED; from 0% to about 5% of a polymer (e.g., polycarboxylate and PVP); 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0 to 5% of minor ingredients (e.g. optical brighteners, photobleaches, perfumes, suds suppressors).

15) A detergent composition in the form of granules having a bulk density of at least 600g/L comprising from about 4% to about 8% (C)₁₂-C₁₈) An alkyl sulfate; from about 11% to about 15% alcohol ethoxylate; from about 1% to about 4% soap; from about 35% to about 45% zeolite MAP or zeolite a; about 2% to about 8% sodium carbonate (e.g., Na)₂CO₃) (ii) a 0% to about 4% soluble silicate (e.g., Na)₂O，2SiO₂) (ii) a About 13% to about 22% sodium percarbonate; 1-8% TAED; 0% to about 3% carboxymethylcellulose (CMC); from 0% to about 3% of a polymer (e.g., polycarboxylate and PVP); 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0 to 3% of minor ingredients (e.g. optical brighteners, phosphates, perfumes).

(16) Detergent formulations as described in 1) to 15) above, which contain stabilized or encapsulated peracids as an additional component or as a replacement for the already mentioned bleaching systems.

17) The detergent compositions described in 1), 3), 7), 9) and 12) above, wherein perborate is replaced with percarbonate.

18) The detergent compositions described above in 1), 3), 7), 9), 12), 14) and 15), further comprising a manganese catalyst. For example, the manganese catalyst is "Efficient catalyst catalysts for low-temperature blowing", Nature 369: 637-639 (1994).

19) Detergent compositions formulated as non-aqueous detergent liquids include liquid nonionic surfactants, e.g., linear alkoxylated primary alcohols, builder systems (e.g., phosphates), enzymes, and bases. The detergent may also include an anionic surfactant and/or a bleach system.

The Bacillus species TS-23 strain alpha-amylase or variant thereof may be added at concentrations conventionally used in detergents. It is presently contemplated that in detergent compositions, the Bacillus species TS-23 strain alpha-amylase or variant thereof may be added in an amount corresponding to 0.00001-1.0mg (calculated as pure enzyme protein) of enzyme per liter of wash liquor.

In another embodiment, the 2, 6- β -D-levan hydrolase may be incorporated into detergent compositions and used for biofilm removal/cleaning present on household and/or industrial textiles/clothing.

For example, the detergent composition may be formulated as a hand or machine laundry detergent composition, including a laundry additive composition suitable for pre-treating soiled fabrics and a rinse-added fabric softener composition, or as a detergent composition for general household hard surface cleaning operations, or as a hand or machine laundry washing operation.

In a particular aspect, the detergent composition can further comprise a2, 6- β -D-levan hydrolase, one or more α -amylases other than bacillus species TS-23 strain α -amylase or variant thereof, and one or more other cleaning enzymes, such as a protease, a lipase, a cutinase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, a laccase, and/or a peroxidase, and/or combinations thereof.

Generally, the properties of the enzyme selected should be compatible with the detergent selected (e.g., pH optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme should be present in an effective amount.

4.2 dishwashing detergent compositions

The alpha-amylases of the present invention may also be used in dishwashing detergent compositions, including the following examples:

1) powdered automatic dishwashing composition

0.4 to 2.5 percent of nonionic surfactant

0 to 20 percent of sodium silicate

3 to 20 percent of sodium disilicate

Sodium tripolyphosphate 20-40%

0 to 20 percent of sodium carbonate

Sodium perborate 2-9%

Tetraacetylethylenediamine (TAED) 1-4%

Sodium sulfate 5-33%

0.0001-0.1% of enzyme

2) Powdered automatic dishwashing composition

1-2% of nonionic surfactant

(e.g., alcohol ethoxylates)

2 to 30 percent of sodium disilicate

Sodium carbonate 10-50%

0 to 5 percent of sodium phosphate

Trisodium citrate dihydrate 9-30%

Nitrilo Trisodium Acetate (NTA) 0-20%

Sodium perborate monohydrate 5-10%

Tetraacetylethylenediamine (TAED) 1-2%

6-25% of polyacrylate polymer

(e.g., maleic/acrylic acid copolymer)

0.0001-0.1% of enzyme

0.1 to 0.5 percent of perfume

5 to 10 percent of water

3) Powdered automatic dishwashing composition

0.5-2.0% of nonionic surfactant

Sodium disilicate 25-40%

30 to 55 percent of sodium citrate

Sodium carbonate 0-29%

0 to 20 percent of sodium bicarbonate

0 to 15 percent of sodium perborate monohydrate

Tetraacetylethylenediamine (TAED) 0-6%

Maleic acid/acrylic acid copolymer 0-5%

1 to 3 percent of clay

0 to 20 percent of polyamino acid

0 to 8 percent of sodium polyacrylate

0.0001-0.1% of enzyme

4) Powdered automatic dishwashing composition

1-2% of nonionic surfactant

Zeolite MAP 15-42%

30 to 34 percent of sodium disilicate

0 to 12 percent of sodium citrate

0 to 20 percent of sodium carbonate

Sodium perborate monohydrate 7-15%

Tetraacetylethylene 0-3%

Diamine (TAED) Polymer 0-4%

Maleic acid/acrylic acid copolymer 0-5%

0 to 4 percent of organic phosphate

1 to 2 percent of clay

0.0001-0.1% of enzyme

Sodium sulfate balance

5) Powdered automatic dishwashing composition

1 to 7 percent of nonionic surfactant

18 to 30 percent of sodium disilicate

Trisodium citrate 10-24%

12 to 20 percent of sodium carbonate

Monopersulfate 15-21%

(2KHSO₅.KHSO₄.K₂SO₄)

Bleaching stabilizer 0.1-2%

Maleic acid/acrylic acid copolymer 0-6%

Diethylene triamine pentaacetate 0-2.5%

Pentasodium salt

0.0001-0.1% of enzyme

Sodium sulfate and water in balance

6) Powder and liquid dishwashing compositions with a cleansing surfactant system

0 to 1.5 percent of nonionic surfactant

Octadecyl dimethylamine N-oxide dihydrate 0-5%

Octadecyl dimethylamine N-oxide dihydrate and

process for preparing hexadecyldimethylamine N-oxide dihydrate

80: 20wt. C18/C16 blend 0-4%

Octadecyl bis (hydroxyethyl) amine N-oxide anhydrate

With hexadecyldi (hydroxyethyl) amine N-oxide anhydrate

70: 30wt. C18/C16 blend 0-5%

C with an average degree of ethoxylation of 3₁₃-C₁₅0 to 10 percent of alkyl ethoxy sulfate

C with an average degree of ethoxylation of 3₁₂-C₁₅0 to 5 percent of alkyl ethoxy sulfate

C with an average degree of ethoxylation of 12₁₃-C₁₅Ethoxylated alcohol 0-5%

C with an average degree of ethoxylation of 9₁₂-C₁₅Ethoxylated alcohol blend 0-6.5%

C with an average degree of ethoxylation of 30₁₃-C₁₅Ethoxylated alcohol blend 0-4%

Sodium disilicate 0-33%

Sodium tripolyphosphate 0-46%

0 to 28 percent of sodium citrate

0 to 29 percent of citric acid

0 to 20 percent of sodium carbonate

Sodium perborate monohydrate 0-11.5%

Tetraacetylethylenediamine (TAED) 0-4%

Maleic acid/acrylic acid copolymer 0-7.5%

Sodium sulfate 0-12.5%

0.0001-0.1% of enzyme

7) Non-aqueous liquid automatic dishwashing composition

Liquid nonionic surfactant (such as alcohol ethoxylate) 2.0-10.0%

Alkali metal silicate 3.0-15.0%

Alkali metal phosphate 20.0-40.0%

Selected from higher ethylene glycol, polyethylene glycol, polyoxide, 25.0-45.0%

Liquid carrier of glycol ether

Stabilizers (e.g. phosphoric acid and C)₁₆-C₁₈Partial esters of alkanols) 0.5 to 7.0%

Suds suppressors (e.g., silicone) 0-1.5%

0.0001-0.1% of enzyme

8) Non-aqueous liquid dishwashing composition

Liquid nonionic surfactant (such as alcohol ethoxylate) 2.0-10.0%

Sodium silicate 3.0-15.0%

Alkali metal carbonate 7.0-20.0%

Sodium citrate 0.0-1.5%

Stabilizing systems (e.g. finely divided silicones and low molecular weight)

Mixture of dialkyl polyglycol ethers) 0.5-7.0%

Low molecular weight polyacrylate polymer 5.0-15.0%

Clay gel thickener (such as bentonite) 0.0-10.0%

Hydroxypropyl cellulose polymer 0.0-0.6%

0.0001-0.1% of enzyme

Selected from higher glycols, polyglycols, polyoxides,

Liquid carrier balance of glycol ether

9) Thixotropic liquid automatic dishwashing composition

C₁₂-C₁₄0 to 0.5 percent of fatty acid

Block copolymer surfactant 1.5-15.0%

0 to 12 percent of sodium citrate

Sodium tripolyphosphate 0-15%

0 to 8 percent of sodium carbonate

0 to 0.1 percent of aluminum tristearate

Sodium cumene sulfonate 0-1.7%

1.32 to 2.5 percent of polyacrylate thickener

2.4 to 6.0 percent of sodium polyacrylate

Boric acid 0-4.0%

Sodium formate 0-0.45%

0 to 0.2 percent of calcium formate

Sodium n-decylbenzene oxide disulfonate 0-4.0%

Monoethanolamine (MEA) 0-1.86%

Sodium hydroxide (50%) 1.9-9.3%

0 to 9.4 percent of 1, 2-propylene glycol

0.0001-0.1% of enzyme

Foam inhibitor, dye, perfume and water

10) Liquid automatic dishwashing composition

Alcohol ethoxylate 0-20%

0 to 30 percent of fatty acid ester sulfonate

Sodium dodecyl sulfate 0-20%

0 to 21 percent of alkyl polyglycoside

Oleic acid 0-10%

Sodium disilicate monohydrate 18-33%

18-33% of sodium citrate dihydrate

Sodium stearate 0-2.5%

0 to 13 percent of sodium perborate monohydrate

Tetraacetylethylenediamine (TAED) 0-8%

Maleic acid/acrylic acid copolymer 4-8%

0.0001-0.1% of enzyme

11) Liquid automatic dishwashing composition containing protected bleach particles

5 to 10 percent of sodium silicate

Tetrapotassium pyrophosphate 15-25%

Sodium tripolyphosphate 0-2%

4 to 8 percent of potassium carbonate

Protected bleach granules, e.g. 5-10% chlorine

Polymer thickener 0.7-1.5%

0 to 2 percent of potassium hydroxide

0.0001-0.1% of enzyme

Balance of water

11) The automatic dishwashing compositions of 1), 2), 3), 4), 6) and 10) above, wherein the perborate is replaced by percarbonate.

12) The automatic dishwashing composition of 1) -6) above, further comprising a manganese catalyst. The manganese catalyst may be, for example, "Efficient catalyst catalysts for low-temperature purification," Nature 369, 1994, pp: 637-639.

4.3 biofilm removal compositions and uses

The composition for removing biofilm may comprise as a major enzyme component a bacillus species TS-23 strain alpha-amylase or variant thereof, e.g. a one-component composition. Alternatively, the composition may comprise a plurality of enzyme activities, such as a plurality of amylases, or a mixture of enzymes comprising any combination of the following enzymes: aminopeptidase, amylase (beta-or alpha-or glucoamylase), carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutamidase, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, and/or xylanase, or any combination thereof, for removing biofilm. Other enzymes can be produced by microorganisms belonging to the genera: aspergillus, Trichoderma, Humicola (e.g., Humicola insolens), and Fusarium (mildew). Illustrative examples of Aspergillus include Aspergillus aculeatus, Aspergillus awamori, Aspergillus niger or Aspergillus oryzae. Examples of Fusarium species include Fusarium bactridioides, f.cerealis, f.crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambuci, Fusarium sarcochroum, Fusarium sulphureum, f.torulosum, Fusarium trichothecioides, and Fusarium venenatum.

Compositions comprising bacillus species TS-23 strain alpha-amylase or variants thereof can be prepared according to methods known in the art and can be in the form of liquid or dry compositions. For example, a composition comprising Bacillus species TS-23 strain alpha-amylase or variant thereof can be in the form of granules or microparticles. The polypeptides to be included in the composition may be stabilized according to methods known in the art.

Examples of uses of the polypeptide compositions are given below. The amount of the composition comprising Bacillus species TS-23 strain alpha-amylase or variant thereof and other conditions for use of the composition can be determined based on methods known in the art.

It is also contemplated that the Bacillus species TS-23 strain alpha-amylase or variant thereof is used in the composition with 2, 6-beta-D-levan hydrolase or variant thereof.

Another aspect is compositions and methods for disrupting and/or removing biofilm. The term "disruption" as used herein is to be understood as hydrolysis of the polysaccharide in the biofilm matrix, which polysaccharide links and binds together individual microbial cells in the biofilm, whereby said microbial cells are able to be released and removed from the biofilm. Biofilms are typically present on surfaces, and disruption of biofilms may be achieved by contacting the surface (e.g., by submerging, covering or spraying the surface) with an aqueous medium containing the bacillus species TS-23 strain alpha-amylase or variant thereof or one or more other enzymes responsible for degrading biofilms (such as, but not limited to, 2, 6-beta-D-levan hydrolase). The composition can be used for hydrolyzing sludges in, for example, pulp and paper mill white water.

