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WO2001088107A2 - Variantes d'alpha-amylase avec une activite 1,6 alteree - Google Patents

Variantes d'alpha-amylase avec une activite 1,6 alteree Download PDF

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Publication number
WO2001088107A2
WO2001088107A2 PCT/DK2001/000323 DK0100323W WO0188107A2 WO 2001088107 A2 WO2001088107 A2 WO 2001088107A2 DK 0100323 W DK0100323 W DK 0100323W WO 0188107 A2 WO0188107 A2 WO 0188107A2
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Prior art keywords
alpha
amylase
variant
amino acid
bacillus
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WO2001088107A3 (fr
Inventor
Allan Svendsen
Christel Thea JØRGENSEN
Bjarne Rønfeldt Nielsen
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Novozymes AS
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Novozymes AS
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Priority to AU2001258229A priority Critical patent/AU2001258229A1/en
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Publication of WO2001088107A3 publication Critical patent/WO2001088107A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38681—Chemically modified or immobilised enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source

Definitions

  • the present invention relates to variants (mutants) of parent Termamyl-like alpha-amylases, which variant exhibits an alteration in alpha-1 , 6-D-glucosidic branch linkage cleavage activity.
  • the invention also relates a DNA construct comprising a DNA sequence encoding the alpha-amylase variant of the invention, an expression vector for recombinant production and a host cell for recombinant production.
  • the invention also relates to various compositions comprising a variant of the invention, especially for use in the liquefaction step of, e.g., a strach convention or ethanol process, and finally to the use of such variants or compositions of the invention for various uses.
  • Alpha-Amylases (alpha-1, 4-glucan-4-glucanohydrolases, E.C. 3.2.1.1) constitute a group of enzymes, which catalyze hydrolysis of starch and other linear and branched 1, 4-glucosidic oligo- and polysaccharides.
  • Protein engineering is increasingly used to alter the properties of enzymes - such as alpha-amylase - to obtain an enzyme with properties tailored for specific applications.
  • the object of the present invention is to provide Termamyl- like amylases which variants in comparison to the corresponding parent alpha-amylase, i.e., un-mutated alpha-amylase, has alpha- amylase activity and exhibits an alteration in alpha-1, 6-D- glucosidic branch linkage cleavage activity of amylopectin and alpha- and beta-limit dextrins.
  • Alpha-amylases with altered activity towards alpha-1, 6-D- glucosidic branch linkages of amylopectin and alpha- and beta- limit dextrins are desired, because such enzyme variants may, if the 1,6-activity is increased, increase the glucose yield in the liquefaction process in connection with, e.g., high fructose corn syrup (HFCS) production, because less panose will be formed. Further, in detergents the degradation of starch into smaller sugar units (less limit dextrins or smaller limit dextrins) facilitates the washing out of the sugar molecules and thus improve the wash performance. If a higher limit dextrins level is desired reduced 1,6-activity is advantageous .
  • HFCS high fructose corn syrup
  • Ala30Asn or A3ON a deletion of alanine in the same position is shown as: Ala30* or A30* and insertion of an additional amino acid residue, such as lysine, is shown as:
  • a deletion of a consecutive stretch of amino acid residues, such as amino acid residues 30-33, is indicated as (30-33)* or
  • Ala30Asp + Glu34Ser or A30N+E34S representing mutations in positions 30 and 34 substituting alanine and glutamic acid for asparagine and serine, respectively.
  • any amino acid residue may be substituted for the amino acid residue present in the position.
  • the alanine may be deleted or substituted for any other amino acid, i.e., any one of: R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.
  • A3OX means any one of the following substitutions : A30R, A30N, A30D, A30C, A30Q, A30E, A30G, A30H, A30I, A30L, A30K, A30M, A30F, A30P, A30S, A30T, A30W, A30Y, or A30 V; or in short: A30R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.
  • the invention relates a variant of a parent Termamyl-like alpha-amylase, comprising an alteration at one or more of the following regions or positions selected from the group of : Region: 186-193, Region: 261-276, Region: 283-293, Region: 334-339, Position: 234, wherein (a) the alteration (s) are independently
  • each position corresponds to a position of the amino acid sequence of the parent Termamyl-like alpha-amylase having the B. lichen ⁇ formis alpha-amylase amino acid sequence of shown in SEQ ID NO: 8.
  • the invention also relates to a number of specific variants; to a DNA construct comprising a DNA sequence encoding an alpha- amylase variant of the invention; a recombinant expression vector which carries a DNA construct of the invention; a cell which is transformed with a DNA construct of the invention or a vector of the invention; composition comprising alpha-amylase variant of the invention; the use of a variant of the invention for specified industrial applications.
  • Figure 1 is an alignment of the amino acid sequences of five parent Termamyl-like alpha-amylases. The numbers on the extreme left designate the respective amino acid sequences as follows:
  • the object of the present invention is to provide Termamyl- like amylases, which variants exhibits an alteration in the alpha-1, 6-D-glucosidic branch linkage cleavage activity of especially amylopectin and alpha- and/or beta- limit dextrins.
