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WO2001064852A1 - Polypeptides possedant une activite de l'alpha-amylase et acides nucleiques codant pour ces polypeptides - Google Patents

Polypeptides possedant une activite de l'alpha-amylase et acides nucleiques codant pour ces polypeptides Download PDF

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Publication number
WO2001064852A1
WO2001064852A1 PCT/DK2001/000133 DK0100133W WO0164852A1 WO 2001064852 A1 WO2001064852 A1 WO 2001064852A1 DK 0100133 W DK0100133 W DK 0100133W WO 0164852 A1 WO0164852 A1 WO 0164852A1
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Prior art keywords
seq
polypeptide
nucleotide sequence
amylase
sequence
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Inventor
Carsten Andersen
Helle Outtrup
Bjarne Roenfeldt Nielsen
Lisbeth Hedegaard Hoeck
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Novozymes AS
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Novozymes AS
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Priority to AU37248/01A priority Critical patent/AU3724801A/en
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    • 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
    • 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
    • C11D3/37—Polymers
    • C11D3/3703—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • C11D3/3719—Polyamides or polyimides
    • 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
    • C11D3/38609—Protease or amylase in solid compositions only
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • 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)

Definitions

  • the present invention relates to polypeptides having ⁇ -amylase activity and nucleotide sequences encoding the polypeptides. Further, the invention relates to variants of the ⁇ -amylases of the invention. The invention also relates to nucleic acid constructs, vectors, host cells and transgenic organisms comprising the nucleotide sequences as well as methods for producing and using the polypeptides.
  • ⁇ -amylase enzymes have been used for a variety of different purposes, the most important of which are starch liquefaction, textile desizing, starch modification in the paper and pulp industry, and for brewing and baking.
  • ⁇ -amylase products examples include Termamyl ® , DuramylTM,
  • WO 95/26397 discloses an ⁇ -amylase from a Bacillus strain which has optimum activity at pH 8.
  • WO 96/23873 describes variants of Bacillus amylases with improved performance under washing conditions.
  • US 5,147,796 describes an alkaline pullulanase having alpha-amylase activity.
  • Fig. 2b of the document shows optimum amylase activity at pH 8-8.5.
  • Tsukamoto A. et al. Biochem. Biophys. Res. Commun. 151 :25-31 (1988) describes a nucleotide sequence of a maltohexaose-producing amylase gene from alkalophilic Bacillus sp. #707.
  • M. Takagi et al., J. Ferment. Bioeng., vol 81 , No. 6, 557-559 (1996) describe an alkaliphilic alpha-amylase-pullulanase from Bacillus sp.
  • the enzyme has optimum amylase activity at pH 9, but the activity drops rapidly at higher pH, and the activity at pH 10 is lower than at pH 7.
  • WO 97/00324 discloses a gene encoding an alkaline liquefying ⁇ - amylase derived from Bacillus sp. strain KSM-AP1378 with the deposited no. FERM BP-3048 suitable for detergents.
  • One object of the present invention is to provide novel ⁇ -amylases with activity in alkaline solutions, especially in alkaline detergent solutions at pH around 7-1 1.
  • the present invention relates to polypeptides having ⁇ -amylase activity selected from the group consisting of: (a) a polypeptide having an amino acid sequence of amino acids 1 to 486 of SEQ ID NO:2;
  • polypeptide encoded by a nucleotide sequence which hybridises under medium-high stringency conditions with (i) the nucleotide sequence 359-1816 of SEQ ID NO:1 , or 359-1816 of SEQ ID NO:3, or 566-2023 of SEQ ID NO:5, (ii) the cDNA sequence of 359-1816 of SEQ ID NO:1 , or 359-1816 of SEQ ID NO:3, or 566- 2023 SEQ ID NO:5, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii);
  • the invention relates to variants of the ⁇ -amylases of the invention having one or more amino acid substituted, inserted and/or deleted compared to
  • the present invention also relates to nucleotide sequences encoding the polypeptides and to nucleic acid constructs, vectors, and host cells and transgenic organisms comprising the nucleotide sequences as well as methods for producing and using the polypeptides.
  • Figure 1 shows the alignment of a number of Bacillus ⁇ -amylases, including AA180, AAG20 and Amrk385 (corresponding to amino acids 1 to 486 of SEQ ID NO:2, 4 and 6, respectively).
  • Figure 2 shows the pH profile of the ⁇ -amylase AA180 as described in
  • Example 4 The activity is shown in relative values. The pH profile is measured at 37°C.
  • Figure 3 shows the temperature profile of the ⁇ -amylase AA180 as described in Example 4. The activity is shown as absorbance per mg protein. The temperature profile is measured at pH 8.5.
  • Figure 4 shows wash performance of AA180 compared to other alpha- amylases.
  • Example 6 shows wash performance of AA180 compared to other alpha- amylases.
  • the polypeptides of the invention has ⁇ -amylase activity.
  • ⁇ -Amylases ( ⁇ -1 ,4- glucan-4-glucanohydrolases, EC 3.2.1.1 ) constitute a group of enzymes that catalyze hydrolysis of starch and other linear and branched 1 ,4-glucosidic oligo- and polysaccharides.
  • ⁇ -amylase activity may e.g. be determined using the Phadebas assay or the pNPG7 assay described below in the "Materials and Methods" section.
  • the ⁇ -amylase activity of the polypeptides of the invention is determined using the Phadebas assay or the pNPG7 assay described below in the "Materials and Methods" section with the assay conditions pH 7.3 and at 37°C.
  • the specific activity is determined using the
  • polypeptides of the invention have a specific activity as determined using the Phadebas assay described below in the "Materials and Methods" section with the assay conditions pH 7.3 and at 37°C which is higher than the specific activity of the ⁇ -amylase SP722 (SEQ ID NO: 15).
  • the polypeptides of the invention have a specific activity as determined using the Phadebas assay described below in the "Materials and Methods" section with the assay conditions pH 7.3 and at 37°C which is at least 5%, least 10% higher, such as at least 20%, at least 25% at least, 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 90%, at least 100% higher than the specific activity of the ⁇ -amylase SP722.