The Bacillus species TS-23 strain alpha-amylase or variant thereof can be present in an amount of 0.0001-10,000mg/L, 0.001-1000mg/L, 0.01-100mg/L, or 0.1-10 mg/L. Other enzymes and enzyme variants may be present in similar or lower amounts.

The process may suitably be carried out at a temperature of from room temperature to about 70 ℃. Suitable temperature ranges include from about 30 ℃ to about 60 ℃, e.g., from about 40 ℃ to about 50 ℃.

Suitable pH for hydrolyzing the biofilm is in the range of about 3.5 to about 8.5. Exemplary pH ranges include pH of about 5.5 to about 8, for example about 6.5 to about 7.5. The contact time or reaction time for the enzyme to effectively remove the biofilm may vary considerably depending on the characteristics of the biofilm and the frequency of treatment of the surface with the enzyme, alone or in combination with other biofilm degrading enzymes (e.g. 2, 6- β -D-levan hydrolase). Exemplary reaction times may include from about 0.25 to about 25 hours, and from about 1 to about 10 hours, such as about 2 hours.

Other biofilm degrading enzymes that may be combined with the bacillus species TS-23 strain alpha-amylase or variant thereof and 2, 6-beta-D-levan hydrolase include, but are not limited to: cellulases, hemicellulases, xylanases, other amylases, including other alpha-amylases, lipases, proteases and/or pectinases.

The Bacillus species TS-23 strain alpha-amylase or variant thereof can also be combined with an antimicrobial agent, such as an enzymatic or non-enzymatic biocide. The enzymatic biocide may be, for example, a composition comprising an oxidoreductase, e.g. a laccase or a peroxidase, in particular a haloperoxidase, and optionally an enhancer, e.g. an alkyl syringate, as described, for example, in international PCT applications WO 97/42825 and DK 97/1273.

The surface from which the biofilm may be removed and/or removed may be a hard surface, which is defined to refer to any surface that is substantially impermeable to microorganisms. Examples of surfaces are surfaces made of metals such as stainless steel alloys, plastics/synthetic polymers, rubber, sheet, glass, wood, paper, textiles, concrete, rock, marble, plaster and ceramic materials, optionally coated with e.g. paint, enamel, polymers etc. Thus, the surface may be a component of a system that supports, transports, processes, or contacts an aqueous solution, such as a water supply system, a food processing system, a cooling system, a chemical processing system, or a pharmaceutical processing system. The compositions and methods of using the compositions are useful for removing biofilm in wood-processing systems, such as the pulp and/or paper industry. Thus, the enzymes and compositions containing the enzymes can be used in conventional cleaning-in-place (C-I-P) systems. The surface may be part of a system unit such as a pipe, tank, pump, membrane, filter, heat exchanger, centrifuge, evaporator, mixer, spray tower, valve, and reactor. The surface may also be an instrument used in medical science and industry (such as a contaminated endoscope, prosthetic device, or medical implant) or a part thereof.

The composition for biofilm removal may also be considered for preventing so-called bio-corrosion occurring when a metal surface, such as a pipe, is attacked by a microbial biofilm, i.e. by disrupting the biofilm, thereby preventing microbial cells in the biofilm from establishing a biofilm environment that corrodes the metal surface to which it is attached.

Other applications for anti-biofilm compositions include oral care. However, the surface may also be of biological origin, such as mucosa, skin, teeth, hair, nails, and the like.

The surface thus contains plaque-bearing teeth (e.g., by incorporating enzymes into the toothpaste) and contaminated contact lenses. Thus, the Bacillus species TS-23 strain alpha-amylase or variant thereof can be used in compositions and methods for the preparation of a medicament for disrupting dental plaque in a human or animal. Another use is self-adhesive membrane disruption of biofilms, such as in the lungs of patients with cystic fibrosis.

Thus, a further aspect of the invention relates to an oral care composition comprising a recombinant enzyme, e.g. a purified enzyme substantially free of any active contaminants. The oral care composition may suitably comprise an amount of a recombinant enzyme.

Other biofilm degrading enzymes for use in oral care compositions include, but are not limited to, 2, 6-beta-D-fructan hydrolase activity in oral care compositions. Contemplated enzyme activities include activities in the following groups of enzymes: a dextranase; mutanase (mutanase); oxidases, such as glucose oxidase, L-amino acid oxidase, peroxidases, such as Coprinus (Coprinus) peroxidase or lactoperoxidase as described in WO 95/10602, haloperoxidases, in particular haloperoxidases derived from Curvularia species (Curvularia), in particular Curvularia rugulosa (C.verruculosa) and Curvularia inequality (C.inaequalis); a laccase; proteases such as papain, acid proteases (e.g., the acid protease described in WO 95/02044), endoglycosidases, lipases, amylases, including amyloglucosidases, such as AMG (Novo Nordisk A/S); antimicrobial enzymes, and mixtures thereof.

The oral care composition can be in any suitable physical form (i.e., powder, paste, gel, liquid, paste, tablet, etc.). "oral care compositions" include compositions that can be used to maintain or improve oral hygiene in the mouth of humans and animals, to prevent dental caries, prevent plaque and tartar formation, remove plaque and tartar, prevent and/or treat dental diseases, and the like. Oral care compositions herein also include at least products for cleaning dentures, artificial teeth, and the like. Examples of oral care compositions include toothpastes, tooth creams, gels or tooth powders, mouthwashes, rinses before and after brushing, chewing gums, lozenges and candies. Toothpastes and tooth gels generally include abrasive polishing materials, foaming agents, flavoring agents, humectants, binders, thickeners, sweeteners, whitening/bleaching/stain removing agents, water and optionally additional enzymes and enzyme combinations.

Mouthwashes, including plaque removing liquids, generally include a water/alcohol solution, a flavor, a humectant, a sweetener, a foaming agent, a colorant, and optionally an additional enzyme or combination of enzymes.

Abrasive polishing materials can also be incorporated into oral care compositions such as dentifrices.

Thus, the abrasive polishing material may comprise alumina and hydrates thereof, such as alpha-alumina trihydrate; magnesium trisilicate; magnesium carbonate; kaolin; aluminosilicates, such as calcined aluminosilicates and aluminosilicates; calcium carbonate; zirconium silicate; also powdered plastics, such as polyvinyl chloride; a polyamide; polymethyl methacrylate; polystyrene; a phenol formaldehyde resin; a melamine formaldehyde resin; a urea-formaldehyde resin; an epoxy resin; powdered polyethylene; a silica xerogel; hydrogels and aerogels, and the like. Also suitable as abrasives are calcium pyrophosphate; a water-insoluble alkaline metaphosphate; dicalcium phosphate and/or its dihydrate, dicalcium orthophosphate; tricalcium phosphate; hydroxyapatite particles, and the like. Mixtures of these materials may also be employed.

Depending on the oral care composition, the abrasive product may be present from about 0% to about 70%, or from about 1% to about 70%, by weight. For toothpastes, the abrasive content is typically in the range of 10% to 70% by weight of the final toothpaste.

Humectants are employed to prevent water loss from, for example, toothpastes. Humectants suitable for use in oral care compositions include the following compounds and mixtures thereof: glycerol; a polyol; sorbitol; polyethylene glycol (PEG); propylene glycol; 1, 3-propanediol; 1, 4-butanediol; hydrogenated partially hydrolyzed polysaccharides, and the like. The humectant is typically present in the toothpaste at from 0% to about 80%, or from about 5% to about 70% by weight.

Silicon dioxide, starch, tragacanth, xanthan gum, Irish moss (Irish moss) extract, alginate, pectin, cellulose derivatives such as hydroxyethyl cellulose, sodium carboxymethyl cellulose and sodium hydroxypropyl cellulose; polyacrylic acid and its salts, polyvinylpyrrolidone, are examples of suitable thickening agents and binders that help stabilize the dentifrice product. Thickeners may be present in toothpaste emulsions and gels in amounts of from about 0.1% to about 20% by weight of the final product, and binders to the extent of from about 0.01 to about 10%.

Anionic, cationic, nonionic, amphoteric and/or zwitterionic surfactants can be used as the foaming agent soap. These may be present at a level of from 0% to about 15%, from about 0.1 to about 13%, or from about 0.25% to about 10% by weight of the final product.

The surfactant is only suitable to the extent that it does not exert an inactivating effect on the alpha-amylase of the Bacillus species TS-23 strain of the present invention or a variant thereof. The surfactants include fatty alcohol sulfates, salts of sulfonated monoacylglycerols or fatty acids having 10 to 20 carbon atoms, fatty acid-albumin condensation products, salts of fatty acid amides and taurines and/or salts of fatty acid isethionates.

Suitable sweeteners include saccharin for pharmaceutical use.

Fragrances such as spearmint are generally present in low levels, for example from about 0.01% to about 5%, especially from about 0.1% to about 5% by weight. The whitening/bleaching agent comprises H₂O₂And may be added in an amount of less than about 5%, or from about 0.25% to about 4%, by weight of the final product. The whitening/bleaching agent may be an enzyme, such as an oxidoreductase. Examples of suitable tooth bleaching enzymes are for example those described in WO 97/06775.

Water is typically added in an amount that imparts a flowable form to, for example, a toothpaste.

Other water-soluble antibacterial agents may also be included, such as chlorhexidine digluconate, aminocaproidine (hexidine), biguanidine (alexidine), Triclosan (Triclosan)) Quaternary ammonium antibacterial compounds, and water-soluble sources of certain metals ions, such as zinc, copper, silver, and tin sources (e.g., zinc chloride, copper chloride, and stannous chloride, and silver nitrate).

Compounds that can act as fluoride sources, pigments/colorants, preservatives, vitamins, pH adjusters, anticaries agents, desensitizers, and the like can also be added.

Biofilm degrading enzymes can provide several benefits when used to clean the oral cavity. Proteases degrade salivary proteins, which adsorb to the tooth surface and form a pellicle (pellicle), the first layer of the plaque that results. Proteases, along with lipases, destroy bacteria by cleaving proteins and lipids that constitute structural components of bacterial cell walls and membranes.

Glucanases and other carbohydrases such as 2, 6-beta-D-levan hydrolase degrade the bacterial produced organic backbone structure forming the bacterial adhesion matrix. Proteases and amylases not only prevent plaque formation, but also prevent mineralization by degrading calcium-binding carbohydrate-protein complexes, thereby preventing tartar development.

Toothpastes may generally comprise the following ingredients (in weight percent of the final toothpaste composition): abrasive to about 70%; humectant: 0% to about 80%; thickening agent: about 0.1% to about 20%; adhesive: about 0.01% to about 10%; and (3) a sweetener: about 0.1% to about 5%; foaming agent: 0% to about 15%; whitening agent: 0% to about 5%; and an enzyme: from about 0.0001% to about 20%.

In one embodiment, the toothpaste has a pH in the range of about 6.0 to about 8.0 and comprises: a) about 10% to about 70% abrasive; b) 0% to about 80% of a humectant; c) 0.1% to about 20% of a thickening agent; d) 0.01% to about 10% of a binder; e) about 0.1% to about 5% of a sweetener; f) 0% to about 15% of a blowing agent; g) 0% to about 5% of a whitening agent and i) about 0.0001% to about 20% of an enzyme.

i) Said enzymes referred to in (b) include bacillus species TS-23 strain alpha-amylase or variants thereof, other biofilm degrading enzymes such as 2, 6-beta-D-levan hydrolase, alone or in combination, and optionally other types of enzymes of the above mentioned type known for use in toothpaste, and the like.

A mouthwash may generally comprise the following ingredients (in weight percent of the final mouthwash composition): 0% to about 20% of a humectant; 0% to about 2% of a surfactant; 0% to about 5% enzyme; 0% to about 20% ethanol; from 0% to about 2% of other ingredients (e.g., flavors, sweetener active ingredients, such as fluoride). The composition may also contain from about 0% to about 70% water.

The mouthwash composition may be buffered with a suitable buffering agent, such as sodium citrate or sodium phosphate at a pH of about 6.0 to about 7.5. Mouthwashes may be in undiluted form (i.e., must be diluted prior to use).

The oral care composition can be produced using any conventional method known in the oral care art.

4.4 starch processing compositions and uses

In another aspect, compositions having the disclosed Bacillus species TS-23 strain alpha-amylase or variant thereof can be used for starch liquefaction or saccharification.

In one aspect, the compositions and use of the compositions in the preparation of sweeteners using starch. Conventional processes for converting starch to fructose syrup typically comprise three sequential enzymatic steps: a liquefaction step, a saccharification step and an isomerization step. In the liquefaction step, starch is degraded to dextrins by the action of an alpha-amylase of strain TS-23 of bacillus species or a variant thereof for a period of about 2 hours at a pH of about 5.5 to about 6.2 and a temperature of about 95 ℃ to about 160 ℃. To ensure optimal enzyme stability under these conditions, 1mM calcium (40ppm free calcium ion) was added. Starch processing is used for alcohol production (e.g., grain liquefaction to fuel or potable alcohol, alcohol brewing), and starch liquefaction is used for sweetener production, sucrose processing, and other food-related starch processing purposes. Other conditions may be used for different Bacillus species TS-23 strain alpha-amylase or variants thereof.

After the liquefaction step, glucoamylase (e.g., AMG) may be added^TM) And debranching enzymes (e.g. isoamylase or pullulanase (e.g. PROMOZYME)) Dextrin is converted to dextrose. Prior to this step, the pH is lowered to a value below about 4.5, the temperature is maintained at a high temperature (above 95 ℃) and the activity of the alpha-amylase of the liquefying Bacillus species TS-23 strain or a variant thereof is denatured. Cooling to 60 deg.C, and optionally adding glucose starchEnzymes and debranching enzymes. The saccharification step is typically conducted for about 24 to about 72 hours.