  • Alpha-amylases which catalyze hydrolysis of starch and other linear and branched 1, 4-glucosidic oligo- and polysaccharides have in general limited activity on the alpha-
  • alpha-1, 6-D- glucosidic branch linkages activity it may be advantageous to decrease the alpha-1, 6-D- glucosidic branch linkages activity.
  • alpha-1, 6-D- glucosidic branch linkages of amylopectin and/or alpha- and/or 5 beta-limit dextrins can be altered by mutating within one or more of the below mentioned regions or positions in Termamyl- like alpha-amylases.
  • the invention relates a variant of a parent Termamyl-like alpha-amylase, comprising an alteration 10 at one or more of the following regions or positions selected from the group of : Region: 186-193, Region: 261-276, Region: 283-293, 15 Region: 334-339, Position 234, wherein (a) the alteration (s) are independently
  • each position corresponds to a position of the amino acid sequence of the parent Termamyl-like alpha-amylase having the B . licheniformis alpha-amylase amino acid sequence of shown in SEQ ID NO: 8.
  • the region mutated is the Region: 186-193.- 30 Specific preferred positions contemplated include one or more of positions 186, 187, 188, 189, 190, 191, 192, 193. Specific mutations include one or more of: E189GASTV;
  • the region mutated is the Region: 35 261-276.
  • Specific preferred positions contemplated include one or more of positions 261, 262, 263, 264, 265, 266, 266, 267, 268,
  • Specific mutations include one or more of: 5 W263GASTV; Q264X; N265GASTV;
  • Specific preferred positions contemplated include one or more of positions 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, lo 293.
  • Specific mutations include one or more of:
  • V286FWY or smaller residues than V e.g., GAS; Y290 smaller residues than Y, e.g., A,N,D, C, Q, E,G,H, I , L, K,M, F, P, S, T, V.
  • the region mutated is the Region: 15 334-339.
  • Specific preferred positions contemplated include one or more of positions 334, 335, 336, 337, 338, 339.
  • Specific mutations include one or more of:
  • position K234 is also contemplated according to the invention, preferably K234X, especially preferably
  • hydrolysis of the individual bond types can be followed during reaction, e.g., hydrolysis of alpha (1_4) - linkages will give a reduction in the integral of the alpha (1_»4) -signals and an equivalent increase in the integral of the signals coming from the reducing ends.
  • hydrolysis of alpha (1_4) - linkages will give a reduction in the integral of the alpha (1_»4) -signals and an equivalent increase in the integral of the signals coming from the reducing ends.
  • Synthetic designed substrates and limit dextrins can be used as substrates.
  • Termamyl ® (B . licehniformis alpha-amylase shown in SEQ ID NO: 8) and Novamyl ® (B . stearothermophilus C599 maltogenic amylase disclosed in EP patent no. 120,693 (Novo Industri A/S) limit dextrins (made from amylopectin) , where the low molecular weight material ( ⁇ 1000 Da) has been removed by ultrafiltration may be used.
  • the incubations can be followed directly in the NMR tube (D 2 0) at 60 °C if enzyme and substrate are pretreated repeatedly with D 2 0.
  • Limit dextrins may be prepared as described by Mottawia et al, Carbohydr. Res. 277 (1995), 109-123 (which is hereby incorporated by reference) or specifically as described in the "Materials & Methods” section.
  • a variant of the invention may in an embodiment be capable of hydrolysing starch and other linear and branched 1, 4-glucosidic oligo- and polysaccharides and may further have altered (i.e., higher or lower) alpha-1, 6-D-glucosidic branch linkage cleavage activity.
  • the variant of the invention is capable of hydrolysing starch and other linear and branched 1, 4-glucosidic oligo- and polysaccharides, but do not have any detectable alpha-1, 6-D-glucosidic branch linkage cleavage activity.
  • a number of alpha-amylases produced by Bacillus spp. are highly homologous (identical) on amino acid level.
  • the identity of a number of Bacillus alpha-amylases can be found in the below Table 1:
  • the B . licheniformis alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 8 (commercially available as TermamylTM) has been found to be about 81% homologous (identical) to the B . amyloliquefaciens alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 10 and about 65% homologous with the B . stearothermophilus alpha- amylase comprising the amino acid sequence shown in SEQ ID NO: 6.
  • Further homologous parent alpha-amylases include SP690 and SP722, respectively, disclosed in WO 95/26397 and further depicted in SEQ ID NO : 2 and SEQ ID NO: 4, respectively, herein.
  • Termamyl-like alpha-amylases are the AA560 alpha-amylase derived from Bacillus sp . and shown in SEQ ID NO: 12, and the #707 alpha-amylase derived from Bacillus sp., shown in SEQ ID NO: 13 and described by Tsukamoto et al . , Biochemical and Biophysical Research Communications, 151 (1988), pp. 25-31.