  • the polypeptides of the invention have a specific activity as determined using the Phadebas assay described below in the "Materials and Methods" section with the assay conditions pH 7.3 and at 37°C which is at least a factor 1 , 3 higher, such as at least a factor 1 ,5 higher than the specific activity of the ⁇ -amylase SP722.
  • polypeptides of the invention having a specific activity determined using the Phadebas assay described below in the "Materials and
  • Methods section with the assay conditions pH 7.3 and at 37°C which is at least 40000 NU/mg, such as at least 45000 NU/mg, or at least 50000 NU/mg, such as about 56000 (NU/mg).
  • ⁇ -amylase AA180 has very high activity under alkaline conditions (measured at pH 8,5) in the temperature range from 37°C to 70°C, cf. Figure 3 and Example 4.
  • ⁇ -amylase AA180 has an excellent washing performance in alkaline detergent solutions, cf. Example 5 disclosed herein.
  • the invention in further aspects relates to polypeptides of the invention having a suitable washing performance in alkaline detergent solutions, e.g. at a pH around 7-11 , and more preferably, at a pH around 8-10.5.
  • the activity at alkaline conditions and medium temperature, particularly relevant for wash and desizing, was found to be superior to other ⁇ -amylases.
  • the polypeptides of the invention have at least 10%, at least 20%, preferably at least 40%, at least 50%, more preferably at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% and most preferably at least 100% of the ⁇ -amylase activity of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:6.
  • the ⁇ -amylase activity is in a preferred embodiment determined using the Phadebas assay described below in the "Mate ⁇ als and Methods" section with the assay conditions pH 7.3 and at 37°C.
  • polypeptides of the invention have a temperature optimum determined using the Phadebas method (pH 8.5) in the range between 50°C to 70°C.
  • the polypeptides of the invention may have a theoretical pi between 6-7 calculated from the amino acid sequence.
  • the present invention relates to polypeptides of the invention, including the mature polypeptides of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO:6, having one or more of the following properties: i. a temperature optimum (see Fig. 3) determined using the Phadebas method
  • pH 8.5 in the range between 50 and 70°C, such as e.g. about 60°C; ii. a theoretical pi calculated to be between 6 and 7, such as about 6.4 to about 6,5; iii. at least 10%, at least 20%, at least 30%, preferably at least 40%, at least 50%, more preferably at least 60%, such as at least 70%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 100% of the ⁇ -amylase activity of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:6. iv.
  • polypeptides of the invention may have one or more of the properties as described herein in connection with the polypeptides of the o inventions.
  • a homology search comparing ⁇ -amylase AA180, AAG20 and Amrk385 provided by the inventors with known sequences showed identities for the mature s protein of AA180, AAG20, Amrk385 with a number of Bacillus amylases in the range 67-90 % on amino acid level determined with Clustal X version 1.8 which can be obtained from: European Molecular Biology Laboratory, EMBL (ftp.embl- heidelberg.de), for multiple alignment with a gap creation penalty of 30, a gap extension penalty of 1 using the "identity" matrice for calculating the degree of o identity.
  • homologous ⁇ -amylases to the sequences of the invention are SP690 (i.e. "NCIB 12512" disclosed in SEQ ID NO: 1 of US patent no. 5,856,164 which has about 89% identity, SP722 (i.e. "NCIB 12513” disclosed in SEQ ID NO: 2 of US patent no. 5,856,164 which has about 87% identity), the mature part (amino 5 acids 31-516) of the ⁇ -amylase obtained from Bacillus sp. KSM-AP1378 disclosed as SEQ ID NO: 2 of WO 97/00324 (which has about 89% identity) and the mature part (amino acid 34-518) of the alpha-amylase obtained from Bacillus sp.
  • SP690 i.e. "NCIB 12512” disclosed in SEQ ID NO: 1 of US patent no. 5,856,164 which has about 89% identity
  • SP722 i.e. "NCIB 12513” disclosed in SEQ ID NO: 2
  • ⁇ -amylases homologous to the nucleotide sequences coding the mature part of the protein of the invention are SP690 (SEQ ID NO:4 of US patent no. 5,856,164 which is about 77% identical, SP722 (SEQ ID NO:5 of US patent no. 5,856,164 which is about 79% identical), the mature part (amino acids 31-516) of the ⁇ -amylase obtained from Bacillus sp. KSM-AP1378 disclosed as SEQ ID NO:2 of WO 97/00324 (which is about 78% identical) and the mature part (amino acid 34- 518) of the alpha-amylase obtained from Bacillus sp. 707 described by Tsukamoto A.
  • the present invention relates to polypeptides having an amino acid sequence which has a degree of identity or similarity to amino acids 1 to 486 of SEQ ID NO:2 (i.e., the mature polypeptide) of at least about 92%, preferably of at least about 93%, such as at least 94%, more preferably at least about 95%, such as at least 96%, even more preferably at least 97%, at least 98%, and most preferably of at least 99% identity or similarity to amino acids 1 to 486 of SEQ ID NO:2, which polypeptides have ⁇ -amylase activity (hereinafter "homologous polypeptides").
  • homologous polypeptides which polypeptides have ⁇ -amylase activity
  • the invention also related to a polypeptide having ⁇ -amylase activity, which polypeptide comprises or consists of an amino acid sequence which has at least 92%, preferably of at least 93%, such as at least 94%, more preferably at least 95%, such as at least 96%, even more preferably at least 97%, at least 98%, and most preferably of at least 99% identity with amino acids 1 to 486 of SEQ ID NO:2, of SEQ ID NO:4 or of SEQ ID NO:6.
  • the homologous polypeptides include polypeptides having an amino acid sequence which differs by at most 38 amino acids, such as at most 35 amino acids, at most 30 amino acids, at most 25 amino acids, at most 20 amino acids, at most 15 amino acids, such as at most 10 amino acids, such as at most 5 amino acids (e.g. 5 amino acids), such as at most four amino acids, e.g. by three amino acids, by two amino acids, or by one amino acid from amino acids 1 to 486 of SEQ ID NO:2, SEQ ID NO:4 or of SEQ ID NO:6.