After the saccharification step, the pH is raised to a value in the range of about 6.0 to about 8.0 (e.g., pH7.5) and calcium is removed by ion exchange. Then using, for example, immobilized glucose isomerase (e.g., Sweetzyme)) Converting the glucose syrup into high fructose syrup.

Improvements in at least one enzyme in the process can be achieved. The Bacillus species TS-23 strain alpha-amylase or variant thereof has a reduced calcium requirement in liquefaction. The addition of free calcium is required to ensure sufficiently high stability of the alpha-amylase of Bacillus species TS-23 strain or variants thereof; however, free calcium strongly inhibits glucose isomerase activity and requires expensive unit operations to remove the calcium to the extent that the level of free calcium drops below 3-5 ppm. If such an operation can be avoided and the liquefaction step can be carried out without the addition of free calcium ions, cost savings can be achieved.

For example, less calcium dependent enzymes that are stable and have high activity at low concentrations of free calcium (< 40ppm) can be utilized in compositions and operations. Such bacillus species TS-23 strain alpha-amylase or variant thereof should have a pH optimum in the pH range of about 4.5 to about 6.5, or in the range of about 4.5 to about 5.5.

In laboratory and industrial settings, bacillus species TS-23 strain alpha-amylase or variants thereof can be used to hydrolyze starch or any maltodextrin (maltodextrine) -containing compound for various purposes. These bacillus species TS-23 strain alpha-amylases, or variants thereof, can be used alone to provide specific hydrolysis, or in combination with other amylases to provide a mixture with a broad spectrum of activity. Exemplary applications include the removal or partial or complete hydrolysis of starch or any maltodextrin containing compound from biological, food, animal feed, pharmaceutical or industrial samples.

Another aspect of the invention relates to compositions and methods of using the compositions in fermentation processes in which a starch substrate is liquefied and/or saccharified in the presence of a bacillus species TS-23 strain alpha-amylase or variant thereof to produce glucose and/or maltose, which is suitable for conversion to a fermentation product by a fermenting microorganism, such as a yeast. Such fermentation processes include processes for producing ethanol for use as a fuel or potable ethanol (potable ethanol), processes for producing beverages, processes for producing desired organic compounds (e.g., citric acid, itaconic acid, lactic acid, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, glucono delta-lactone, or sodium erythorbate), ketones, amino acids (e.g., glutamic acid, monosodium glutamate), and more complex compounds that are difficult to prepare synthetically (e.g., antibiotics such as penicillin, tetracycline), enzymes, vitamins (e.g., riboflavin, vitamin B12, beta-carotene), and hormones).

The starch to be processed may have a highly refined starch quality, e.g. at least 90%, at least 95%, at least 97% or at least 99.5% pure. Alternatively, the starch may be a relatively crude starch-containing material comprising milled whole grain, including non-starch fractions such as embryo residues and fiber. The raw material, such as whole grain, is milled to open up its structure, allowing further processing. Two milling methods can be used: wet milling and dry milling. In addition, corn grits such as ground corn grits can be used.

The dry-milled grain will contain significant amounts of non-starch saccharide compounds in addition to starch. When such heterogeneous feedstocks are processed by jet cooking, the bacillus species TS-23 strain tends to achieve only partial gelatinization of starch. Since the Bacillus species TS-23 strain alpha-amylase or variant thereof has a high activity on ungelatinized starch, the enzyme has advantages for use in processes involving dry-milled starch liquefaction and/or saccharification jet cooking.

Also, the need for glucoamylase in the saccharification step is greatly reduced due to the excellent hydrolytic activity of the Bacillus species TS-23 strain alpha-amylase or variant thereof. This allows saccharification to be carried out at very low levels of glucoamylase activity. The glucoamylase is either not present or, if present, is present in an amount of no more than or even less than 0.5AGU/gDS, or no more than or even less than 0.4AGU/gDS, or no more than or even less than about 0.3AGU/g DS, or less than 0.1AGU, e.g., no more than or even less than 0.05AGU/g DS of starch substrate. "DS" is the unit of enzyme added per gram of dry solid substrate. The enzyme protein is either absent, expressed in mg, or present in an amount of no more than or even less than about 0.5mg EP/g DS, or no more than or even less than about 0.4mg EP/g DS, or no more than or even less than about 0.3mg EP/g DS, or no more than or even less than about 0.1mg EP/g DS (e.g., no more than or even less than about 0.05mg EP/g DS or no more than or even less than 0.02mg EP/g DS of starch substrate). Glucoamylases may be derived from strains of Aspergillus species, pachycosis species (Talaromyces sp.), chrysosporium species (Pachykytospora sp.), or Trametes species (Trametes sp.), of which illustrative examples are Aspergillus niger (Aspergillus niger), nigrospora cassiicola (Talaromyces emersonii), Trametes annulata (Trametes cingulata), or chrysosporium paperii (pachycosporia).

The method may comprise a) contacting a starch substrate with a bacillus species TS-23 strain alpha-amylase or variant thereof comprising a catalytic module having alpha-amylase activity and a carbohydrate-binding module, e.g. a polypeptide as described in the first aspect; b) incubating the starch substrate with the enzyme for a time and at a temperature sufficient to convert at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even at least 99.5% w/w of the starch substrate to fermentable sugars; c) fermenting to produce a fermentation product; and d) optionally recovering the fermentation product. In step b) and/or c) of the process, the enzyme having glucoamylase activity is either absent or present in an amount of 0.001-2.0AGU/g DS, 0.01-1.5AGU/g DS, 0.05-1.0AGU/g DS, 0.01-0.5AGU/g DS. The enzyme having glucoamylase activity may be either absent or present in an amount of no more than or even less than 0.5AGU/g DS, or no more than or even less than 0.4AGU/g DS, or no more than or even less than 0.3AGU/g DS, or no more than or even less than 0.1AGU/g DS, for example no more than or even less than 0.05AGU/g DS, of the starch substrate. The enzyme protein is either absent, expressed in mg, or present in an amount of no more than or even less than 0.5mgEP/g DS, or no more than or even less than 0.4mgEP/g DS, or no more than or even less than 0.3mgEP/g DS, or no more than or even less than 0.1mgEP/g DS (e.g., no more than or even less than 0.05mgEP/g DS or no more than or even less than 0.02mgEP/g DS of the starch substrate). In the method, steps a), b), c) and/or d) may be performed individually or simultaneously.

In another aspect, the method comprises: a) contacting the starch substrate with a yeast cell transformed to express a bacillus species TS-23 strain alpha-amylase or a variant thereof, said variant comprising a catalytic module having alpha-amylase activity and a carbohydrate-binding module; b) incubating the starch substrate with the yeast for a time and at a temperature sufficient to convert at least 90% w/w of the starch substrate to fermentable sugars; c) fermenting to produce ethanol; and d) optionally recovering the ethanol. In the method, steps a), b) and c) may be performed separately or simultaneously.

In yet another aspect, the method comprises hydrolyzing gelatinized syrup or granular starch, particularly hydrolyzing granular starch to a soluble starch hydrolysate at a temperature below the initial gelatinization temperature of said granular starch. In addition to contacting with a polypeptide comprising a catalytic module having alpha-amylase activity and a carbohydrate-binding module, the starch may also be contacted with any one or more of the fungal alpha-amylases (EC 3.2.1.1), described below, and one or more of the enzymes of the beta-amylases (EC 3.2.1.2) and glucoamylases (EC 3.2.1.3), described below. In another aspect, other starch hydrolyzing or debranching enzymes, such as isoamylase (EC 3.2.1.68) or pullulanase (EC 3.2.1.41), may be added to the Bacillus species TS-23 strain alpha-amylase or variant thereof.

In one embodiment, the process is carried out at a temperature below the initial gelatinization temperature. Such methods are often performed at least 30 ℃, at least 31 ℃, at least 32 ℃, at least 33 ℃, at least 34 ℃, at least 35 ℃, at least 36 ℃, at least 37 ℃, at least 38 ℃, at least 39 ℃, at least 40 ℃, at least 41 ℃, at least 42 ℃, at least 43 ℃, at least 44 ℃, at least 45 ℃, at least 46 ℃, at least 47 ℃, at least 48 ℃, at least 49 ℃, at least 50 ℃, at least 51 ℃, at least 52 ℃, at least 53 ℃, at least 54 ℃, at least 55 ℃, at least 56 ℃, at least 57 ℃, at least 58 ℃, at least 59 ℃, or at least 60 ℃. The pH at which the process is carried out may be in the range of about 3.0 to about 7.0, or about 3.5 to about 6.0, or about 4.0 to about 5.0. In one aspect, methods involving fermentation, e.g., at temperatures around 32 deg.C (e.g., 30-35 deg.C), e.g., using yeast, are contemplated.

In another aspect, the method includes simultaneous saccharification and fermentation, e.g., at a temperature of 30-35 ℃ (e.g., about 32 ℃), e.g., to produce ethanol using yeast or to produce the desired organic compound using other suitable fermenting organisms.

In the above fermentation process, the ethanol content reaches at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, for example, at least about 16% ethanol.

The starch slurry used in any of the above aspects may have from about 20% to about 55% dry solid particulate starch, from about 25% to about 40% dry solid particulate starch, or from about 30% to about 35% dry solid particulate starch. After contact with the bacillus species TS-23 strain alpha-amylase or variant thereof, the enzyme converts soluble starch in granular starch in an amount of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to a soluble starch hydrolysate.

In another embodiment, the bacillus species TS-23 strain alpha-amylase or a variant thereof comprising a catalytic module having alpha-amylase activity and a carbohydrate-binding module, such as a polypeptide as described in the first aspect, is used in a method for liquefying, saccharifying gelatinized starch (such as, but not limited to, gelatinization by jet cooking). The method may include fermentation to produce a fermentation product, such as ethanol. Such processes for producing ethanol from starch-containing feedstocks by fermentation include: (i) liquefying the starch-containing feedstock with a polypeptide comprising a catalytic module having alpha-amylase activity and a carbohydrate-binding module, e.g., a polypeptide of the first aspect; (ii) saccharifying the resulting liquefied mash (mash); and (iii) fermenting the material obtained in step (ii) in the presence of a fermenting organism. Optionally, the process further comprises recovering the ethanol. The process of saccharification and fermentation may be carried out as a simultaneous saccharification and fermentation process (SSF). During the above fermentation, the ethanol content reaches at least about 7%, at least about 8%, at least about 9%, at least about 10%, such as at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, such as at least about 16% ethanol.

The starch to be processed in the method of the above aspect may in particular be from tubers, roots, stems, legumes, cereals or whole grains. More specifically, the granular starch may be obtained from corn, corn cobs, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice, peas, beans, bananas, or potatoes. Waxy and non-waxy types of corn and barley are also contemplated.

The above compositions can be used to liquefy and/or saccharify gelatinized or granular starch, and partially gelatinized starch. Partially gelatinized starch is starch that is gelatinized to some extent, i.e., wherein a portion of the starch is irreversibly swollen and gelatinized while a portion of the starch is still present in the granular state.

The above composition may comprise an acid alpha-amylase variant present in an amount of 0.01-10.0AFAU/g DS or 0.1-5.0AFAU/g DS or 0.5-3.0AFAU/AGU or 0.3-2.0AFAU/g DS. The composition may be used in any of the starch processes described above.

As used herein, the term "liquefaction" in the form of nouns or verbs refers to the process of converting starch into dextrins with shorter chain lengths and less viscosity. Typically, this process involves gelatinisation of the starch, with or after addition of the bacillus species TS-23 strain alpha-amylase or variant thereof. Other liquefaction inducing enzymes may also be added.

As used herein, the term "primary liquefaction" refers to the liquefaction step when the temperature of the slurry is raised to or near its gelatinization temperature. Following the temperature increase, the slurry is passed through a heat exchanger or injector to a temperature of about 200-300F, such as 220-235F. Following the application of the heat exchanger or eductor temperature, the slurry is held at this temperature for a period of 3 to 10 minutes. This step of maintaining the slurry at 200-.

As used herein, the term "secondary liquefaction" refers to a liquefaction step when the slurry is allowed to cool to room temperature following the primary liquefaction (heating to 200-. This cooling step may be from 30 minutes to 180 minutes (3 hours), for example from 90 minutes to 120 minutes (2 hours).

As used herein, the term "minutes of secondary liquefaction" refers to the time elapsed from the start of secondary liquefaction to the time when DE is measured.

On the other hand, it is contemplated to additionally apply a beta-amylase in a composition comprising the Bacillus species TS-23 strain alpha-amylase or a variant thereof, the beta-amylase (EC 3.2.1.2) being an exo-maltogenic amylase, which catalyzes the hydrolysis of 1, 4-alpha-glucosidic bonds in amylose, amylopectin and related glucose polymers, thereby releasing maltose.

Beta-amylases have been isolated from a variety of plants and microorganisms (W.M.Fogarty and C.T.Kelly, PROGRESS IN INDUSTRIAL MICROBIOLOGY, Vol.15, p.112-115, 1979). These beta-amylases are characterized by having a temperature optimum in the range of 40 ℃ to 65 ℃ and a pH optimum in the range of about 4.5 to about 7.0. Contemplated beta-amylases include, but are not limited to: SPEZYME from barleyBBA 1500、SPEZYMEDBA、OPTIMALTME、OPTIMALTBBA (Genencor International Inc.) and NOVOZYM^TMWBA (Novozymes A/S).