  • the Termamyl-like alpha-amylase referred to are KSM AP1378 is disclosed in WO 97/00324 (from KAO Corporation, JP) .
  • Termamyl-like alpha-amylases include the alpha-amylase produced by the B . licheniformis strain described in EP 0252666 (ATCC 27811) , and the alpha- amylases identified in WO 91/00353 and WO 94/18314.
  • Termamyl-like alpha-amylases are comprised in the products sold under the following tradenames : OptithermTM and TakathermTM (available from Solvay) ; MaxamylT (available from Gist-brocades (DSM) /Genencor) , Spezym AATM and Spezyme Delta AATM (available from Genencor) , and KeistaseTM available from Daiwa) , PURASTARTM ST 5000E, PURASTRARTM HPAM L (from Genencor Int.) .
  • alpha-amylases Because of the substantial homology found between these alpha-amylases, they are considered to belong to the same class of alpha-amylases, namely the above-mentioned class of "Termamyl-like alpha-amylases" .
  • Termamyl-like alpha-amylase is intended to indicate an alpha-amylase, which, at the amino acid level, exhibits a substantial identity to TermamylTM, i.e., the B . licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 8 herein.
  • alpha-amylases which has the amino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12 and 13 herein are considered to be "Termamyl-like alpha- amylase" .
  • Other Termamyl-like alpha-amylases are alpha-amylases i) which displays at least 60%, such as at least 70%, e.g., at least 75%, or at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% homology (identity) with at least one of the amino acid sequences shown in SEQ ID NOS: 2,4,6,8,10,12, and 13, and/or ii) is encoded by a DNA sequence which hybridizes to the DNA sequences encoding the above-specified alpha-amylases which are apparent from SEQ ID NOS: 1,3,5,7,9, and of the present specification (which encoding sequences encode the amino acid sequences shown in SEQ
  • the term "homology” may be determined as the degree of "identity” between the two sequences indicating a derivation of the first sequence from the second.
  • the homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (described above) .
  • GAP uses the method of Needleman/Wunsch/Sellers to make alignments.
  • a structural alignment between, e.g., Termamyl ® (SEQ ID NO: 8) and a Termamyl-like alpha-amylase may be used to identify equivalent/corresponding positions in other Termamyl-like alpha- amylases.
  • One method of obtaining said structural alignment is to use the Pile Up programme from the GCG package using default values of gap penalties, i.e., a gap creation penalty of 3.0 and gap extension penalty of 0.1.
  • Other structural alignment methods include the hydrophobic cluster analysis (Gaboriaud et al . , (1987), FEBS LETTERS 224, pp. 149-155) and reverse threading (Huber, T; Torda, AE, PROTEIN SCIENCE Vol. 7, No. 1 pp. 142- 149 (1998) .
  • oligonucleotide probe used in the characterisation of the Termamyl-like alpha-amylase in accordance with property ii) above may suitably be prepared on the basis of the full or partial nucleotide or amino acid sequence of the alpha-amylase in question.
  • Suitable conditions for testing hybridisation involve pre- soaking in 5xSSC and prehybridizing for 1 hour at -40 °C in a solution of 20% formamide, 5xDenhardt ' s solution, 50mM sodium phosphate, pH 6.8, and 50mg of denatured sonicated calf thymus DNA, followed by hybridisation in the same solution supplemented with lOO M ATP for 18 hours at ⁇ 40°C, followed by three times washing of the filter in 2xSSC, 0.2% SDS at 40°C for 30 minutes (low stringency) , preferred at 50°C (medium stringency) , more preferably at 65°C (high stringency) , even more preferably at ⁇ 75°C (very high stringency) . More details about the hybridisation method can be found in Sambrook et al . , Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.
  • derived from is intended not only to indicate an alpha-amylase produced or producible by a strain of the organism in question, but also an alpha-amylase encoded by a DNA sequence isolated from such strain and produced in a host organism transformed with said DNA sequence.
  • the term is intended to indicate an alpha-amylase, which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the alpha-amylase in question.
  • the parent alpha-amylase may be a variant of a naturally occurring alpha- amylase, i.e., a variant, which is the result of a modification (insertion, substitution, deletion) of one or more amino acid residues of the naturally occurring alpha-amylase.
  • Termamy-like alpha-amylases may be used as the parent (i.e., backbone) alpha-amylase.
  • the parent alpha-amylase is derived from B . licheniformis, e.g., one of those referred to above, such as the B . licheniformis alpha- amylase having the amino acid sequence shown in SEQ ID NO: 8.
  • the parent alpha-amylase (i.e., backbone alpha-amylase) may also be a hybrid alpha-amylase, i.e., an alpha-amylase, which comprises a combination of partial amino acid sequences derived from at least two alpha-amylases.
  • the parent hybrid alpha-amylase may be one, which on the basis of amino acid homology (identity) and/or DNA hybridization (as defined above) can be determined to belong to the Termamyl- like alpha-amylase family.