  • the term "differ” includes both deletion, substitution and addition of amino acid, respectively. It is also understood that such polypeptides may further be part of a fusion polypeptide, i.e. have an amino acid sequence fused to the N-terminal and/or the C-terminal part.
  • the invention also relates to polypeptides having an amino acid sequence which has a degree of identity or similarity - to the mature ⁇ -amylase encoded by the ⁇ -amylase encoding part of the nucleotide sequence cloned into a plasmid fragment present in E.coli pCa168-Amrk385, NN049553, deposited at DSMZ, accession number DSM 13763 - of at least about 92%, preferably of at least about 93%, such as at least 94%, more preferably at least about 95%, such as at least 96%, even more preferably at least 97%, at least 98%, and most preferably of at least 99%, which polypeptides have ⁇ -amylase activity (hereinafter "homologous polypeptides").
  • the amino acid sequence of AA180 amino acid 1-486 of SEQ ID NO:2
  • amino acid AA180 amino acid 1-486 of SEQ ID NO:2
  • AAG20 (amino acid 1-486 of SEQ ID NO:4) are identical.
  • the amino acid sequence of Amrk385 (amino acid 1-486 of SEQ ID NO:6) differs from AA180 and AAG20 in only two amino acid positions, namely position 90 and position 420.
  • SEQ ID NO:5 as disclosed herein is interchanged with SEQ ID NO:18 and SEQ ID NO:6 as disclosed herein is interchanged with SEQ ID NO:19.
  • polypeptides of the invention also encompass the mature ⁇ -amylase encoded by the nucleotide sequence contained in Escherichia coli, pCa168- Amrk385, NN049553 deposited at DSMZ, (the deposit was made by Novo Nordisk A/S and is assigned to Novozymes A/S), with accession number DSM 13763 and accession date 5 October 2000 and variants thereof as disclosed herein also in connection with the variants of the mature sequence of SEQ ID NO:2.
  • polypeptides of the invention may have one or more of the properties as disclosed herein.
  • One embodiment relates to polypeptides having an amino acid sequence which has a degree of identity or similarity to amino acids 1 to 486 of SEQ ID NO:2 (i.e., the mature polypeptides) of at least about 92%, such as at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least 97%, at least 98%, or of at least 99%, which polypeptides have ⁇ -amylase activity, and which polypeptides have at least 10%, such as at least 20%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 85%, and preferably at least 100% of the ⁇ -amylase activity of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:6
  • the ⁇ -amylase activity is in a preferred embodiment determined using the Phadebas assay described below in the "Materials and Methods" section with the assay conditions pH 7.3 and at 37°C.
  • the degree of similarity in connection with comparing amino acid sequences takes into account conservative amino acid substitution.
  • similarity in this context is also some times called term “homology”.
  • the amino acid sequence identity may be determined as the "degree of identity” between the two sequences indicating a derivation of the first sequence from the second.
  • the “degree of similarity” also takes into account e.g. conservative amino acid substitution when calculating the degree of similarity.
  • the identity and the similarity may suitably be determined by means of computer programs known in the art.
  • GAP provided in GCG version 8 (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453) may be used for a pairwise alignment of the sequences and calculation of the degree of identity using the default settings GAP creation penalty of 5.0 and GAP extension penalty of 0.3, default scoring matrix for identity.
  • Clustal X version 1.8 which can be obtained from: European Molecular Biology Laboratory, EMBL (ftp.embl-heidelberg.de), may be used for multiple alignment with a gap creation penalty of 30 and a gap extension penalty of 1 using the "identity" matrice for calculating the degree of identity.
  • the Clustal X program is described in the manuscript: Thompson,J.D., Gibson.T.J., Plewniak.F., Jeanmougin.F. and Higgins, D.G. (1997) "The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.” Nucleic Acids Research, 24:4876-4882.
  • the comparison of different amino acid sequences and DNA sequences may also be done by using the program "ALIGN” to calculate individual amino acid and DNA homologies.
  • the program "ALIGN” makes an optimal global alignment of two sequences and is useful for both protein and DNA alignments, utilizing a full Smith- Waterman alignment.
  • the default scoring matrices BLOSUM50 and the identity matrix are used for protein and DNA alignments, respectively.
  • the penalty for the first residue in a gap is -12 for proteins and -16 for DNA, while the penalty for additional residues in a gap is -2 for proteins and -4 for DNA.
  • ALIGN may be provided from the FASTA package version v20u6 (William R. Pearson, Department of Biochemistry, Box 440, Jordan Hall University of Virginia, Charlottesville, VA, USA). The program is a slightly modified version of one taken from E. Myers and W. Miller. The algorithm is described in E. Myers and W. Miller, Optimal Alignments in Linear Space” (CABIOS (1988) 4:11-17).
  • Multiple alignments of protein sequences may be done using "Clustal W” (Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680). Multiple alignment of DNA sequences may be done using the protein alignment as a template, replacing the amino acids with the corresponding codon from the DNA sequence.
  • the polypeptides of the present invention comprise the mature amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 or an allelic variant thereof; or a fragment thereof that has ⁇ -amylase activity.
  • Amino acid 1-486 of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6 show the mature part the ⁇ - amylases of the invention.
  • a fragment of SEQ ID NO:2, SEQ ID NO: 4 or SEQ ID NO:6 are polypeptides having one or more amino acids deleted from the amino and/or carboxyl terminus of the mature part of these amino acid sequence.
  • allelic variant denotes any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences.
  • An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
  • amino acid sequences of the homologous polypeptides may differ from the amino acid sequence of the mature part of SEQ ID NO:2, SEQ ID NO: 4 or SEQ ID NO:6 by an insertion or deletion of one or more amino acid residues and/or the substitution of one or more amino acid residues by different amino acid residues.
  • the amino acid changes are of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
  • conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine).
  • Amino acid substitutions which do not generally alter the specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York.
  • the most commonly occurring exchanges are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Gln/Glu, Asn/Gln, Leu/He, Leu/Val as well as these in reverse.
  • Polypeptides encoded by nucleotide sequences of the present invention also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the invention, including variants of the mature polypeptide of SEQ ID NO:2; 4 and 6 or fragment thereof.