Another enzyme contemplated for use in the composition is glucoamylase (EC 3.2.1.3). The glucoamylase is derived from a microorganism or a plant. Exemplary glucoamylases are of fungal or bacterial origin. Exemplary bacterial glucoamylases are Aspergillus glucoamylases, particularly Aspergillus niger G1 or G2 glucoamylase (Boel et al, EMBO J.3 (5): 1097-1102(1984)) or variants thereof, such as those disclosed in WO 92/00381 and WO 00/04136; aspergillus awamori glucoamylase (WO 84/02921); aspergillus oryzae glucoamylase (Agric. biol. chem., 55 (4): 941-949(1991)) or variants or fragments thereof.

Other contemplated aspergillus glucoamylase variants include variants that enhance thermostability: G137A and G139A (Chen et al (1996), prot. Eng.9: 499-; D257E and D293E/Q (Chen et al, prot. Eng.8: 575-582 (1995)); n182(Chen et al, biochem. J.301: 275-281 (1994)); disulfide bond, A246C (Fierobe et al, Biochemistry, 35: 8698-; and Pro residues were introduced at positions A435 and S436 (Li et al ProteinEng.10: 1199-1204 (1997)). Other contemplated glucoamylases include those of the genus Talaromyces, particularly those derived from Talaromyces emersonii (WO 99/28448), Talaromyces leycettanus (U.S. Pat. No. RE 32,153), Talaromyces duponti, Talaromyces thermophilus (Talaromyces thermophilus) (U.S. Pat. No.4,587,215). Bacterial glucoamylases contemplated include those from the genus Clostridium (Clostridium), particularly C.thermoamylolyticum (EP135138) and C.thermohydrosulfuricum (WO 86/01831). Examples of glucoamylases include those derived from Aspergillus oryzae. Commercial glucoamylases are also contemplated, e.g., AMG200L、AMG 300L、SAN^TMSUPER and AMG^TM E(Novozymes)；OPTIDEX300 (available from Genencor International Inc.); AMIGASEAnd AMIGASEPLUS(DSM)；G-ZYMEG900(Enzyme Bio-Systems)；G-ZYMEG990ZR (aspergillus niger glucoamylase and low protease content).

The glucoamylase may be added in an amount of 0.02-2.0AGU/g DS or 0.1-1.0AGU/g DS (e.g., 0.2AGU/g DS).

Other enzymes and enzyme variants may be included in the composition. In addition to the bacillus species TS-23 strain alpha-amylase or variant thereof, one or more alpha-amylases can be used, or other enzymes discussed herein can be further included.

Another enzyme that may optionally be added is a debranching enzyme, such as isoamylase (EC 3.2.1.68) or pullulanase (EC 3.2.1.41). Isoamylase hydrolyzes the alpha-1, 6-D-glucosidic branch bonds in amylopectin and beta-limit dextrins and can be distinguished from pullulanase by the inability of isoamylase to attack prallulose glucans and their limited effect on alpha-limit dextrins. The debranching enzyme may be added in an effective amount as is well known to those skilled in the art.

The exact composition of the product of the process depends on the enzyme combination used and the type of granular starch processed. For example, the soluble hydrolysate can be maltose having a purity of at least about 85%, at least about 90%, at least about 95.0%, at least about 95.5%, at least about 96.0%, at least about 96.5%, at least about 97.0%, at least about 97.5%, at least about 98.0%, at least about 98.5%, at least about 99.0%, or at least about 99.5%. Alternatively, the soluble starch hydrolysate may be glucose, or the starch hydrolysate has a DX (percentage of glucose to total dissolved dry solids) of at least 94.5%, at least 95.0%, at least 95.5%, at least 96.0%, at least 96.5%, at least 97.0%, at least 97.5%, at least 98.0%, at least 98.5%, at least 99.0%, or at least 99.5%. The method may include tailoring syrup products, such as tailoring syrups comprising mixtures of glucose, maltose, DP3 and DPn for the production of ice cream, cakes, candies, canned fruit.

The two milling methods are: wet milling and dry milling. In the dry milling process, whole grains are milled and used. The wet milling process provides good separation of germ and flour (starch granules and protein), which is commonly used when starch hydrolysates are used in syrup production. Both dry and wet milling methods are well known in the starch processing art and are equally contemplated for use with the disclosed compositions and methods. The process may be carried out in an ultrafiltration system, wherein the retentate is kept recirculated in the presence of enzymes, raw starch and water, and wherein the permeate is a soluble starch hydrolysate. Equally contemplated is a process carried out in a continuous membrane reactor with ultrafiltration membranes, wherein the retentate is kept recycled in the presence of enzymes, raw starch and water, and wherein the permeate is a soluble starch hydrolysate. Also contemplated is a process carried out in a continuous membrane reactor with a microfiltration membrane, wherein the retentate is kept recirculated in the presence of enzymes, raw starch and water, and wherein the permeate is a soluble starch hydrolysate.

In one aspect, the soluble starch hydrolysate of the process is converted to a high fructose starch-based syrup (HFSS), such as High Fructose Corn Syrup (HFCS). This conversion can be achieved by using glucose isomerase, and by immobilizing the glucose isomerase on a solid support. Contemplated isomerases include the commercial SWEETZYMEIT(Novozymes A/S)；G-ZYMEIMGI and G-ZYMEG993，KETOMAXG-ZYMEG993(Rhodia)，G-ZYMEG993 liquid and GENSWEETIGI (Genencor International Inc.).

On the other hand, the soluble starch hydrolysates produced by these processes can be used to produce fuel or potable ethanol. In the process of the third aspect, the fermentation may be carried out simultaneously or separately/sequentially with the hydrolysis of the granular starch slurry. When fermentation and hydrolysis are carried out simultaneously, the temperature may be between 30 ℃ and 35 ℃, in particular between 31 ℃ and 34 ℃. The process may be carried out in an ultrafiltration system, wherein the retentate is kept recirculated in the presence of enzymes, raw starch, yeast nutrients and water, and wherein the permeate is an ethanol containing liquid. Equally contemplated is a process carried out in a continuous membrane reactor with ultrafiltration membranes, wherein the retentate is kept recirculated in the presence of enzymes, raw starch, yeast nutrients and water, and wherein the permeate is an ethanol containing liquid.

The soluble starch hydrolysate of the process may also be used in the production of fermentation products, which include fermentation of the treated starch into a fermentation product, such as citric acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, glucono delta-lactone or sodium erythorbate.

The starch hydrolyzing activity of the Bacillus species TS-23 strain alpha-amylase or variant thereof can be determined, for example, using potato starch as a substrate. This method is based on the degradation of modified potato starch by the enzyme, and the reaction is followed by mixing a sample of the starch/enzyme solution with an iodine solution. A dark blue color formed initially, but during starch degradation, the blue color became weaker and gradually turned to reddish brown, which was compared to a colored glass standard.

5. Method of producing a composite material

5.1 Filter Screen experiment

The experiments discussed below can be used to screen for at high or low pH and/or at Ca as compared to the parent alpha-amylase²⁺AmyTS23 alpha-amylase variants with altered stability under depleted conditions.

5.2 high pH Filter screening experiments

Libraries of Bacillus species were plated on multiple layers of cellulose acetate (OE 67, Schleicher & Schuell, Dassel, Germany) and cellulose nitrate filters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plates containing 10. mu.g/ml kanamycin at 37 ℃ for at least 21 hours. The cellulose acetate layer was placed on a TY agar plate.

After plating but before incubation the individual filter layers were specifically labelled with needles to enable the positive variants to be located on the filters, the nitrocellulose filter with the bound variant was transferred to a vessel containing glycine-NaOH buffer, pH8.6-10.6 and incubated for 15 minutes at room temperature (which can vary from 10-60 ℃). Cellulose acetate filters with colonies were stored at room temperature on TY plates until use. After incubation, residual activity was detected on plates containing 1% agarose, 0.2% starch in glycine-NaOH buffer (pH 8.6-10.6). The plates with nitrocellulose filters were labelled in the same way as the filter sandwich described above and incubated for 2 hours at room temperature. After removal of the filter, the plates were stained with 10% compound iodine solution (Lugol solution). On a dark blue background, variants of degraded starch were detected as white spots and then identified on the storage plates. The positive variants were rescreened twice under the same conditions as the first screen.

5.3 Low calcium Filter screening experiments

A pool of Bacillus species was plated on multiple layers of cellulose acetate (OE 67, Schleicher & Schuell, Dassel, Germany) and cellulose nitrate filters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plates containing the relevant antibiotic (e.g.kanamycin or chloramphenicol) at 37 ℃ for at least 21 hours. The cellulose acetate layer was placed on a TY agar plate.

After plating but before incubation the individual filter layers were specifically labelled with needles to enable the positive variants to be located on the filters, the nitrocellulose filters with the bound variants were transferred to containers containing carbonate/bicarbonate buffer, pH8.5-10, with different EDTA concentrations (0.001mM-100 mM). The filters were incubated for 1 hour at room temperature. Cellulose acetate filters with colonies were stored at room temperature on TY plates until use. After incubation, residual activity was detected on plates containing 1% agarose, 0.2% starch in carbonate/bicarbonate buffer (pH 8.5-10). The plates with nitrocellulose filters were labelled in the same way as the filter sandwich described above and incubated for 2 hours at room temperature. After removal of the filter, the plates were stained with 10% compound iodine solution (Lugol solution). On a dark blue background, variants of degraded starch were detected as white spots and then identified on the storage plates. The positive variants were rescreened twice under the same conditions as the first screen.

5.4 Low pH Filter Membrane assay

The Bacillus species library was plated on multiple layers of cellulose acetate (OE 67, Schleicher & Schuell, Dassel, Germany) and cellulose nitrate filters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) at 37 ℃ for at least 21 hours on TY agar plates containing 10. mu.g/ml chloramphenicol. The cellulose acetate layer was placed on a TY agar plate.

After plating but before incubation, each filter sandwich was specially marked with a needle to locate the positive variant on the filter, and the nitrocellulose filter with the bound variant was transferred to a container containing citrate buffer, ph4.5 and incubated at 80 ℃ for 20 minutes (when screening variants on the wild type background (backbone)) or at 85 ℃ for 60 minutes (when screening variants of the parent alpha-amylase). Cellulose acetate filters with colonies were stored at room temperature on TY plates until use. After incubation, residual activity was detected on an experimental plate containing 1% agarose, 0.2% starch in citrate buffer (ph 6.0). The plates with nitrocellulose filters were labelled in the same way as the filter sandwich described above and incubated for 2 hours at 50 ℃. After removal of the filter membrane, the plates were stained with 10% compound iodine solution. On a dark blue background, variants of degraded starch were detected as white spots and then identified on the storage plates. The positive variants were rescreened twice under the same conditions as the first screen.

5.5 Secondary screening

Rescreened positive transformants were picked from the storage plates and tested in a secondary plate screen. Positive transformants were grown in 5mL LB + chloramphenicol at 37 ℃ for 22 hours. Each positive transformant and the Bacillus culture of clones expressing the corresponding background as a control were incubated at 90 ℃ in citrate buffer (pH4.5) and sampled at 0, 10, 20, 30, 40, 60 and 80 minutes. Spot 3 μ L of sample on the experimental plate. The test plates were stained with 10% compound iodine solution. Improved variants are considered to be variants with higher residual activity (detected as halos on the experimental plate) compared to background. Nucleotide sequencing was used to determine the improved variants.

5.6 stability experiments of unpurified variants

The stability of the variants can be tested as follows: the Bacillus cultures expressing the variants to be analyzed were grown in 10ml LB + chloramphenicol at 37 ℃ for 21 hours. 800. mu.L of the culture was mixed with 200. mu.L of citrate buffer (pH 4.5). Multiple 70 mu corresponding to sample time point numberL aliquots are prepared in PCR tubes and incubated at 70 ℃ or 90 ℃ for various times (typically 5, 10, 15, 20, 25 and 30 minutes) in a PCR instrument. The 0 minute samples were not incubated at high temperature. By transferring 20. mu.L of sample to 200. mu.L of alpha-amylase PNP-G₇The activity of the samples was determined in the substrate MPR3(Boehringer Mannheim Cat No. 1660730), as described in the section "alpha-amylase activity assay" below. Results are plotted as percent activity (relative to activity at the 0 minute time point) versus time, or as percent residual activity after incubation for a certain time.

5.7 fermentation and purification of alpha-Amylase variants

Bacillus subtilis strains containing the relevant expression plasmids were fermented and purified as follows: strains taken from stocks stored at-80 ℃ were streaked onto LB agar plates containing 10. mu.g/ml kanamycin and grown overnight at 37 ℃. Colonies were transferred to 100mL PS-1 medium supplemented with 10. mu.g/mL chloramphenicol in 500mL shake flasks.

Composition of PS-1 Medium

Granulated sugar (Pearl sugar) 100g/l

40g/l soybean powder

Na₂HPO₄，12H₂O 10g/l

Pluronic^TM PE 6100 0.1g/l

CaCO₃ 5g/l

The culture was cultured at 37 ℃ for 5 days with shaking at 270 rpm.

Cells and cell debris were removed from the fermentation broth by centrifugation at 4500rpm for 20-25 minutes. The supernatant was filtered to give a completely clear solution. The filtrate was concentrated and washed on a UF-filter (10000 cut-off membrane) and the buffer was changed to 20mM acetate buffer, pH 5.5. The UF-filtrate was loaded onto S-Sepharose F.F. and eluted with the same buffer phase containing 0.2M NaCl. The eluate was dialyzed against 10mM Tris, ph9.0 and loaded onto Q-sepharose f.f., eluting with a linear gradient of NaCl from 0 to 0.3M in 6 column volumes. Fractions containing activity (measured using Phadebas experiment) were collected, pH adjusted to pH7.5, and residual color was removed by 0.5% W/vol.