  • the hybrid alpha- amylase is typically composed of at least, one part of a Termamyl-like alpha-amylase and part (s) of one or more other alpha-amylases selected from Termamyl-like alpha-amylases or non-Termamyl-like alpha-amylases of microbial (bacterial or fungal) and/or mammalian origin.
  • the parent hybrid alpha-amylase may comprise a combination of partial amino acid sequences deriving from at least two Termamyl-like alpha-amylases, or from at least one Termamyl-like and at least one non-Termamyl-like bacterial alpha-amylase, or from at least one Termamyl-like and at least one fungal alpha-amylase.
  • the Termamyl-like alpha-amylase from which a partial amino acid sequence derives may be any of those specific Termamyl-like alpha-amylase referred to herein.
  • the parent alpha-amylase may comprise a C- terminal part of an alpha-amylase derived from a strain of JB.
  • the parent alpha-amylase may comprise at least 430 amino acid residues of the C-terminal part of the B . licheniformis alpha-amylase, and may, e.g., comprise a) an amino acid segment corresponding to the 37 N-terminal amino acid residues of the B . amyloliquefaciens alpha-amylase having the amino acid sequence shown in SEQ ID NO: 10 and an amino acid segment corresponding to the 445 C-terminal amino acid residues of the B .
  • licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO : 8 , or a hybrid Termamyl-like alpha-amylase being identical to the Termamyl sequence, i.e., the Bacillus licheniformis alpha-amylase (BLA) shown in SEQ ID NO : 8, except that the N-terminal 35 amino acid residues (of the mature protein) has been replaced by the N-terminal 33 residues of BAN (mature protein), i.e., the Bacillus amyloliquefaciens alpha-amylase (BAN) shown in SEQ ID NO: 10; or b) an amino acid segment corresponding to the 68 N- terminal amino acid residues of the B .
  • BLA Bacillus licheniformis alpha-amylase
  • stearothermophilus alpha- amylase having the amino acid sequence shown in SEQ ID NO: 6 and an amino acid segment corresponding to the 415 C-terminal amino acid residues of the 23.
  • licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 8.
  • the parent Termamyl-like alpha-amylase is a hybrid alpha-amylase of SEQ ID NO: 8 and SEQ ID NO: 10.
  • the parent hybrid Termamyl-like alpha-amylase may be a hybrid alpha-amylase comprising the 445 C-terminal amino acid residues of the 23. licheniformis alpha-amylase shown in SEQ ID NO: 4 and the 37 N- terminal amino acid residues of the alpha-amylase derived from 23.
  • amyloliquefaciens shown in SEQ ID NO: 5 which may suitably further have the following mutations: H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 8) .
  • the latter mentioned hybrid is referred to as LE174.
  • Another suitable parent hybrid alpha-amylase is the one previously described in WO 96/23874 (from Novozymes) constituting the N-terminus of BAN, Bacillus amyloliquefaciens alpha-amylase (amino acids 1-300 of the mature protein) and the C-terminus from Termamyl ® (amino acids 301-483 of the mature protein) .
  • Other specifically contemplated parent alpha-amylase include LE174 with fewer mutations, i.e., the right above mentioned hydrid having the following mutations
  • the parent Termamyl-like alpha amylase is LE174, SP722, or AA560 including any of LE174+G48A+T49I+G107A+I201F; LE174+M197L
  • the particularly interesting amino acid substitutions are those that increase the mobility around the active site of the enzyme. This is accomplished by changes that disrupt stabilizing interaction in the vicinity of the active site, i . e. , within preferably 10A or 8A or 6A or 4A from any of the residues constituting the active site.
  • Val to Ala or Gly lie or Leu to Val, Ala, or Gly
  • Thr to Ser are expected to cause increased flexibility in the active site region either by the introduction of cavities or by the structural rearrangements that fill the space left by the mutation. It may be preferred that a variant of the invention comprises one or more modifications in addition to those outlined above. Thus, it may be advantageous that one or more Proline residues present in the part of the alpha-amylase variant which is modified is/are replaced with a non-Proline residue which may be any of the possible, naturally occurring non-Proline residues, and which preferably is an Alanine, Glycine, Serine, Threonine, Valine or Leucine.
  • one or more Cysteine residues present among the amino acid residues with which the parent alpha-amylase is modified is/are replaced with a non- Cysteine residue such as Serine, Alanine, Threonine, Glycine, Valine or Leucine.
  • a variant of the invention may - either as the only modification or in combination with any of the above outlined modifications - be modified so that one or more Asp and/or Glu present in an amino acid fragment corresponding to the amino acid fragment 185-209 of SEQ ID NO: 10 is replaced by an Asn and/or Gin, respectively. Also of interest is the replacement, in the Termamyl-like alpha-amylase, of one or more of the Lys residues present in an amino acid fragment corresponding to the amino acid fragment 185-209 of SEQ ID NO: 10 by an Arg.