  • a fused polypeptide is produced by fusing a nucleotide sequence (or a portion thereof) encoding another polypeptide to a nucleotide sequence (or a portion thereof) of the present invention.
  • Techniques for producing fusion polypeptides include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator.
  • the present invention relates to a nucleic acid comprising or consisting of a nucleotide sequence which nucleotide sequence has a degree of identity to nucleotide 359 to 1816 of SEQ ID NO:1 or to nucleotide 359 to 1816 of SEQ ID NO:3 or to nucleotide 566 to 2023 of SEQ ID NO:5 (i.e., nucleotides encoding the mature polypeptides) of at least about 85%, preferably of at least about 90%, more preferably of at least about 95%, even more preferably at least 97%, at least 98%, and most preferably at least 99%, which nucleotide sequence encodes a polypeptide with ⁇ -amylase activity (hereinafter "homologous nucleotide").
  • homologous nucleotide a polypeptide with ⁇ -amylase activity
  • the homologous nucleotide includes nucleotides encoding an amino acid sequence which differs by at most 10 amino acids, such at most 5 amino acids, such as by 5 amino acids, by at most four amino acids, more preferably by at most three amino acids, even more preferably by at most two amino acids, and most preferably by one amino acid from amino acids 1 to 486 of SEQ ID NO:2, SEQ ID NO:4 or of SEQ ID NO:6.
  • the nucleotide sequence identity may be determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second.
  • the identity may suitably be determined by means of computer programs known in the art.
  • GAP provided in GCG version 9 (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453) may be used for a pairwise alignment of the sequences and calculation of the degree of identity using the default settings GAP creation penalty of 5.0 and GAP extension penalty of 0.3, default scoring matrix for identity.
  • Clustal X version 1.8 which can be obtained from EUROPEAN MOLECULAR BIOLOGY LABORATORY, EMBL (ftp.embl-heidelberg.de), may be used for multiple alignment with a gap creation penalty of 15, a gap extension penalty of 6.66 using the IUC scoring matrice for calculating the degree of identity.
  • Different DNA sequences may also be compared by using the program "align" as described herein to calculate individual DNA homologies.
  • the present invention relates to polypeptides having ⁇ -amylase activity which are encoded by nucleotide sequences which hybridise under (a) medium-high stringency conditions, even more preferably (b) high stringency conditions, and most preferably (c) very high stringency conditions with a nucleic acid probe which hybridises under the same conditions with (i) the nucleotide sequence of SEQ ID NO:1 , SEQ ID NO: 3 or SEQ ID NO:5, (ii) the cDNA sequence of SEQ ID NO:1 , SEQ ID NO: 3 or SEQ ID NO:5, (iii) a subsequence of (i) or (ii), or (iv) a complementary strand of (i), (ii), or (iii) (J.
  • the subsequence of SEQ ID NO:1 , SEQ ID NO: 3 or SEQ ID NO:5 may be at least 100 nucleotides or preferably at least 200 nucleotides. Moreover, the subsequence may encode a polypeptide fragment that has ⁇ -amylase activity. The polypeptides may also be allelic variants or fragments of the polypeptides that have ⁇ -amylase activity.
  • medium to very high stringency conditions are defined as pre-hybridisation and hybridisation at 42°C in 5X SSPE, 0.3% SDS, 200 ⁇ g/ml sheared and denatured salmon sperm DNA, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures.
  • the carrier material is finally washed three times each for 15 minutes using 2 x SSC (Standard Saline Citrate), 0.2% SDS preferably at least at 55°C (medium stringency), preferably at least at 60°C (medium-high stringency), more preferably at least at 65°C (high stringency), and most preferably at least at 70°C (very high stringency).
  • 2 x SSC Standard Saline Citrate
  • 0.2% SDS preferably at least at 55°C (medium stringency), preferably at least at 60°C (medium-high stringency), more preferably at least at 65°C (high stringency), and most preferably at least at 70°C (very high stringency).
  • the conditions for determining hybridisation between a nucleotide probe and a homologous DNA or RNA sequence may involve pre-soaking of the filter containing the DNA fragments or RNA to hybridise in 5 x SSC (standard saline citrate) for 10 min, and pre-hybridisation of the filter in a solution of 5 x SSC (Sambrook et al. 1989), 5 x Denhardt's solution (Sambrook et al. 1989), 0.5 % SDS and 100 ⁇ g/ml of denatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed by hybridisation in the same solution containing a random-primed (Feinberg, A. P. and Vogelstein, B.
  • 5 x SSC standard saline citrate
  • nucleotide sequence of SEQ ID NO:1 , SEQ ID NO: 3 or SEQ ID NO:5 or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO:2, SEQ ID NO: 4 or SEQ ID NO:6 or a fragment thereof, may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having ⁇ -amylase activity from strains of different genera or species according to methods well known in the art.
  • probes can be used for hybridisation with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein.
  • probes can be considerably shorter than the entire sequence, but should be at least 15, preferably at least 25, and more preferably at least 35 nucleotides in length. Longer probes can also be used. Both DNA and RNA probes can be used.
  • the probes are typically labelled for detecting the corresponding gene (for example, with 32 P, 3 H, 35 S, biotin, or avidin). Such probes are encompassed by the present invention.
  • genomic DNA or cDNA library prepared from such other organisms may be screened for DNA that hybridises with the probes described above and which encodes a polypeptide having ⁇ -amylase activity.
  • Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques.
  • DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material.
  • the carrier material is used in a Southern blot.
  • hybridisation indicates that the nucleotide sequence hybridises to a nucleic acid probe corresponding to the nucleotide sequence shown in SEQ ID NO:1 , SEQ ID NO: 3 or SEQ ID NO:5, its complementary strand, or a subsequence thereof, under medium to very high stringency conditions as described herein. Molecules to which the nucleic acid probe hybridises under these conditions are detected using X-ray film.
  • a polypeptide of the present invention may be obtained from any organism, such as in particular a microorganism of any genus.
  • the term "obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by the nucleotide sequence is produced naturally by the source or by a cell in which the nucleotide sequence from the source has been inserted.