5.8 specific Activity assay

Using PHADEBASThe specific activity was determined in the experiment (Pharmacia) and recorded as "activity/mg enzyme". The manufacturer's instructions were followed (see also "alpha-amylase activity assay" below).

5.9 isoelectric Point determination

pI is determined by isoelectric focusing (e.g., Pharmacia, Amphiline, pH 3.5-9.3).

5.10 increased stability assay

A50 ml propylene tube was charged with 10ml of the desired detergent. After appropriate dilution of AmyTS23t and AmyTS23t Δ RS to draw them into the respective tubes containing the detergent, each was measured at 180 ppm. The detergents with the respective mutant enzymes were vortexed for 30 seconds and then placed on a Rotamix (ATR RKVS model) for 10 minutes. Draw 100 microliters of detergent containing the mutant enzyme at a dilution of 1: 651. The initial activity of the mutants was tested on Konelab, Model20XT using a blocked P-Nitro-Phenyl-maltoheptose (BlockP-Nitro-Phenyl-Maltoheptaose (PBNPG 7 blocked)) substrate. The detergent samples were then incubated in a thermostated incubator set at 37 ℃. Samples were taken on days 1,2, 4,7 and 17 and tested for enzyme activity.

5.11 alpha-Amylase Activity assay

5.11.1 Phadebas experiment

To use PHADEBASMethod of using the sheet as a substrate to measure the alpha-amylase activity. Phadebas tablet (PHADEBAS)Amylase assay, supplied by Pharmacia Diagnostic) contains a cross-linked insoluble blue starch polymer which has been mixed with bovine serum albumin and buffer substances and tableted.

For each individual measurement, the measurement was carried out in a medium containing 5ml of 50mM Britton-Robinson buffer (50mM acetic acid, 50mM phosphoric acid, 50mM boric acid, 0.1mM CaCl₂Adjusted to the desired pH with NaOH) was suspended one piece in a tube. The test is carried out in a water bath at the temperature of interest. The alpha-amylase to be tested was diluted in X ml of 50mM Britton-Robinson buffer. 1ml of this alpha-amylase solution was added to 5ml of 50mM Britton-Robinson buffer. The alpha-amylase hydrolyzes starch to give soluble blue fragments. The absorbance of the blue solution measured with a spectrophotometer at 620nm is a function of the alpha-amylase activity.

It is important that the absorbance at 620nm measured after 10 or 15 minutes (detection time) incubation is in the range of 0.2-2.0 absorbance units at 620 nm. Within this absorbance range there is a linear relationship between activity and absorbance (Lambert-beer law). The dilution of the enzyme must therefore be adjusted to meet this criterion. At a given set of conditions (temperature, pH, reaction time, buffer conditions), 1mg of a given alpha-amylase will hydrolyze a certain amount of the substrate and will produce a blue color. The color intensity was measured at 620 nm. The measured absorbance is directly proportional to the specific activity (activity/mg of pure alpha-amylase protein) of the measured alpha-amylase at the given set of conditions.

5.11.2Alternative method

Use of PNP-G for alpha-amylase Activity₇Method for determination of a substrate. PNP-G₇Is an abbreviation for p-nitrophenyl-alpha-D-maltoheptoside, a block oligosaccharide capable of being cleaved by an endo-amylase. After lysis, the alpha-glucosidase contained in the kit digests the substrate, releasing free PNP molecules, which are yellow and therefore capable of functioning at λ405nm (400-. Containing PNP-G₇Kits of substrate and alpha-glucosidase were produced by Boehringer-Mannheim (cat # 1054635).

To prepare the reagent solution, 10ml of substrate/buffer was added to 50ml of enzyme/buffer according to the manufacturer's recommendations. Transfer 20 microliter of sample to a 96-well microtiter plate and incubate at 25 ℃ to perform the assay. 200 microliters of reagent solution pre-equilibrated at 25 ℃ was added. The solutions were mixed and pre-incubated for 1 min, and absorbance was measured at OD 405nm every 30 seconds on a microplate reader for 4 min.

The slope of the time-dependent absorbance curve is directly proportional to the activity of the tested alpha-amylase at the given condition setting.

5.12Determination of enzyme Performance in detergent compositions

5.12.1Conditions of the United states

The Dose Efficiency Curve (DEC) of the mutant enzyme of interest was measured using a Terg-o-meter, american testing center, Hoboken, new jersey-to simulate the wash test under american laundry conditions at 20 ℃ with standard detergents such as liquid AATCC2003 and/or powder AATCC1993 (american association of textile chemistry and color). The corresponding DEC of the equivalent alpha-amylase was then determined to compare the stain removal performance of the mutant enzymes of the invention. The process was repeated at 40 ℃. Typically, 4 samples of CS-28 rice starch contaminated (CFT, Netherlands) were placed in a steel container of a Terg-o-meter filled with 1 liter of deionized water and 1.5g of liquid AATCC. When powder AATCC was used, 1.5g of this detergent powder was weighed out on an analytical balance (Model PM4800, Mettler Instrument Corp., Highston, N.J.08520) and then added to a Terg-o-meter. The test was repeated twice at the same time. Unless otherwise stated, the assay was run for 12 minutes, washing for 3 minutes. After washing, the samples were air dried and the reflectance of the samples tested was determined using Chroma Meter model CR-410 from Konica Minolta. The collected data were processed with appropriate statistical analysis.

5.12.2European Condition

The Dose Efficiency Curve (DEC) of the mutant enzyme of interest was measured using a Lander-O-meter, manufactured by Atlas, Inc. -to simulate the wash test under European wash conditions at 40 ℃ with standard European test detergents, IECA and IEC A containing bleach (TAED-tetraacetylethylenediamine acetate) and sodium perborate. The corresponding DEC curves for comparable mutant enzymes were then determined to compare the stain removal performance of the mutant enzymes of the invention. The process is repeated at higher washing temperatures if necessary. Typically, four samples of EMPA 161, corn starch (EMPA, Switzerland) were placed in steel containers containing 250ml of deionized water containing 6.8g/L IEC A detergent or 8.0g/L IEC A detergent containing bleach. The test was repeated twice at the same time. Unless otherwise stated, the assay was run for 45 minutes, washing for 5 minutes. After washing, the samples were air dried and the reflectance of the samples tested was determined using a ChromaMeter Model CR-410. The collected data were processed with appropriate statistical analysis.

5.12.3 micro-sample method for evaluating detergent compositions

There are many kinds of alpha-amylase cleaning assays. Exemplary descriptions of detecting cleanliness include the following.

A "sample" is a piece of material, such as a fabric, on which stains have been applied. The material may be, for example, a fabric made of cotton, polyester or a mixture of natural and synthetic fibers. The sample may also be paper, such as filter paper or nitrocellulose, or a piece of hard material, such as ceramic, metal or glass. For alpha-amylase, stains are starch based, but may also include blood, milk, ink, grass, tea, wine, spinach, gravy, chocolate, egg, cheese, clay, pigments, oils or mixtures of these compounds.

A "small sample" is a portion cut from a sample using a single well perforation device, or a custom-made 96-well perforation device (the multi-well perforation pattern matching that of a standard 96-well microtiter plate), orThe portion removed from the sample is otherwise removed. The sample may be textile, paper, metal or other suitable material. Small samples may be fixed by dirt before or after placement in the wells of a 24-, 48-or 96-well microtiter plate. A "small sample" can also be made by applying a stain to a small piece of material. For example, the small sample may be a piece of soiled fabric with a diameter of 5/8 "or 0.25". The custom punch is designed to deliver 96 samples to all wells of a 96-well plate simultaneously. The device can deliver more than one sample per well by simply loading the same 96-well plate multiple times. It is contemplated that the multi-well perforation device can be used to simultaneously deliver samples to plates of any format, including but not limited to 24-well, 48-well, and 96-well plates. In another conceivable approach, the soiled test platform may be a bead formed of metal, plastic, glass, ceramic, or other suitable material, coated with a soil substrate, for testing cleaning compositions for materials other than textiles. One or more soil coated beads are then placed into wells of a 96-, 48-, or 24-well plate or wells of a larger format plate containing the appropriate buffer and enzyme. In this case, the released soil can be detected by direct absorbance measurement or in the supernatant after the secondary chromogenic reaction. Analysis of the released contaminants may also be performed by mass spectrometry. A further microscreening assay may be to deliver and fix a sample (e.g. indigo dyed denim fabric) into the pores of a multiwell plate and add particles, such as sand or larger particles, e.g. sieved to include 6-8 or 9 gauge stone particles, and shake the multiwell plate to cause abrasion of the sample by the added particles. This assay can be used in the evaluation of cellulase in stonewashing applications. The effectiveness of the enzyme can be measured by color release into the reaction buffer (e.g., the released indigo is dissolved in dimethyl sulfoxide, and A is measured₆₀₀Absorbance) or reflectance measurements of abraded samples.

When e.g. an untreated BMI (blood/milk/ink) sample is washed in a detergent without bleach, a large proportion of the ink is released even without the aid of proteases. The addition of proteases leads to a small increase in ink release, which is difficult to quantify in large backgrounds. One aspect of the present invention provides a treatment regimen that allows one to control the degree of fixation of a stain. As a result, samples can be produced that release unequal amounts of stain, for example, when washed in the absence of the enzyme being detected. The use of the immobilized sample resulted in a significant improvement in the signal to noise ratio in the wash test. Furthermore, by varying the degree of fixation, stains can be produced that give the best results under different cleaning conditions.

Samples of stains with known "strength" on a variety of material types are commercially available (EMPA, St. Gallen, Switzerland; wfk- -Testgewell GmbH, Krefeld Germany; or Center for Test Materials, Vlardingen, The Netherlands) and/or can be made by The practitioner (Morris and Prato, Textile Research Journal 52 (4): 280286 (1982)). Other test samples include, but are not limited to, blood/milk/ink (BMI) stains on cotton-containing fabrics, spinach stains on cotton-containing fabrics, or grass stains on cotton-containing fabrics, and chocolate/milk/soot stains on cotton-containing fabrics.

BMI stains can be fixed on cotton with 0.0003% to 0.3% hydrogen peroxide. Other combinations include grass or spinach stains fixed with 0.001% -1% glutaraldehyde, gelatin and Coomassie Brilliant blue dye fixed with 0.001% -1% glutaraldehyde, or chocolate, milk and soot fixed with 0.001% -1% glutaraldehyde.

The sample may also be agitated during incubation with the enzyme and/or detergent formulation. Wash performance data was dependent on the orientation (horizontal versus vertical) of the samples in the wells, especially in the 96-well plate. This indicates that the mixing is insufficient during the incubation. Although there are many ways to ensure adequate agitation during incubation, a plate clamp can be constructed that clamps the microtiter plate between two aluminum plates. This can be accomplished simply by, for example, placing an adhesive plate seal over the wells and then clamping the two aluminum plates to the 96-well plate with any type of suitable, commercially available clamp. It can then be placed in a commercial incubation shaker. Setting the shaker at about 400 rpm results in very effective mixing, while the plate holder effectively prevents leakage or cross-contamination.

Trinitrobenzenesulfonic acid (TNBS) can be used to quantify the amino concentration in the wash solution. This can be used as a measure of the amount of protein removed from the sample (see, e.g., Cayot and Taintuurier, anal. biochem. 249: 184-200 (1997)). However, if the detergent or enzyme sample results in the formation of unusually small peptide fragments (e.g., due to the presence of peptidases in the sample), one will obtain a greater TNBS signal, i.e., more "noise".

Another way to determine the wash performance on blood/milk/ink or other stains is based on the release of ink. Proteolysis of proteins on the sample results in the release of ink particles, which can be quantified by measuring the absorbance of the wash solution. The absorbance can be measured at any wavelength between 350 and 800 nm. The measurement wavelength was 410nm or 620 nm. The wash liquor can also be examined to determine wash performance on stains containing grass, spinach, gelatin or coomassie brilliant blue. For these stains, exemplary wavelengths include 670nm for spinach or grass stains and 620nm for gelatin or Coomassie Brilliant blue stains. For example, aliquots of the wash solution (e.g., typically 100-. It is then placed in a spectrophotometer and the absorbance is read at the appropriate wavelength.

The system can also be used to assay enhanced enzymes and/or detergent compositions for dishwashing, for example by using blood/milk/ink stains on suitable substrates such as cloth, plastics or ceramics.

In one aspect, BMI stains are fixed on cotton by applying 0.3% hydrogen peroxide to a BMI/cotton sample for 30 minutes at 25 ℃, or by applying 0.03% hydrogen peroxide to a BMI/cotton sample for 30 minutes at 60 ℃. A small sample of approximately 0.25 "was cut from the BMI/cotton sample described above and placed in a well of a 96-well microtiter plate. A mixture of known detergent composition and an enzyme such as a variant protein is placed into each well. After placing the adhesive plate seal on top of the microtiter plate, the microtiter plate was clamped together with an aluminum plate and agitated on an orbital shaker at about 250rpm for about 10-60 minutes. At the end of this time, the supernatant was transferred to the wells of a new microtiter plate and the absorbance of the ink at 620nm was measured. Similarly, cotton-fixed spinach or grass stains can be detected by applying 0.01% glutaraldehyde to a spinach/cotton sample or grass/cotton sample at 25 ℃ for 30 minutes. Chocolate, milk and/or soot stains may also be detected. Other blood/milk/ink tests and conditions are described in U.S. patent 7,122,334 (Genencor International, Inc.).