  • the DNA sequence encoding a parent alpha-amylase may be isolated from any cell or microorganism producing the alpha- amylase in question, using various methods well known in the art.
  • a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the alpha-amylase to be studied.
  • homologous, labeled oligonucleotide probes may be synthesized and used to identify alph -amylase-encoding clones from a genomic library prepared from the organism in question.
  • oligonucleotide probe containing sequences homologous to a known alpha-amylase gene could be used as a probe to identify alpha-amylase-encoding clones, using hybridization and washing conditions of lower stringency.
  • Yet another method for identifying ⁇ am ⁇ lase-encoding clones would involve inserting fragments of genomic DNA into an ex- pression vector, such as a plasmid, transforming ⁇ -amylase- negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for alpha-amylase, thereby allowing clones expressing the alpha-amylase to be identified.
  • an ex- pression vector such as a plasmid
  • transforming ⁇ -amylase- negative bacteria with the resulting genomic DNA library
  • the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g., the phosphoroamidite method described by S.L. Beaucage and M.H. Caruthers, Tetrahedron Letters 22, 1981, pp. 1859-1869, or the method described by Matthes et al . , The EMBO J. 3, 1984, pp. 801-805.
  • the phosphoroamidite method oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.
  • the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, the fragments corresponding to various parts of the entire DNA sequence) , in accordance with standard techniques.
  • the DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or R.K. Saiki et al., Science 239, 1988, pp. 487-491.
  • mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligonucleotide synthesis.
  • a single-stranded gap of DNA, bridging the alpha-amylase-encoding sequence is created in a vector carrying the alpha-amylase gene.
  • the synthetic nucleotide, bearing the desired mutation is annealed to a homologous portion of the single-stranded DNA.
  • PCR-generated fragment containing the desired mutation introduced by using a chemically synthesized DNA strand as one of the primers in the PCR reactions.
  • a DNA fragment carrying the mutation may be isolated by cleavage with restriction endonucleases and reinserted into an expression plasmid.
  • Random mutagenesis is suitably performed either as localised or region-specific random mutagenesis in at least three parts of the gene translating to the amino acid sequence shown in question, or within the whole gene.
  • a further aspect of the present invention relates to a method for generating a variant of a parent alpha-amylase, e.g., wherein the variant exhibits altered or increased thermal stability relative to the parent, the method comprising: (a) subjecting a DNA sequence encoding the parent - amylase to random mutagenesis,
  • step (b) expressing the mutated DNA sequence obtained in step (a) in a host cell
  • Step (a) of the above method of the invention is preferably performed using doped primers .
  • the random mutagenesis may be performed by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis.
  • the random mutagenesis may be performed by use of any combination of these mutagenizing agents.
  • the mutagenizing agent may, e . g. , be one which induces transitions, transversions, inversions, scrambling, deletions, and/or insertions.
  • Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N' -nitro-N-nitrosoguanidine (MNNG) , O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS) , sodium bisulphite, formic acid, and nucleotide analogues.
  • UV ultraviolet
  • MNNG N-methyl-N' -nitro-N-nitrosoguanidine
  • EMS ethyl methane sulphonate
  • sodium bisulphite formic acid
  • nucleotide analogues examples include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N' -nitro-N-nitrosoguanidine (MNNG) , O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS) , sodium bisulphite,
  • the oligonucleotide may be doped or spiked with the three non-parent nucleotides during the synthesis of the oligonucleotide at the positions, which are to be changed.
  • the doping or spiking may be done so that codons for unwanted amino acids are avoided.
  • the doped or spiked oligonucleotide can be incorporated into the DNA encoding the alpha-amylase enzyme by any published technique, using, e . g. , PCR, LCR or any DNA polymerase and ligase as deemed appropriate.
  • the doping is carried out using "constant random doping", in which the percentage of wild-type and mutation in each position is predefined.
  • the doping may be directed toward a preference for the introduction of certain nucleotides, and thereby a preference for the introduction of one or more specific amino acid residues.
  • the doping may be made, e . g. , so as to allow for the introduction of 90% wild type and 10% mutations in each position.
  • An additional consideration in the choice of a doping scheme is based on genetic as well as protein-structural constraints.
  • the doping scheme may be made by using the DOPE program, which, inter alia, ensures that introduction of stop codons is avoided.
  • PCR-generated mutagenesis When PCR-generated mutagenesis is used, either a chemically treated or non-treated gene encoding a parent alpha-amylase is subjected to PCR under conditions that increase the mis- incorporation of nucleotides (Deshler 1992; Leung et al . , Technique, Vol.l, 1989, pp. 11-15).
  • a mutator strain of E. coli (Fowler et al . , Molec. Gen. Genet., 133, 1974, pp. 179-191), S . cereviseae or any other microbial organism may be used for the random mutagenesis of the DNA encoding the alpha-amylase by, e . g.
  • mutator strain containing the parent glycosylase into the mutator strain
  • the mutated plasmid may be subsequently transformed into the expression organism.