  • a polypeptide of the invention may also be obtained from transgenic plant expressing said polypeptide.
  • a polypeptide of the present invention may be a bacterial polypeptide.
  • the polypeptide may be a gram positive bacterial polypeptide such as a Bacillus polypeptide, e.g., a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide; or a Streptomyces polypeptide, e.g., a Streptomyces lividans or Streptomyces murinus polypeptide; or a gram negative bacterial polypeptide, e.g., an E. coli or a Pseudomonas sp. polypeptide.
  • Bacillus polypeptide e.g.,
  • the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.
  • polypeptides may be identified and obtained from other sources including microorganisms from nature (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms from natural habitats are well known in the art.
  • the nucleotide sequence may then be derived by similarly screening a genomic or cDNA library of another microorganism. Once a nucleotide sequence encoding a polypeptide has been detected with the probe(s), the sequence may be cloned by utilizing techniques which are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).
  • polypeptide of the invention may be an isolated polypeptide.
  • an "isolated polypeptide” in this context refers to a polypeptide which is substantially free of other non- ⁇ -amylase polypeptides, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably at least about 60% pure, even more preferably at least about 80% pure, most preferably at least about 90% pure, at least about 95% pure, at least about 97% pure, such as about 100% pure, as determined by SDS-PAGE, this is also termed “substantially pure”.
  • the polypeptide of the invention may be "essentially pure.
  • the term "essentially pure” in this context refers to a polypeptide of the invention which is essentially free of other polypeptides which are present in the native organism, i.e. the species in which it is present naturally, i.e. the organism of origin, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably at least about 60% pure, even more preferably about at least 80% pure, most preferably about at least 90% pure, and even most preferably about at least 95% pure, such as 100% pure with respect to other polypeptides present in the native organism.
  • the polypeptides of the invention may also be expressed in transgenic plants comprising a gene coding for a polypeptide of the invention.
  • a transgenic plant capable of expression a polypeptide of the invention and the use of such plant for the manufacturing of a polypeptide of the invention.
  • the invention also relates to a transgenic plant, plant part or plant cell which has been transformed with a DNA sequence encoding the polypeptides of the invention so as to express and produce this enzyme in recoverable quantities.
  • the enzyme may be recovered from the plant or plant part.
  • the plant or plant part containing the recombinant enzyme may be used as such, e.g. for food/feed of the transgenic plant material.
  • the transgenic plant may be dicotyledonous or monocotyledonous, for short a dicot or a monocot.
  • monocot plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as festuca, lolium, temperate grass, such as Agrostis, and cereals, e.g. wheat, oats, rye, barley, rice, sorghum and maize (corn).
  • dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous (family Brassicaceae), such as cauliflower, oil seed rape and the closely related model organism Arabidopsis thaliana.
  • plant parts are stem, callus, leaves, root, fruits, seeds, and tubers.
  • plant tissues such as chloroplast, apoplast, mitochondria, vacuole, peroxisomes and cytoplasm are considered to be a plant part.
  • any plant cell, or any tissue origin, is considered to be a plant part.
  • the invention also relates to a transgenic plant, plant part or plant cell capable of producing the polypeptide of the invention, said transgenic plant, plant part or plant cell comprising a nucleotide sequence encoding said polypeptide.
  • a transgenic plant, plant part or plant cell capable of producing the polypeptide of the invention, said transgenic plant, plant part or plant cell comprising a nucleotide sequence encoding said polypeptide.
  • progeny, seeds, parts, fruits, flowers of a plant of the invention claim, said progeny, seeds, parts, fruits, flowers comprising a nucleotide sequence of the invention encoding the polypeptide of the invention.
  • Contemplated is also a method for the production of a plant comprising regenerating the plant cell of the invention to a plant.
  • FIG. 1 For purposes of a) transforming a plant cell with the nucleotide sequence of the invention.
  • FIG. 1 For purposes of the production of a plant comprising the steps of: (a) transforming a plant cell with the nucleotide sequence of the invention; and (b) regenerating the plant cell of step a) to a plant; optionally further comprising the step c) of crossing the regenerated plant with a second plant having a desired genotype to form a hybrid, and optionally d) backcrossing said hybrid and its progeny with the plant having the desired genotype to obtain
  • the transgenic plant or plant cell expressing the enzyme of the invention may be constructed in accordance with methods known in the art.
  • the plant or plant cell may be constructed by incorporating one or more expression constructs encoding the enzyme of the invention into the plant host genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.
  • An example of expression of amylase in plant is: Czihal, A et al. (1999): “Gene farming in Plants: Expression of heatstable Bacillus amylase in transgenic legume seeds.” Journal of Plant Physiology, Vol. 155 (2) pp. 183-189.
  • 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,A,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.
  • the invention also relates to a mutant of the ⁇ -amylases shown in SEQ ID NO: 2, SEQ ID NO:4 or SEQ ID NO: 6, including of the mature sequences thereof.
  • the mutant ⁇ -amylase of the invention is characterized by the fact that one or more of the methionine amino acid residues is exchanged with any amino acid residue except for Cys and Met.
  • the amino acid residues to replace the methionine amino acid residue are the following: Ala, Arg, Asn, Asp, Gin, Glu, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.
  • a preferred embodiment of the mutant ⁇ -amylase of the invention is characterized by the fact that one or more of the methionine amino acid residues is (are) exchanged with a Leu, Thr, Ala, Gly, Ser, lie, or Asp amino acid residue, preferably a Leu, Thr, Ala, or Gly amino acid residue.
  • a very satisfactory activity level and stability in the presence of oxidizing agents may be obtained.
  • one or more of the methionines in the following position may be replaced or deleted using any suitable technique known in the art, including especially site directed mutagenesis and gene shuffling.
  • positions in SEQ ID NO: 2 are: 9, 10, 105, 202, 208, 262, 310, 383, 431 , 441 and positions in SEQ ID NO: 4 are: 9, 10, 105, 202, 208, 262, 310, 383, 431 , 441 and positions in SEQ ID NO: 6 are: 9, 10, 105, 202, 208, 262, 310, 383, 431 , 441.