5.13 LAS sensitivity assay

The variants were incubated with different concentrations of LAS (linear alkyl benzene sulfonate; Nansa 1169/P) for 10 min at 40 ℃.

Using PhadebasMeasuring or using PNP-G₇Other methods of substrate determination of residual activity.

LAS was diluted in 0.1M, pH7.5 phosphate buffer.

The following concentrations were used: 500ppm, 250ppm, 100ppm, 50ppm, 25ppm and 10ppm or no LAS.

The variants were diluted to a concentration of 0.01-5mg/l in different LAS buffers in a total volume of 10ml and incubated in a temperature controlled water bath for 10 min. The incubation was stopped by transferring a small aliquot into cold detection buffer. It is important that the LAS concentration is below 1ppm during the activity measurement in order not to affect the activity measurement.

Then using the above PHADEBASMeasurement or other methods, residual activity was determined in duplicate for each sample.

Activity was determined after blank subtraction.

LAS-free activity was 100%.

The present application is organized into sections for ease of reading; however, the reader should understand that the description in one section may be used in other sections. In this manner, the headings for the various sections of this disclosure should not be construed as limiting.

To further illustrate the compositions and methods of this invention and their advantages, the following specific examples are given. It is to be understood that these examples are provided solely for the purpose of illustrating the compositions and methods of the present invention and are not to be construed as limiting the scope thereof in any way.

Examples

Throughout the specification, the following abbreviations are used: wt% (weight percent); deg.C (degrees Celsius); h₂O (water); dH₂O or DI (deionized water); dIH₂O (deionized water, Milli-Q filtration); g or gm (gram); μ g (μ g); mg (milligrams); kg (kilogram); μ L and μ L (microliters); mL and mL (milliliters); mm (millimeters); μ m (micrometers); m (moles per liter); mM (millimoles per liter); μ M (micromoles per liter); u (unit); MW (molecular weight); sec (seconds); min(s) (min); hr(s) (hours); DO (dissolved oxygen); W/V (weight to volume); W/W (weight ratio); V/V (volume ratio); genencor (Danisco US Inc, division of Genencor, palo alto, ca); ncm (Newton centimeters) and ETOH (ethanol); eq (equivalents); n (normal); DS or DS (dry solids content).

Example 1

Expression of AmyTS23 in Bacillus subtilis

To examine the full-length expression of AmyTS23, a synthetic DNA sequence as shown in FIG. 3 (prepared by Geneart, Rangeberg, Germany) was cloned into the pHPLT vector (see, e.g., WO2005111203 and Solingen et al (2001) extreme patents 5: 333-341) after the LAT (Bacillus licheniformis amylase) promoter,fused in frame with the signal peptide encoding LAT (FIG. 5), and transformed into 9 protease-deficient Bacillus subtilis strains (degU)^Hy32, oppA, Δ spoII3501, amyE:: xylRPxylAcomK-ermC, Δ aprE, Δ nprE, Δ epr, Δ ispA, Δ bpr, Δ vpr, Δ wprA, Δ mpr-ybfJ, Δ nprB) (see, e.g., US publication No. 20050202535A 1). Neomycin (10. mu.g/ml) resistant transformants secreted AmyTS23 amylase, which could be judged by the formation of halos after iodine staining on starch plates (see WO 2005111203). One of these amylase-positive transformants was selected and named BG6006(pHPLT-AmyTS 23). Cultures of this strain are typically grown for 60-72 hours at 37 degrees, 250rpm in the following medium (per liter): 10g Soytone (Soytone), 75g glucose, 7.2g urea, 40mM MOPS, 4mM Tricine, 3mM dipotassium hydrogen phosphate, 21.4mM KOH, 50mM NaCl, 276. mu.M potassium sulfate, 528. mu.M magnesium chloride, 50. mu.M sodium citrate dihydrate, 100. mu.M calcium chloride dihydrate, 14. mu.M ferrous sulfate heptahydrate, 5.9. mu.M manganese sulfate dihydrate, 5.7. mu.M zinc sulfate monohydrate, 2.9. mu.M copper chloride dihydrate, 4.2. mu.M cobalt chloride hexahydrate, 4.5. mu.M sodium molybdate dihydrate. For a1 liter volume of medium, all ingredients except soy peptone were mixed in 500mL, filter sterilized, and then added to an aliquot of 2 × soy peptone that had been autoclaved. Trace metals and citrate can be made into 100 x or 1000 x stock solutions. Buffer, potassium hydroxide, sodium chloride, potassium sulfate, magnesium chloride and trace metals can be prepared as a 10 × stock solution. After mixing all ingredients, the pH was adjusted to 7.3. The culture broth was supplemented with 20mM calcium chloride before use.

The cultures express amylase in two main forms. The high molecular weight form was observed at the 66kDa molecular weight standard in a 10% SDS-PAGE gel. A small molecular weight form was observed at 55 kDa.

The high molecular weight components were separated from the medium by the following method: 500mL of this medium was treated with a fixed volume of 10mL of beta-cyclodextrin-agarose affinity matrix resin (which was synthesized using standard protocols from beta-cyclodextrin (Sigma Aldrich Cat. No. c4767) and epoxy-activated Sepharose-6B (GEHealthcare, N.J.Cat. No. 17-0480-01)), with gentle agitationStirring overnight at 4 deg.C, collecting the resin, and adding a solution containing 2mM calcium chloride (CaCl)₂) Washed with 25mM bis-Tris propane buffer (pH 8.5). The high molecular weight enzyme was eluted by washing the resin with the same buffer supplemented with 50mM beta-cyclodextrin. Fractions were analyzed by SDS-PAGE and fractions containing the enzyme were collected and dialyzed to remove beta-cyclodextrin. The enzyme protein concentration was estimated by gel densitometry using Oxam amylase (Genencor) as protein standard.

Example 2

Expression of AmyTS23t in Bacillus subtilis

To detect expression of genetically truncated AmyTS23(AmyTS23t), the synthetic DNA shown in fig. 4 was cloned into pHPLT and transformed into a 9 protease-deficient bacillus subtilis strain as described in example 1. The neomycin resistant transformants secreted AmyTS23t amylase, which was judged by the formation of halos after iodine staining on starch plates. One of these amylase positive transformants was selected and named BG6006 (pME622.1). The strain was cultured as described in example 1 to produce AmyTS23t amylase. The culture supernatant was examined by SDS-PAGE and the expected molecular weight of 55kDa was produced.

NH was added to 500mL of culture₄SO₄To a final concentration of 1M, to partially purify the amylase protein. Then, 10mL of a fixed volume of benzene-agarose resin was added and the resulting mixture was gently stirred at 4 ℃ overnight. Collecting the resin and using a solution containing 1M NH₄SO₄And 2mM calcium chloride (CaCl)₂) Washed with 25mM bis-Tris propane buffer (pH 8.5). By not containing NH₄SO₄The enzyme activity was eluted with the same buffer as in (1). Fractions were analyzed by SDS-PAGE, fractions containing enzyme were collected and dialyzed to remove residual NH₄SO₄. Enzyme protein concentration was estimated by gel densitometry using OxAm amylase (Genencor International, Inc.) as a protein standard.

Example 3

AmyTS23 in a clean screening assay

Partially purified full-length AmyTS23 as described in example 1 was analyzed in a 96-well CS28 orange-dyed rice starch-soiled fabric sample micro application cleaning experiment. To perform this experiment, a 96-well plate was loaded with 1/4-inch size fabric samples cut from fabric pre-washed in room temperature water for 1 hour and air-dried. This washing removes a large amount of loosely bound stains. Optionally, these samples are also pre-washed after loading into the plate. Both methods produced similar results. The selected buffer is added to the wells of the plate, which is temperature equilibrated to the preferred temperature. In this example, the experiment was carried out in 25mM HEPES (pH8.0) and 25mM CAPS (pH10.3) buffer, and incubated at 20 or 40 ℃. After this temperature equilibration period, the enzyme is added to the desired concentration and incubation is continued for 30 minutes to 1 hour while shaking at 750rpm in an Eppendorf Thermomix temperature-controlled box. Enzyme performance was judged by the amount of enzyme dependent color release into solution. The color release was measured in a 488nm quantitative spectrophotometer. For additional information on this experiment, see U.S. patent 7,122,334.

The cleaning data for this enzyme in this experiment are shown in FIG. 6(20 ℃) and FIG. 7(40 ℃). Full-length AmyTS23(AmyTS23fl) showed efficient stain removal performance at ph8.0, but also showed surprising stain removal performance at ph 10.3.

The data indicate that AmyTS23fl exhibited better performance than the control (OxAm) at both pH values.

The sample experiment can be modified in several ways to suit different purposes. The 96-well assay is well suited for high throughput clean assays where absorbance is measured spectrophotometrically after incubation of the enzyme with the sample, while, for example, 24-well plates with samples cut to fit the 24-well plate can be used to wash larger samples whose reflectance can be measured by techniques well known in the art. Both measurements of supernatant absorbance and sample reflectance show almost complete correlation.

Reaction of washed samplesThe correlation between the reflectance and the absorbance of the supernatant is very high, and the coefficient of determination r is very high²Is 0.99. The experiment can in principle be scaled up to 384 well plates. The experiment can be performed with any soiled sample and, in addition to the CS28 sample, CS26, CS27 and CS29 samples (e.g., corn starch, potato starch, tapioca starch, Testfabrics, inc., WestPittiston, PA, respectively) can be tested to demonstrate the effectiveness of the measurements as described in example 3. The experiments were also performed on detergent compositions and at different temperatures and different pH values. These experiments were adapted from us patent 7,122,334.

Example 4

Clean screening assay for AmyTS23t

The partially purified truncated AmyTS23(AmyTS23t) described in example 2 was analyzed in a 96-well CS28 orange-stained, rice starch-soiled fabric sample reapplication cleaning experiment as described in example 3. The cleaning data for this enzyme in this experiment are shown in FIG. 8(20 ℃) and FIG. 9(40 ℃). The data show that AmyTS23t exhibited superior performance at both phs than the control amylase (OxAm, an amylase commercially available from Genencor). Comparison of fig. 6 and 8 clearly shows that truncated AmyTS23 has superior performance at 20 ℃ than the AmyTS23 full length molecule. The truncated molecule is therefore a more optimal molecule for laundry applications.

Example 5

Expression of AmyTS23 variants in Bacillus subtilis

In this example, the construction of a bacillus subtilis strain expressing the AmyTS23t variant is illustrated. A synthetic DNA fragment 056426 (produced by Geneart GmbH, Josef-Engert-Strass 11, D-93053 Regensburg, Germany) containing the codon-optimized AmyTS23 gene was used as template DNA (FIG. 3). The pHPLT vector (Solingen et al, extreme patents 5: 333-341, [2001]) containing the Bacillus licheniformis alpha-amylase (LAT) promoter and LAT signal peptide (preLAT) before the PstI and HpaI restriction sites for cloning was used for expression of the AmyTS23t variant.

Three DNA fragments were prepared by PCR using the following DNA primers:

1.AmyTS23t with codon CGG at position 180 and codon AGC deletion at position 181 (AmyTS23 t. DELTA.RS);

2.AmyTS23t having codon 201 ATG substituted with CTG (AmyTS23t (M201L);

3.AmyTS23t with substitution of ATG at codon 201 by CTG, CGG at codon 180 and deletion of AGC at codon 181 (AmyTS23t (M201L + Δ RS)

These DNA primers were synthesized and desalted using Sigma (Sigma-Aldrich Chemie B.V., Postbus 27, 3330AA Zwijndrecht, The Netherlands).

For all PCR reactions described below, DNA primers (forward and reverse) were used at a final concentration of 0.2. mu.M, and 0.1-10ng of DNA template (DNA fragment 056426 or pDNA pHPLT). In addition, all PCR reactions were performed in 50. mu.L volumes using Finnzymes (FinnzymesOY, Keilaranta 16A, 02150 Espoo, Finland) and Phusion high fidelity DNA polymerase (Cat. No. F-530L). Meanwhile, all PCR reaction mixtures contained 10. mu.L of 5 XPPhusionHF buffer, 1. mu.L of 10mM dNTP mix, 0.75. mu.L of Phusion DNA polymerase (2 units/. mu.L), 1. mu.L of 100% DMSO, and autoclaved deionized water, supplemented to a final volume of 50. mu.L. The PCR program was carried out using an MJ Research PTC-200 Peltier thermal cycler (MJ Research, 590 Lincoln Street, Waltham, MA 02451, USA) under conditions as described in the Finnzymes (manufacturer's instructions): 98 ℃ for 30 seconds, 30 cycles (98 ℃ for 10 seconds, 55 ℃ for 20 seconds, 72 ℃ per kb for 22 seconds), 72 ℃ for 5 minutes.

1.Generation of AmyTS23t Δ RS:

two PCR reactions were performed: primers TS-delRS-FW and pHPLT-HpaI-RV were used on the synthesized DNA fragment 056426; and primers TS-delRS-RV and pHPLT-PstI-FW on the synthetic DNA fragment 056426. To fuse the two DNA fragments produced, 1. mu.L of unpurified PCR mixture from the two PCR reactions was added to a third PCR reaction sample to which primers pHPLT-PstI-FW and pHPLT-HpaI-RV were added.