  • the DNA sequence to be mutagenized may be conveniently present in a genomic or cDNA library prepared from an organism expressing the parent alpha-amylase.
  • the DNA sequence may be present on a suitable vector such as a plasmid or a bacteriophage, which as such may be incubated with or otherwise exposed to the utagenising agent.
  • the DNA to be mutagenized may also be present in a host cell either by being integrated in the genome of said cell or by being present on a vector harboured in the cell.
  • the DNA to be mutagenized may be in isolated form. It will be understood that the DNA sequence to be subjected to random mutagenesis is preferably a cDNA or a genomic DNA sequence.
  • telomere amplification may be performed in accordance with methods known in the art, the presently preferred method being PCR-generated amplification using oligonucleotide primers prepared on the basis of the DNA or amino acid sequence of the parent enzyme .
  • the mutated DNA is expressed by culturing a suitable host cell carrying the DNA sequence under conditions allowing expression to take place.
  • the host cell used for this purpose may be one which has been transformed with the mutated DNA sequence, optionally present on a vector, or one which was carried the DNA sequence encoding the parent enzyme during the mutagenesis treatment.
  • suitable host cells are the following: gram positive bacteria such as Bacillus subtilis, Bacillus licheniformis , Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus , Bacillus alkalophilus , Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, Streptomyces lividans or Streptomyces murinus; and gram-negative bacteria such as E. coli .
  • gram positive bacteria such as Bacillus subtilis, Bacillus licheniformis , Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus , Bacillus alkalophilus , Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, Str
  • the mutated DNA sequence may further comprise a DNA sequence encoding functions permitting expression of the mutated DNA sequence .
  • the random mutagenesis may be advantageously localized to a part of the parent alpha-amylase in question. This may, e.g., be advantageous when certain regions of the enzyme have been identified to be of particular importance for a given property of the enzyme, and when modified are expected to result in a variant having improved properties. Such regions may normally be identified when the tertiary structure of the parent enzyme has been elucidated and related to the function of the enzyme.
  • the localized, or region-specific, random mutagenesis is conveniently performed by use of PCR generated mutagenesis techniques as described above or any other suitable technique known in the art.
  • the DNA sequence encoding the part of the DNA sequence to be modified may be isolated, e . g. , by insertion into a suitable vector, and said part may be subsequently subjected to mutagenesis by use of any of the mutagenesis methods discussed above.
  • Alternative methods for providing variants of the invention include gene-shuffling method known in the art including the methods, e.g., described in WO 95/22625 (from Affymax Technologies N.V.) and WO 96/00343 (from Novozymes A/S) .
  • a DNA sequence encoding the variant produced by methods described above, or by any alterna- tive methods known in the art can be expressed, in enzyme form, using an expression vector which typically includes control 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 an alpha-amylase variant of the invention may be any vector, which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or an artificial chromosome.
  • 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.
  • the DNA sequence should be operably connected to a suitable 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 .
  • suitable promoters for directing the transcription of the DNA sequence encoding an alpha-amylase variant of the invention, especially in a bacterial host are the promoter of the lac operon of E.
  • the Streptomyces coelicolor agarase gene dagA promoters the promoters of the Bacillus licheniformis alpha- amylase gene (amyL) , the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM) , the promoters of the Bacillus amyloliquefaciens alpha-amylase (amyQ) , the promoters of the Bacillus subtilis xylA and xylB genes etc.
  • useful promoters are those derived from the gene encoding A .
  • oryzae TAKA amylase Rhizomucor miehei aspartic proteinase, A . niger neutral alpha- amylase, A . niger acid stable alpha-amylase, A . niger glu- coamylase, Rhizomucor miehei lipase, A . oryzae alkaline protease, A . oryzae triose phosphate isomerase or A. nidulans acetamidase .
  • the expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, poly- adenylation sequences operably connected to the DNA sequence encoding the alpha-amylase variant of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
  • the vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBHO, pE194, pAMBl and pIJ702.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B . subtilis or B . licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance.
  • a selectable marker e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B . subtilis or B . licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance.
  • the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, e.g., as described in WO 91/17243.
  • Bacillus ⁇ -amylases mentioned herein comprise a preregion permitting secretion of the expressed protease into the culture medium. If desirable, this preregion may be replaced by a different preregion or signal sequence, conveniently accom- plished by substitution of the DNA sequences encoding the respective preregions .
  • the procedures used to ligate the DNA construct of the invention encoding an alpha-amylase variant, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al . , Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989) .
  • the cell of the invention either comprising a DNA construct or an expression vector of the invention as defined above, is advantageously used as a host cell in the recombinant production of an alpha-amylase variant of the invention.
  • the cell may be transformed with the DNA construct of the invention encoding the variant, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome.
  • This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination.
  • the cell may be transformed with an expression vector as described above in connection with the different types of host cells.
  • the host cell of the invention may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g., a bacterial or a fungal (including yeast) cell.