  • mutant ⁇ -amylase of the invention is characterized by the fact that the methionine amino acid residue at position 202 is exchanged with any of amino acid residue except for Cys and Met , preferably with a
  • contemplated preferred mutations include deletion of one, two or more residues of amino acids R181 , G182, T183, G184, K185, A186, N260, N261 or substitution of one or more of these residues.
  • a preferred mutation is the deletions of T183-G184, N260 and/or N261.
  • Particularly relevant mutations are substitutions of A186 with Gly, Arg, Asn, Asp, Cys, Gin, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.
  • a particularly preferred substitution is A186R.
  • N195F substitution of N195 with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.
  • a particularly interesting substitution is N195F.
  • deletion of T183-A184+N195F deletion of T183-G184+A186R, deletion of T183-A184+A186R+N195F, A186R+N195F
  • deletion of T183-A184+ deletion of N260+N195F deletion of T183-G184+ deletion of N260+A186R, deletion of T183-A184+ deletion of N260+A186R+N195F
  • deletion of T183-A184+ deletion of N261 +N195F deletion of T183-G184+ deletion of N261 +A186R, deletion of T183- A184+ deletion of N261+A186R+N195F and deletion of N260+A186R+N195F and deletion of N261 +A186R+N195F
  • deletion of T183-A184+N195F+M202L deletion of T183-G184+A186R+M202L, deletion of T183-A186R+N195F
  • Nucleotide sequences The present invention also relates to nucleotide sequences which encode a polypeptide of the present invention.
  • the nucleotide sequence is as set forth in SEQ ID NO:1 , SEQ ID NO:3 or SEQ ID NO:5.
  • the nucleotide sequence is the mature polypeptide coding region of SEQ ID NO: 1 , SEQ ID NO:3 or SEQ ID NO:5.
  • the present invention also encompasses nucleotide sequences which encode a polypeptide having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 which differ from SEQ ID NO: 1 , SEQ ID NO:3 or SEQ ID NO:5 by virtue of the degeneracy of the genetic code.
  • the present invention also relates to subsequences of SEQ ID NO:1 , SEQ ID NO:3 or SEQ ID NO:5 which encode fragments of SEQ ID NO: 2, SEQ ID NO:4 or SEQ ID NO:6, respectively, that have ⁇ -amylase activity.
  • Subsequences of SEQ ID NO: 1 , SEQ ID NO:3 or SEQ ID NO:5 are nucleotide sequences encompassed by SEQ ID NO:1 , SEQ ID NO:3 or SEQ ID NO:5 except that one or more nucleotides from the 5' and/or 3' end have been deleted.
  • nucleotide sequences of the invention also encompasses the nucleotide sequence coding for Amrk385 contained in Escherichia coli, pCa168-Amrk385, NN049553 deposited at DSMZ, with accession number: DSM 13763 and accession date 5 October 2000 and variant thereof as described herein.
  • the present invention also relates to mutant nucleotide sequences comprising at least one mutation in the mature polypeptide coding sequence of SEQ ID NO:1 , SEQ ID NO:3 or SEQ ID NO:5, in which the mutant nucleotide sequence encodes a polypeptide which consists of amino acids 1 to 486 of SEQ ID NO: 2, SEQ ID NO:4 or SEQ ID NO:6.
  • the techniques used to isolate or clone a nucleotide sequence encoding a polypeptide include isolation from genomic DNA, preparation from cDNA, or a combination thereof.
  • the cloning of the nucleotide sequences of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York.
  • nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleotide sequence-based amplification (NASBA) may be used.
  • LCR ligase chain reaction
  • LAT ligated activated transcription
  • NASBA nucleotide sequence-based amplification
  • the nucleotide sequence may be cloned from a strain of Bacillus, or another or related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the nucleotide sequence.
  • the nucleotide sequence of the invention may be an isolated nucleotide sequence.
  • isolated nucleotide sequence in this context refers to a nucleotide sequence of the invention which is essentially free of other nucleotide sequences, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably at least about 60% pure, even more preferably at least about 80% pure, and most preferably at least about 90% pure as determined by agarose electrophoresis.
  • the nucleotide sequence may be essentially pure.
  • essentially pure in this context refers to a nucleotide sequence of the invention which is essentially free of other components which are present in the native organism, i.e. the organism of origin.
  • an isolated nucleotide sequence can be obtained by standard cloning procedures used in genetic engineering to relocate the nucleotide sequence from its natural location to a different site where it will be reproduced.
  • the cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleotide sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleotide sequence wiii be replicated.
  • the nucleotide sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
  • the present invention also relates to nucleotide sequences which have a degree of identity to the mature polypeptide coding sequence of SEQ ID NO:1 (i.e., nucleotides 359 to 1816) or SEQ ID NO: 3 (i.e., nucleotide 359 to 1816) or SEQ ID NO: 5 (i.e., nucleotide 559 to 2016) of at least about 82% identity on DNA level, preferably at least about 85%, more preferably at least about 90%, more preferably at least about 93%, more preferably at least about 95%, even more preferably at least about 97%, such as at least 98%, or at least about 99% identity, which encodes an active polypeptide.
  • SEQ ID NO:1 i.e., nucleotides 359 to 1816
  • SEQ ID NO: 3 i.e., nucleotide 359 to 1816
  • SEQ ID NO: 5 i.e., nucleotide 559 to 2016
  • the DNA sequence identity (sometimes also termed homology) may be determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second.
  • the identity 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 provides in the GCG program package (described above).
  • GAP uses the method of Needleman/Wunsch/Sellers to make alignments.
  • Clustal X version 1.8 which can be obtained from: EUROPEAN
  • MOLECULAR BIOLOGY LABORATORY may be used for multiple alignment with a gap creation penalty of 15, a gap extension penalty of 6.66 using the IUC scoring matrice for calculating the degree of identity.
  • Clustal X described by Thompson,J.D., Gibson.T.J., Plewniak,F., Jeanmougin.F. and Higgins, D.G. (1997) "The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.”
  • Nucleic Acids Research, 24:4876-4882. is a Windows compatible version of Clustal W described in Thompson, J.D., Higgins, D.G. and Gibson, TJ. (1994) CLUSTAL W: "Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice.”