The amplified linear 1.5kb DNA fragment was purified (using Qiagen)Qiaquick PCR purification kit, cat No. 28106) and digested with PstI and HpaI restriction enzymes. Subsequently, the AmyTS23t Δ RS (also referred to herein as AmyTS23t Δ RS) DNA fragment and pHPLT pDNA (in the 50 ng/. mu.l range, and digested with PstI and HpaI) were purified (using Qiagen Qiaquick)PCR purification kit, cat No. 28106) and then ligated at the PstI and HpaI ends. The reaction conditions are as follows:

4 μ L of purified, PstI and HpaI digested AmyTS23t Δ RS DNA fragment; 2 μ L of purified, PstI and HpaI digested pHPLT DNA fragment; 8 μ L T4DNA ligase buffer (Invitrogen Cat No. 46300-018); 25 μ L of sterilized distilled water and 1 μ L T4DNA ligase 1 unit/. mu.L (Invitrogen cat No. 15224-017). The ligation reaction was carried out at 20 ℃ for 16-20 hours.

The ligation mixture was then transformed into Bacillus subtilis strains (aprE, nprE, epr, ispA, bpr) and (degU)^Hy32, oppA, spoIIE3501, amyE:: xylRPxylAcomK-ermC, (vpr, wprA, mpr-ybfJ, nprB). Transformation into B.subtilis was described in WO 02/14490. Selection on agar plates containing Heart infusion agar (Difco, cat. No. 244400) and 10mg/L neomycinThe Bacillus subtilis transformant. Selective growth of the Bacillus subtilis transformants with the pHPLT-AmyTS23 t. delta. RS vector was carried out in shake flasks as described in example 1. Secreted AmyTS23t Δ RS amylases were produced with starch hydrolyzing activity, which could be visualized by loading the culture supernatants on starch agar plates followed by iodine staining.

Generation of AmyTS23t (M201L):

the same experimental protocol as "generation of AmyTS23t Δ RS" was performed except for the first two PCR reactions:

two PCR reactions were performed: PCR was performed on the synthesized DNA fragment 056426 with the primers TS-M201L-FW and pHPLT-HpaI-RV; and PCR was performed on the synthesized DNA fragment 056426 using the primers TS-M201L-RV and pHPLT-PstI-FW.

Generation of AmyTS23t (M201L) -RS deletion:

the same experimental protocol as "generation of AmyTS23t Δ RS" was performed except for the first two PCR reactions:

two PCR reactions were performed: PCR was performed on the synthesized DNA fragment 056426 with primers TS-delRS/M201L-FW and pHPLT-HpaI-RV; and PCR was performed on the synthesized DNA fragment 056426 with the primers TS-delRS/M201L-RV and pHPLT-PstI-FW.

Example 6

AmyTS23t Δ RS improved stability in detergents

In the increased stability assay, the stability of AmyTS23t and AmyTS23t Δ RS was assayed in MOPS buffer, inactivated Tide (Tide) and exemplary detergent (exemplary formulation a), respectively, at 37 ℃. Enzyme samples were incubated in inactivated liquid tide or exemplary formulation a liquid detergent at 37 ℃ and the remaining activity was determined over time in the Megazyme assay. The results are shown in FIG. 10. Only AmyTS23t Δ RS remained stable in the presence of one of the two detergent bases (inactivated tide and example a detergent) without any additional additives. As shown in fig. 10, AmyTS23t lost most of the activity after the first day of the 37 ℃ increased stability assay and lost all activity after two days. Under the same conditions, AmyTS23t Δ RS was stable and approximately 90% of the original enzyme activity was maintained after 17 days.

TABLE 6-1

Example 7

Oxidative stability of AmyTS23 and AmyTS23 mutants

Different amylase reactions differ for the reaction of exposure to peracetic acid (PAA). Thus, this example was used to determine the oxidative stability of AmyTS23 and AmyTS23 mutant amylases. The conditions are summarized as follows:

pressure Condition Megazyme assay

30mM enzyme blocked PNPG7

25mM borate, pH 8.6525 mM BTP/CaCl₂，pH 6.9

1mM PAA，40C，5min 40℃45min kinetic

Quenching: 25mM BTP, pH8.5

Buffer exchange on a 1mL rotary desalting column in 25mM borate buffer, pH8.64, 2mM Ca⁺²To prepare an enzyme diluent. A volume of 5. mu.L of peracetic acid was added to 25. mu.L of the enzyme solution to give 0-1mM peracetic acid. The samples were incubated in a PCR instrument (DNA Engine, BioRad) for 5 minutes at 40 ℃. The reaction was stopped with 25mM BTP, pH 8.5. Residual amylase activity was determined using a standard amylase assay kit from Megazyme (Wicklow, Ireland).

As shown in fig. 11, TS23t (M201L) has greater than 100% stability at low PAA concentrations and then decreases at high PAA concentrations. TS23t (M201L + Δ RS) increased stability by 25% at low PAA concentrations, then dropped to below 100% at high PAA concentrations, and finally maintained oxidative stability at high PAA concentrations. TS23t, TS23t Δ RS, and Amy 707 were unstable in the presence of PAA, with stability dropping to baseline at low concentrations.

Example 8

Cleaning performance in detergents

Dose-effectiveness curves of selected concentrations of AmyTS23t Δ RS were generated using the methods described in section 5.12.1 of the present invention. The performance evaluation was carried out with a tergitometer at 20 ℃ and 40 ℃. Dose-effectiveness curves for Stainzyme and Stainzyme Plus were generated under the same conditions. As can be seen from the data (fig. 12), AmyTS23t Δ RS is significantly better than both Stainzyme products at 20 ℃ and moderately better than both Stainzyme products at 40 ℃. This data demonstrates the unique benefits of AmyTS23t Δ RS as a unique high performance cold water enzyme.

Example 9

Amylase production in Bacillus subtilis

In this example, the production of Bacillus species TS-23t and variants thereof in Bacillus subtilis is illustrated. The transformation is carried out by methods known in the art (see, for example, WO 02/14490). Briefly, the gene encoding the parent amylase was cloned into a pHPLT expression vector containing the LAT Promoter (PLAT), a sequence encoding the LAT signal peptide (preLAT), followed by PstI and HpaI restriction enzyme sites for cloning.

The coding regions for the LAT signal peptide are shown below:

atgaaacaacaaaaacggctttacgcccgattgctgacgctgttatttgcgctcatcttcttgctgcctcattctgcagcttcagca(SEQ ID NO：5).

the amino acid sequence of the LAT signal peptide is shown below:

MKQQKRLYARLLTLLFALIFLLPHSAASA(SEQ ID NO：6).

the coding region of the mature AmyTS-23t amylase is shown in FIG. 4.

The amino acid sequence of the mature AmyTS-23t amylase used as the basis for preparing the variant libraries described herein is shown in FIG. 2.

The PCR product was purified using Qiaquik columns from Qiagen and then resuspended in 50. mu.L of deionized water. 50 μ L of the purified DNA was digested with HpaI (Roche) and PstI (Roche), and the resulting DNA was resuspended in 30 μ L of deionized water. 10-20 ng/. mu.L of DNA was cloned into plasmid pHPLT using PstI and HpaI cloning sites. The ligation mixture was directly transformed into competent Bacillus subtilis cells (genotype: Δ vpr, Δ wprA, Δ mpr-ybfJ, Δ nprB). The Bacillus subtilis cell has a susceptibility gene (comK) located downstream of the xylose-inducible promoter, so that xylose is used to induce susceptibility to DNA binding and uptake (see Hahn et al, mol. Microbiol., 21: 763-.

The elements of plasmid pHPLT-AmyS include: pUB110 is a DNA fragment derived from Plasmid pUB110 (McKenzie et al, Plasmid 15: 93-103, [1986 ]). The plasmid characteristics include: ori-pUB 110-the origin of replication of pUB 110; neo ═ pUB110 neomycin resistance gene; plat ═ the transcriptional promoter of amylase from bacillus licheniformis; Pre-LAT ═ signal peptide from amylase from bacillus licheniformis; SAMY 425 ss-truncate the coding region of the Amy TS-23 gene sequence (substituted with the coding region of each truncated Amy TS-23 variant expressed in this study); and terminator the transcription terminator of amylase from bacillus licheniformis.

Amylase expression-2 mL scale.

The Bacillus subtilis clone containing the AmyTS23t expression vector was replicated using a 96-well steel replicator, transferred from glycerol stock to a 96-well plate (BD, 353075) (containing 150. mu.L of LB medium and 10. mu.g/ml neomycin) and grown overnight at 220 rpm in a wet box at 37 ℃. A100. mu.L aliquot from the overnight culture was used to inoculate 2000. mu.L of defined medium containing 10. mu.g/mL neomycin in a 5mL plastic culture tube. The medium was a fortified semi-defined medium based on MOPS buffer, containing urea as the main nitrogen source, glucose as the main carbon source, and supplemented with 1% soy peptone and 5mM calcium for robust cell growth. The tubes were incubated at 37 ℃ and 250rpm for 72 hours. After incubation, the culture broth was centrifuged at 3000 Xg for 10 min. The supernatant was transferred into a 15mL polypropylene conical tube and 80. mu.L of each sample was added to a 96-well plate for protein quantification.

Generation of a Combinatorial Charge Library (Combinatorial Charge Library) of Bacillus species AmyTS23 t.

A plurality of protein variants having a variety of physical properties of interest are selected from existing libraries or generated by site-directed mutagenesis techniques known in the art (see, e.g., U.S. patent application serial nos. 10/576,331, 11/581,102, and 11/583,334). This defined set of probe proteins is then tested in the assay of interest.

AmyTS23t is a truncated form of the Bacillus TS-23 α -amylase (see Lin et al, 1998, Production and properties of a raw-stable-degrading amylase from the therophilic and alkaline Bacillus sp.TS-23, Biotechnol.appl.biochem.28: 61-68). Shown herein is AmyTS23t Bacillus subtilis strain (degU) deleted in various proteases^Hy32, oppA, spoII3501, amyE:xylRPxylAcomK-ermC, aprE, nprE, epr, ispA, bpr, vpr, wprA, mpr-ybfJ, nprB) (see also US publication No. 20050202535A 1). AmyTS23t plasmid DNA isolated from transformed B.subtilis cells was sent to DNA2.0 (Menlo Park, CA) for use as a template for CCL construction. DNA2.0 was claimed to make the parent construct for CCL by introducing the following 7 mutations to AmyTS23t, which was thus named AmyTS23t-7mut, these 7 mutations being: Q98R, M201L, S243Q, R309A, Q320R, Q359E and K444E. The variants were provided as glycerol stocks in 96-well plates. The DNA2.0 company was then asked to generate a library of positions at each of the four sites in the AmyTS23t-7mut amylase shown in Table 9-1.

The AmyTS23t-7mut combinatorial charge library was designed by identifying the following four residues in AmyTS23t-7 mut: gln 87, Asn225, Asn272 and Asn 282. By forming all combinations of three possibilities (wild-type, arginine or aspartic acid) for each site, a four-site, 81-member CCL is formed.

TABLE 9-1 AmyTS23t-7mut CCL variants

Variant #	Q87	N225	N272	N282	Delta charge
						Parent 1	-	-	-	-	0
2	Q87E	N225E	N272E	N282E	-4
						3	Q87E	N225E	N272E	N282R	-2
4	Q87E	N225E	N272E	-	-3
						5	Q87E	N225E	N272R	N282E	-2
6	Q87E	N225E	N272R	N282R	0
						7	Q87E	N225E	N272R	-	-1
8	Q87E	N225E	-	N282E	-3
						9	Q87E	N225E	-	N282R	-1
10	Q87E	N225E	-	-	-2
						11	Q87E	N225R	N272E	N282E	-2
12	Q87E	N225R	N272E	N282R	0
						13	Q87E	N225R	N272E	-	-1
14	Q87E	N225R	N272R	N282E	0
						15	Q87E	N225R	N272R	N282R	+2
16	Q87E	N225R	N272R	-	+1
						17	Q87E	N225R	-	N282E	-1
18	Q87E	N225R	-	N282R	+1
						19	Q87E	N225R	-	-	0
20	Q87E	-	N272E	N282E	-3
						21	Q87E	-	N272E	N282R	-1
22	Q87E	-	N272E	-	-2
						23	Q87E	-	N272R	N282E	-1

Variant #	Q87	N225	N272	N282	Delta charge
						24	Q87E	-	N272R	N282R	+1
25	Q87E	-	N272R	-	0
						26	Q87E	-	-	N282E	-2
27	Q87E	-	-	N282R	0
						28	Q87E	-	-	-	-1
29	Q87R	N225E	N272E	N282E	-2
						30	Q87R	N225E	N272E	N282R	0
31	Q87R	N225E	N272E	-	-1
						32	Q87R	N225E	N272R	N282E	0
33	Q87R	N225E	N272R	N282R	+2
						34	Q87R	N225E	N272R	-	+1
35	Q87R	N225E	-	N282E	-1
						36	Q87R	N225E	-	N282R	+1
37	Q87R	N225E	-	-	0
						38	Q87R	N225R	N272E	N282E	0
39	Q87R	N225R	N272E	N282R	+2
						40	Q87R	N225R	N272E	-	+1
41	Q87R	N225R	N272R	N282E	+2
						42	Q87R	N225R	N272R	N282R	+4
43	Q87R	N225R	N272R	-	+3
						44	Q87R	N225R	-	N282E	+1
45	Q87R	N225R	-	N282R	+3
						46	Q87R	N225R	-	-	+2
47	Q87R	-	N272E	N282E	-1
						48	Q87R	-	N272E	N282R	+1
49	Q87R	-	N272E	-	0
						50	Q87R	-	N272R	N282E	+1
51	Q87R	-	N272R	N282R	+3
						52	Q87R	-	N272R	-	+2
53	Q87R	-	-	N282E	0
						54	Q87R	-	-	N282R	+2
55	Q87R	-	-	-	+1
						56	-	N225E	N272E	N282E	-3

Variant #	Q87	N225	N272	N282	Delta charge
						57	-	N225E	N272E	N282R	-1
58	-	N225E	N272E	-	-2
						59	-	N225E	N272R	N282E	-1
60	-	N225E	N272R	N282R	+1
						61	-	N225E	N272R	-	0
62	-	N225E	-	N282E	-2
						63	-	N225E	-	N282R	0
64	-	N225E	-	-	-1
						65	-	N225R	N272E	N282E	-1
66	-	N225R	N272E	N282R	+1
						67	-	N225R	N272E	-	0
68	-	N225R	N272R	N282E	+1
						69	-	N225R	N272R	N282R	+3
70	-	N225R	N272R	-	+2
						71	-	N225R	-	N282E	0
72	-	N225R	-	N282R	+2
						73	-	N225R	-	-	+1
74	-	-	N272E	N282E	-2
						75	-	-	N272E	N282R	0
76	-	-	N272E	-	-1
						77	-	-	N272R	N282E	0
78	-	-	N272R	N282R	+2
						79	-	-	N272R	-	+1
80	-	-	-	N282E	-1
						81	-	-	-	N282R	+1

Example 10

Performance parameter

And detecting the rice micro sample.