  • a microbial cell e.g., a bacterial or a fungal (including yeast) cell.
  • suitable bacteria are Gram-positive bacteria such as Bacillus subtilis, Bacillus licheniformis , Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus , Bacillus alkalophilus, Bacillus amyloliquefaciens , Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyces lividans or Streptomyces murinus, or gramnegative bacteria such as E. coli .
  • the trans- formation of the bacteria may, for instance, be effected by protoplast transformation or by using competent cells in a manner known per se .
  • the yeast organism may favorably be selected from a species of Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae .
  • the filamentous fungus may advantageously belong to a species of Aspergillus, e.g., Aspergillus oryzae or Aspergil - lus 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 manner known per se . A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023.
  • the present invention relates to a method of producing an alpha-amylase variant of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the variant and recovering the variant from the cells and/or culture medium.
  • the medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the alpha-amylase variant of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection) .
  • the alpha-amylase variant secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
  • alpha-amylase variants of this invention possess valuable properties allowing for a variety of industrial applications.
  • enzyme variants of the invention are applicable as a component in washing, dishwashing and hard surface cleaning detergent compositions.
  • Variant of the invention with altered properties may be used for starch processes, in particular starch conversion, especially liquefaction of starch (see, e . g. , US 3,912,590, EP patent publications Nos. 252 730 and 63 909, and WO 99/19467.
  • variants of the invention are also particularly useful in the production of sweeteners and ethanol from starch, and/or for textile desizing.
  • variants of the invention may suitably be incorporated in detergent compositions.
  • Detergent compositions comprising a variant of the invention may additionally comprise one or more other enzymes, such as a lipase, cutinase, protease, cellulase, peroxidase or laccase, and/or another alpha-amylase.
  • enzymes such as a lipase, cutinase, protease, cellulase, peroxidase or laccase, and/or another alpha-amylase.
  • Alpha-amylase variants of the invention may be incorporated in detergents at conventionally employed concentrations. It is at present contemplated that a variant of the invention may be incorporated in an amount corresponding to 0.00001-10 mg (calculated as pure, active enzyme protein) of alpha-amylase per liter of wash/dishwash liquor using conventional dosing levels of detergent.
  • TERMAMYL ® B . licheniformis alpha-amylase shown in SEQ ID NO : 8.
  • NOVAMYL ® B. stearothermophilus C599 maltogenic amylase disclosed in EP patent no. 120,693 (available from Novo Nordisk)
  • Bacillus subtilis SHA273 see WO 95/10603
  • PJEl contains the gene encoding a variant of SP722 alpha- amylase (SEQ ID NO: 4) : viz. deletion of 6 nucleotides corresponding to amino acids D183-G184 in the mature protein. Transcription of the JE1 gene is directed from the amyL promoter.
  • the plasmid further more contains the origin of replication and cat-gene conferring resistance towards kanamycin obtained from plasmid pUBHO (Gryczan, TJ et al . (1978), J. Bact. 134:318-329) .
  • the E. coli/ Bacillus shuttle vector pDorKlOl (described below) can be used to introduce mutations without expression of alpha-amylase in E. coli and then be modified in such way that the alpha-amylase is active in Bacillus.
  • the vector was constructed as follows: The JE1 encoding gene (SP722 with the deletion of D183-G184) was inactivated in pJEl by gene interruption in the Pstl site in the 5 'coding region of the SEQ ID NO: 4: SP722 by a 1.2 kb fragment containing an E. coli origin of replication.
  • This fragment was PCR amplified from the pUC19 (GenBank Accession #:X02514) using the forward primer: 5 '-gacctgcagtcaggcaacta-3 " (SEQ ID NO: 14) and the reverse primer: 5'-tagagtcgacctgcaggcat-3 ' ' (SEQ ID NO: 15) .
  • the PCR amplicon and the pJEl vector were digested with Pstl at 37°C for 2 hours.
  • the pJEl vector fragment and the PCR fragment were ligated at room temperature, for 1 hour and transformed in E. coli by electrotransformation.
  • the resulting vector is designated pDorKlOl.
  • Fermentation and purification may be performed by methods well known in the art .
  • the enzyme solutions (of a chosen activity, e.g., 10-100 NU) are diluted with D 2 0 and freeze-dried. The samples are re- dissolved in D 2 0 (0.5 mL) and freeze-dried. Samples containing 25 mg of substrate in D 2 0 (0.5 mL) are freeze-dried before re- dissolved (D 2 0, 0.5 mL) and freeze-dried. Finally the enzymes are dissolved in D 2 0 (1 mL) and added to each sample of substrate. The solutions are transferred to NMR tubes and incubated at 60 °C. 1 H NMR spectra are recorded currently at 60 °C on a Varian Mercury 400 MHz instrument (5 mm inverse probe head, 32 scan) .