  • Modification of a nucleotide sequence encoding a polypeptide of the present invention may be performed for synthesis of polypeptides substantially similar to the polypeptide.
  • the term "substantially similar" to the polypeptide refers to non- naturally occurring forms of the polypeptide.
  • These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g., variants that differ in specific activity, stability, pH optimum, temperature optimum or the like.
  • the variant sequence may be constructed on the basis of the nucleotide sequence presented as the polypeptide encoding part of SEQ ID NO:1 , SEQ ID NO:3 or SEQ ID NO:5, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the polypeptide encoded by the nucleotide sequence, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence.
  • nucleotide substitution see, e.g., Ford et al., 1991 , Protein Expression and Purification 2: 95-107.
  • amino acid residues essential to the activity of the polypeptide encoded by the nucleotide sequence of the invention may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for ⁇ -amylase activity to identify amino acid residues that are critical to the activity of the molecule.
  • Sites of substrate-enzyme interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labelling (see, e.g., de Vos et al., 1992, Science 255: 306-312; Smith et ai, 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).
  • the present invention also relates to nucleotide sequences encoding a polypeptide of the present invention, which hybridise under medium stringency conditions, preferably medium-high stringency conditions, more preferably high stringency conditions, and most preferably very high stringency conditions with a nucleic acid probe which hybridises under the same conditions with the nucleotide sequence of SEQ ID NO:1 , SEQ ID NO:3 or SEQ ID NO:5 or its complementary strand; or allelic variants and subsequences thereof (Sambrook et al., 1989, supra), as defined herein.
  • the present invention also relates to nucleotide sequences produced by (a) hybridising a DNA under medium, medium-high, high, or very high stringency conditions with the sequence of SEQ ID NO:1 , SEQ ID NO:3 or SEQ ID NO:5, or their complementary strands, or a subsequence thereof; and (b) isolating the nucleotide sequence.
  • the subsequence is preferably a sequence of at least 100 nucleotides such as a sequence which encodes a polypeptide fragment which has ⁇ - amylase activity.
  • the present invention further relates to methods for producing a mutant nucleotide sequence, comprising introducing at least one mutation into the mature polypeptide coding sequence of SEQ ID NO: 1 , SEQ ID NO:3 or SEQ ID NO:5 or a subsequence thereof, wherein the mutant nucleotide sequence encodes a polypeptide which consists of 1 to 486 of SEQ ID NO: 2, SEQ ID NO:4 or SEQ ID NO:6 or a fragment thereof which has ⁇ -amylase activity.
  • the introduction of a mutation into the nucleotide sequence to exchange one nucleotide for another nucleotide may be accomplished by site- directed mutagenesis using any of the methods known in the art. Particularly useful is the procedure which utilizes a supercoiled, double stranded DNA vector with an insert of interest and two synthetic primers containing the desired mutation.
  • the oligonucleotide primers, each complementary to opposite strands of the vector, extend during temperature cycling by means of Pfu DNA polymerase. On incorporation of the primers, a mutated plasmid containing staggered nicks is generated.
  • Dpn ⁇ is specific for methylated and hemimethylated DNA to digest the parental DNA template and to select for mutation-containing synthesized DNA.
  • Other procedures known in the art may also be used. These other procedures include gene shuffling, e.g., as described in WO 95/22625 (from Affymax Technologies N.V.) and WO 96/00343 (from Novo Nordisk A/S)._
  • the present invention also relates to nucleic acid constructs comprising a nucleotide sequence of the present invention operably linked to one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
  • Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • Nucleic acid construct is defined herein as a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature.
  • nucleic acid construct is synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence of the present invention.
  • coding sequence is defined herein as a portion of a nucleotide sequence which directly specifies the amino acid sequence of its protein product.
  • the boundaries of the coding sequence are generally determined by a ribosome binding site (prokaryotes) or by the ATG start codon (eukaryotes) located just upstream of the open reading frame at the 5' end of the mRNA and a transcription terminator sequence located just downstream of the open reading frame at the 3' end of the mRNA.
  • a coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleotide sequences.
  • a nucleotide sequence encoding a polypeptide of the present invention may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the nucleotide sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleotide sequences utilizing recombinant DNA methods are well known in the art.
  • control sequences is defined herein to include all components which are necessary or advantageous for the expression of a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide.
  • control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator.
  • the control sequences include a promoter, and transcriptional and translational stop signals.
  • the control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide.
  • linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide.
  • operably linked is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the expression of a polypeptide.
  • the control sequence may be an appropriate promoter sequence, a nucleotide sequence which is recognized by a host cell for expression of the nucleotide sequence.
  • the promoter sequence contains transcriptional control sequences which mediate the expression of the polypeptide.
  • the promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • Suitable promoters for directing the transcription of the nucleic acid constructs of the present invention are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha- amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75: 3727-3731 ), as well as the tac promoter (DeBoer et al., 1983), as well as the tac promoter (De
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription.
  • the terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.
  • the control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway.
  • the 5' end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide.
  • the 5' end of the coding sequence may contain a signal peptide coding region which is foreign to the coding sequence.
  • the foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region.
  • the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide.
  • any signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention.
  • Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
  • regulatory system It may also be desirable to add regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host cell.
  • regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems.
  • yeast the ADH2 system or GAL1 system may be used.
  • filamentous fungi the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences.
  • Other examples of regulatory sequences are those which allow for gene amplification.
  • these include the dihydrofolate reductase gene which is amplified in the presence of methotrexate, and the metaliothionein genes which are amplified with heavy metals.
  • the nucleotide sequence encoding the polypeptide would be operably linked with the regulatory sequence.
  • the present invention also relates to recombinant expression vectors comprising a nucleotide sequence of the present invention, a promoter, and transcriptional and translational stop signals.
  • the various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites.
  • the nucleotide sequence of the present invention may be expressed by inserting the nucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression.
  • the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
  • the recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleotide sequence.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vectors may be linear or closed circular plasmids.
  • 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, an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
  • the vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells.
  • a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
  • Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance.
  • Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3.
  • a selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (omithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), as well as equivalents thereof.