Test detergents were prepared as described elsewhere herein. The apparatus used included: NewBrunswick Innova 4230 shaker/incubator and Spectramax (340 type) MTP reader. MTP was purchased from Corning (3641 type). A swatch of aged rice starch (CS-28) with orange pigment was taken from the center of the test material (Vlardingen, Netherlands). The fabric was washed with water and then cut into 0.25 inch round micro swatches. Two microtiter plates were placed in each well of a 96-well microtiter plate. Detergents were tested at 20 ℃ (north america) or 40 ℃ (western europe) equilibrium. To each well containing the MTP of the micro swatch was added 190 μ L of detergent solution. To this mixture was added 10. mu.L of diluted enzyme solution. The MTP was sealed with adhesive foil, placed in an incubator and shaken at 750rpm for 1 hour at the desired test temperature (typically 20 ℃ or 40 ℃). After incubation, 150 μ L of solution was taken per well and transferred to fresh MTP. The MTP reading was done at 488nm using a SpectraMax MTP reader to quantify the cleaning effect. A blank control was included, as well as a control containing both the microtablets and detergent but no enzyme.

Detergent heat inactivation

Heat inactivation of commercial detergent formulations serves to destroy the enzymatic activity of any protein component while retaining the properties of the non-enzymatic components. Thus, the method is suitable for preparing commercially available detergents for detecting enzyme variants of the compositions and methods of the invention. For the North American (NA) and Western European (WE) robust liquid laundry (HDL) detergents, heat inactivation was performed by placing a pre-weighed amount of liquid detergent (in glass bottles) in a water bath at 95 ℃ for 2 hours. Incubation time for heat inactivation of North American (NA) and Japanese (JPN) strong granular laundry (HDG) detergents required 8 hours, and Western European (WE) HDG detergents required 5 hours. The incubation time for heat-inactivated NA and WE automatic dishwashing detergent (ADW) was 8 hours. Detergents are purchased from local supermarkets. The percentage of inactivation was accurately determined by testing the detergent in 5 minutes of dissolution without heat treatment and after heat treatment. The enzyme activity was tested in an AAPF assay with 1mg/mL AAPF.

Detergent working solutions were prepared from heat-inactivated stock solutions for detection of enzyme activity in heat-inactivated detergents. To this detergent solution, appropriate amounts of water hardness (6gpg or 12gpg) and buffer were added to satisfy the required conditions (Table 10-1). Vortex or invert the vial to mix the solution.

Abbreviation: baojie corporation (P & G); and Reckitt Benckiser (RB).

And (4) calculating enzyme performance.

The resulting absorbance was corrected for a blank value (i.e., the value obtained in incubating the microsheet in the absence of enzyme). The absorbance obtained is a measure of the hydrolytic activity. The results are shown in tables 10-2 and 10-3. Enzyme performance was assessed using heat-inactivated detergents as described above. "winners" are defined as those having a Performance Index (PI) greater than 1. PI is the ratio of the residual activity of the mutant to the residual activity of the wild type.

Table 10-2: TS23t-7mut CCL-CS-28 Rice micro sample piece winning, tide 2x

Variant #	87	225	272	282	Relative charge	PI
							11	Q87E	N225R	N272E	N282E	-2	1.24
12	Q87E	N225R	N272E	N282R	0	1.20
							13	Q87E	N225R	N272E		-1	1.16
14	Q87E	N225R	N272R	N282E	0	1.15
							17	Q87E	N225R		N282E	-1	1.34
18	Q87E	N225R		N282R	1	1.26
							19	Q87E	N225R			0	1.34
20	Q87E		N272E	N282E	-3	1.17
							21	Q87E		N272E	N282R	-1	1.34
22	Q87E		N272E		-2	1.13

Variant #	87	225	272	282	Relative charge	PI
							27	Q87E			N282R	0	1.22
28	Q87E				-1	1.22
							29	Q87R	N225E	N272E	N282E	-2	1.44
30	Q87R	N225E	N272E	N282R	0	1.15
							31	Q87R	N225E	N272E		-1	1.36
35	Q87R	N225E		N282E	-1	1.15
							40	Q87R	N225R	N272E		1	1.27
44	Q87R	N225R		N282E	1	1.38
							45	Q87R	N225R		N282R	3	1.21
47	Q87R		N272E	N282E	-1	1.65
							48	Q87R		N272E	N282R	1	1.52
49	Q87R		N272E		0	1.28
							50	Q87R		N272R	N282E	1	1.10
53	Q87R			N282E	0	1.47
							54	Q87R			N282R	2	1.25
55	Q87R				1	1.51
							64		N225E			-1	1.15
65		N225R	N272E	N282E	-1	1.26
							66		N225R	N272E	N282R	1	1.22
67		N225R	N272E		0	1.19
							74			N272E	N282E	-2	1.21
76			N272E		-1	1.13
							80				N282E	-1	1.27
81				N282R	1	1.49

Tables 10 to 3: TS-23t-7mut CCL CS-28 rice micro-starch-like chip, Persil

Variant #	87	225	272	282	Relative charge	PI
							4	Q87E	N225E	N272E	0	-3	1.13
6	Q87E	N225E	N272R	N282R	0	1.11
							9	Q87E	N225E		N282R	-1	1.20
10	Q87E	N225E		0	-2	1.17
							11	Q87E	N225R	N272E	N282E	-2	1.41

Variant #	87	225	272	282	Relative charge	PI
							13	Q87E	N225R	N272E	0	-1	1.40
14	Q87E	N225R	N272R	N282E	0	1.28
							15	Q87E	N225R	N272R	N282R	2	1.13
16	Q87E	N225R	N272R	0	1	1.17
							17	Q87E	N225R		N282E	-1	1.51
18	Q87E	N225R		N282R	1	1.47
							19	Q87E	N225R		0	0	1.48
20	Q87E		N272E	N282E	-3	1.46
							21	Q87E		N272E	N282R	-1	1.40
22	Q87E		N272E	0	-2	1.42
							25	Q87E		N272R	0	0	1.18
26	Q87E			N282E	-2	1.54
							27	Q87E			N282R	0	1.47
28	Q87E			0	-1	1.40
							29	Q87R	N225E	N272E	N282E	-2	1.46
30	Q87R	N225E	N272E	N282R	0	1.59
							31	Q87R	N225E	N272E	0	-1	1.14
34	Q87R	N225E	N272R	0	1	1.29
							35	Q87R	N225E		N282E	-1	1.47
36	Q87R	N225E		N282R	1	1.62
							37	Q87R	N225E		0	0	1.53
38	Q87R	N225R	N272E	N282E	0	1.13
							39	Q87R	N225R	N272E	N282R	2	1.13
40	Q87R	N225R	N272E	0	1	1.17
							41	Q87R	N225R	N272R	N282E	2	1.31
44	Q87R	N225R		N282E	1	1.26
							47	Q87R		N272E	N282E	-1	1.45
48	Q87R		N272E	N282R	1	1.50
							49	Q87R		N272E	0	0	1.17
50	Q87R		N272R	N282E	1	1.16
							53	Q87R			N282E	0	1.21
54	Q87R			N282R	2	1.30

Variant #	87	225	272	282	Relative charge	PI
							55	Q87R			0	1	1.33
56		N225E	N272E	N282E	-3	1.29
							57		N225E	N272E	N282R	-1	1.12
58		N225E	N272E	0	-2	1.41
							59		N225E	N272R	N282E	-1	1.16
61		N225E	N272R	0	0	1.20
							66		N225R	N272E	N282R	1	1.27
67		N225R	N272E	0	0	1.34
							71		N225R		N282E	0	1.17
73		N225R		0	1	1.12
							74			N272E	N282E	-2	1.29
75			N272E	N282R	0	1.24
							76			N272E	0	-1	1.20
78			N272R	N282R	2	1.18
							79			N272R	0	1	1.11
80				N282E	-1	1.11
							81				N282R	1	1.33

Example 11

Combined LAS/chelator stability

This example describes the determination of the relationship of protein charge to stability in a reaction medium containing an anionic surfactant and a chelating agent. The test amylases were incubated in the presence of 0.1% LAS (sodium dodecylbenzenesulfonate) and 10mM EDTA and LAS stability was measured by measuring residual activity by the BODIPY method as described above. The BODIPY-starch assay was used to determine alpha-amylase activity for both stressed and unstressed samples. Residual LAS and EDTA from the compression plate did not affect the BODIPY-starch experiments.

The reagents used included: control buffer (50mM HEPES, 0.005% Tween-80, pH8.0) and compression buffer (50mM HEPES, 0.1% (w/v) LAS (dodecylbenzenesulfonic acid sodium salt, Sigma D-2525), 10mM EDTA, pH8.0). Enzyme variants (20ppm) were diluted 1: 20 into 96-well unbound flat-bottom plates containing control buffer or pressurized buffer and mixed. The control plate was incubated at room temperature, while the compression plate was immediately placed at 37 ℃ for 30-60 minutes (depending on the stability of the enzyme to be tested). After incubation, the enzyme activity was determined in the BODIPY-starch assay for amylase. The fraction of residual or remaining activity is equal to the reaction rate of the pressurized sample divided by the reaction rate of the control sample. Both the parent enzyme and the variant were stable for 60 minutes in control buffer.

Table 11-1 lists data for variants with enhanced LAS/EDTA stability as a function of net charge change relative to wild-type TS-23t-7mut for the library containing 80 variants. This library was designed and constructed according to the method described in example 2 to cover several net charges relative to the parent TS-23t-7mut molecule. A Performance Index (PI) greater than 1 indicates that the variant has higher specific activity on this starch substrate (maize starch) than the S242Q parent.

Table 11-1: TS23t-7mut CCL-LAS/EDTA stability outperforming

For ASP and FNA, its LAS/EDTA stability is charge-dependent (see WO/2008/153925, 6.6.2008, Genencor attorney docket No. 30974 WO-2). Adding negative charges increases stability. However, even if there is one or two more positive charges than the parent, it is possible to find in our method that the arrangement of the charge mutations confers a higher stability than the parent or the same stability as the parent. For larger enzymes, such as TS23 t' shown in fig. 13, this approach is also effective, and the adverse effect on stability from the addition of positive charges can be compensated for by the optimized charge arrangement that increases stability.

All publications and patents mentioned hereinabove are incorporated by reference. Various modifications and alterations to this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention. While the compositions and methods of this invention have been described in connection with specific preferred embodiments, it should be understood that the compositions and methods of this invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the compositions and methods of the present invention which are obvious to those skilled in the art are intended to be within the scope of the following claims.

Claims (13)

1.A polypeptide consisting of SEQ ID NO: 1, wherein the variant is a variant of a parent AmyTS-23 a-amylase represented by SEQ ID NO: 2 and comprises deletions of residues R180 and S181, and/or comprises a substitution of residue M201L at position 201, wherein said amino acid residue positions are referred to SEQ ID NO: 1.

2. The variant of claim 1, wherein the variant has alpha-amylase activity.

3. The variant of claim 1 or 2, wherein said variant has increased cleaning activity in cold water for removal of starch stains compared to the parent amylase.

4. The variant of claim 3, wherein said variant has increased detergent stability as compared to a parent amylase.

5. The variant of claim 1, further comprising a substitution of residue M201L at position 201, wherein said amino acid residue position is referenced to SEQ ID NO: 1.

6. The variant of claim 5, wherein said variant has increased oxidative stability as compared to a parent amylase.

7. A nucleic acid encoding a variant according to any preceding claim.

8. An expression vector comprising the nucleic acid of claim 7 under the control of a suitable promoter.

9. A host cell comprising the expression vector of claim 8.

10. A composition for hand or automatic dishwashing, which comprises the variant of any of claims 1-6.

11. A laundry detergent additive comprising the variant of any of claims 1-6.

12. A method of removing starch from a fabric comprising: incubating the fabric in the presence of the variant of any of claims 1-6, and

wherein the incubation removes starch from the fabric.

13. A method of processing starch, the method comprising incubating a fabric in the presence of the variant of any of claims 1-6, and

wherein said incubating hydrolyzes said starch.