  • NOVAMYL ® and TERMAMYL ® Limit Dextrins The NOVAMYL ® limit dextrin is prepared from waxy maize starch (100 % amylopectin) by gelatinization at 80°C and treatment with NOVAMYL ® for 4 hours at 60 °C. The enzyme is inactivated at 100 °C (10 minutes) before smaller dextrins are removed by ultrafiltration. The dextrin is then be obtained as white fibers after freeze-drying.
  • the Termamyl limit dextrin is prepared by a similar procedure; gelatinisation at 100°C, incubation for 2 hours at 90°C, inactivation, ultrafiltration and freeze-drying. See also Mottawia et al , Carbohydr. Res. 277 (1995), 109-123 (purification and beta-amylase treatment excluded in the present procedure) .
  • Phadebas assay Alpha-amylase activity is determined by a method employing PHADEBAS ® tablets as substrate.
  • Phadebas tablets (PHADEBAS ® Amylase Test, supplied by Pharmacia Diagnostic) contain a cross- linked insoluble blue-colored starch polymer, which has been mixed with bovine serum albumin and a buffer substance and tabletted.
  • the measured 620 nm absorbance after 10 or 15 minutes of incubation is in the range of 0.2 to 2.0 absorbance units at 620 nm. In this absorbance range there is linearity between activity and absorbance (Lambert-Beer law) .
  • the dilution of the enzyme must therefore be adjusted to fit this criterion.
  • a specified set of conditions temp., pH, reaction time, buffer conditions
  • 1 mg of a given alpha- amylase will hydrolyze a certain amount of substrate and a blue colour will be produced.
  • the colour intensity is measured at 620 nm.
  • the measured absorbance is directly proportional to the specific activity (activity/mg of pure alpha-amylase protein) of the alpha-amylase in question under the given set of conditions. 2.
  • Alpha-amylase activity is determined by a method employing the PNP-G 7 substrate.
  • PNP-G ? which is a abbreviation for p- nitrophenyl-alpha
  • D-maltoheptaoside is a blocked oligosaccharide which can be cleaved by an endo-amylase .
  • Kits containing PNP-G substrate and alpha-Glucosidase is manufactured by Boehringer-Mannheim (cat. No. 1054635) .
  • To prepare the substrate one bottle of substrate (BM)
  • Glucosidase one bottle of alpha-Glucosidase (BM 1462309) is added to 45 ml buffer (BM1442309) .
  • the working solution is made by mixing 5 ml alpha-Glucosidase solution with 0.5 ml substrate.
  • the assay is performed by transforming 20 micro 1 enzyme solution to a 96 well microtitre plate and incubating at 25°C.
  • the slope of the time dependent absorption-curve is directly proportional to the specific activity (activity per mg enzyme) of the alpha-amylase in question under the given set of conditions .
  • the random mutagenesis may be carried out as follows:
  • Suitable dope algorithms for use in step 6 are well known 5 in the art.
  • One algorithm is described by Tomandl , D. et al . , Journal of Computer-Aided Molecular Design, 11 (1997), pp. 29- 38) .
  • Another algorithm, DOPE is described in the following:
  • the dope program lo The "DOPE” program is a computer algorithm useful to optimize the nucleotide composition of a codon triplet in such a way that it encodes an amino acid distribution, which resembles most the wanted amino acid distribution. In order to assess which of the possible distributions is the most similar is to the wanted amino acid distribution, a scoring function is needed. In the "Dope” program the following function was found to be suited:
  • x t ' s are the obtained amounts of amino acids and groups of amino acids as calculated by the program
  • y t ' s are the wanted amounts of amino acids and groups of amino acids as defined by the user of the program (e.g. specify which of the
  • 25 20 amino acids or stop codons are wanted to be introduced, e.g., with a certain percentage (e.g. 90% Ala, 3% lie, 7% Val), and w ⁇ ' s are assigned weight factors as defined by the user of the program (e.g., depending on the importance of having a specific amino acid residue inserted into the
  • N is 21 plus the number of amino acid groups as defined by the user of the program.
  • 0 is defined as being 1.
  • the scores of the new composition and the current composition are compared by the use of the function s as described above. If the new score is higher or equal to the score of the current composition, the new composition is kept and the current composition is changed to the new one. If the new score is smaller, the probability of keeping the new composition is exp ⁇ 1000 ⁇ new_score - current _score) ⁇ .
  • a cycle normally consists of 1000 iterations as described above in which d is decreasing linearly from 1 to 0.
  • One hundred or more cycles are performed in an optimization process.
  • the nucleotide composition resulting in the highest score is finally presented.
  • V286F,W,Y,G,A,S Y290A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,V;

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Abstract

L'invention concerne des variantes d'alpha-amylases du type Termamyl de base, ces variantes possédant une activité d'alpha-amylase et montrant une altération de l'activité d'hydrolyse de liaison ramifiée alpha-1,6-D-glucosidique de l'amylopectine et de dextrines limites.
PCT/DK2001/000323 2000-05-12 2001-05-10 Variantes d'alpha-amylase avec une activite 1,6 alteree Ceased WO2001088107A2 (fr)

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