  • amdS acetamidase
  • argB omithine carbamoyltransferase
  • bar phosphinothricin acetyltransferase
  • hygB hygromycin phosphotransferase
  • niaD nitrate reduct
  • the vectors of the present invention preferably contain an element(s) that permits stable integration of the vector into the host cell genome or autonomous replication of the vector in the cell independent of the genome of the cell.
  • the vector may rely on the nucleotide sequence encoding the polypeptide or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomologous recombination.
  • the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell. The additional nucleotide sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s).
  • the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1 ,500 base pairs, preferably 400 to 1 ,500 base pairs, and most preferably 800 to 1 ,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination.
  • the integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell.
  • the integrational elements may be non-encoding or encoding nucleotide sequences.
  • the vector may be integrated into the genome of the host cell by non-homologous recombination.
  • the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host celi in question.
  • bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAM ⁇ l permitting replication in Bacillus.
  • origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1 , ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.
  • the origin of replication may be one having a mutation which makes its functioning temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75: 1433).
  • More than one copy of a nucleotide sequence of the present invention may be inserted into the host cell to increase production of the gene product.
  • An increase in the copy number of the nucleotide sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleotide sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleotide sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
  • the present invention also relates to recombinant host cells, comprising a nucleotide sequence of the invention, which are advantageously used in the recombinant production of the polypeptides.
  • a vector comprising a nucleotide sequence of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier.
  • the term "host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
  • the host cell may be a unicellular microorganism, e.g., a prokaryote, or a non- unicellular microorganism, e.g., a eukaryote.
  • Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus, or gram negative bacteria such as E.
  • a Bacillus cell e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus
  • the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell.
  • the Bacillus cell is an alkalophilic Bacillus. Also contempleted host cells are Pseudomonas species.
  • the introduction of a vector into a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), using competent ceils (see, e.g., Young and
  • the present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a strain, which in its wild-type form is capable of producing the polypeptide, to produce a supernatant comprising the polypeptide; and (b) recovering the polypeptide.
  • the strain is of the genus
  • the present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a host cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
  • the present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a host cell under conditions conducive for production of the polypeptide, wherein the host cell comprises a mutant nucleotide sequence having at least one mutation in the mature polypeptide coding region of SEQ ID NO:1 , SEQ ID NO:3 or SEQ ID NO:5, wherein the mutant nucleotide sequence encodes a polypeptide which consists of amino acids 1 to 486 of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:5, and (b) recovering the polypeptide.
  • the present invention relates to compositions comprising a polypeptide of the present invention.
  • the compositions are enriched in a polypeptide of the present invention.
  • the term "enriched" indicates that the ⁇ -amylase activity of the composition has been increased, e.g., with an enrichment factor of 1.1.
  • the composition may comprise a polypeptide of the invention as the major enzymatic component, e.g., a mono-component composition.
  • the composition may comprise multiple enzymatic activities, such as an 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,
  • the additional enzyme(s) may be producible by means of a microorganism belonging to the genus Aspergillus, preferably Aspergillus aculeatus, Aspergillus awamori, Aspergillus niger, or Aspergillus oryzae, or Trichoderma, Humicola, preferably Humicola insolens, or Fusarium, preferably Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, or Fu
  • polypeptide compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition.
  • the polypeptide composition may be in the form of a granulate or a microgranulate.
  • the polypeptide to be included in the composition may be stabilized in accordance with methods known in the art.
  • polypeptide compositions of the invention examples are given below of preferred uses of the polypeptide compositions of the invention.
  • the dosage of the polypeptide composition of the invention and other conditions under which the composition is used may be determined on the basis of methods known in the art.
  • the ⁇ -amylases of the invention are well suited for use in a variety of industrial processes, in particular the enzyme finds potential applications as a component in detergents, e.g., laundry and hard surface cleaning detergent compositions, but it may also be useful in the production of sweeteners and ethanol from starch.
  • the present invention is also directed to methods for using the polypeptides having ⁇ -amylase activity in detergent compositions, in particular laundry detergent compositions, dishwash detergent compositions and for hard surface cleaning.
  • polypeptides of the invention may be used in conventional starch- converting processes, such as liquefaction and saccharification processes de-scribed in US Patent No. 3,912,590 and EP patent publications Nos. 252,730 and 63,909.
  • the alkaline ⁇ -amylases of the invention may also be used in the production of lignocellulosic materials, such as pulp, paper and cardboard, from starch reinforced waste paper and cardboard, especially where repulping occurs at pH above 7 and where amylases can facilitate the disintegration of the waste material through degradation of the reinforcing starch.
  • the ⁇ -amylases of the invention is especially useful in a process for producing a papermaking pulp from starch-coated printed paper.
  • the process may be performed as described in WO 95/14807, comprising the following steps: a) disintegrating the paper to produce a pulp, b) treating with a starch-degrading enzyme before, during or after step a), and c) separating ink particles from the pulp after steps a) and b).
  • the ⁇ -amylases of the invention may also be very useful in modifying starch where enzymatically modified starch is used in papermaking together with alkaline fillers such as calcium carbonate, kaolin and clays. With the alkaline ⁇ -amylases of the invention it becomes possible to modify the starch in the presence of the filler thus allowing for a simpler integrated process.
  • the ⁇ -amylases of the invention may also be very useful in textile desizing.
  • ⁇ -amylases are traditionally used as auxiliaries in the desizing process to facilitate the removal of starch-containing size which has served as a protective coating on weft yarns during weaving. Complete removal of the size coating after weaving is important to ensure optimum results in the subsequent processes, in which the fabric is scoured, bleached and dyed. Enzymatic starch breakdown is preferred because it does not involve any harmful effect on the fiber material.
  • the desizing processing is sometimes combined with the scouring and bleaching steps.
  • non-enzymatic auxiliaries such as alkali or oxidation agents are typically used to break down the starch, because traditional ⁇ -amylases are not very compatible with high pH levels and bleaching agents.
  • the non-enzymatic breakdown of the starch size does lead to some fiber damage because of the rather aggressive chemicals used. Accordingly, it would be desirable to use the ⁇ -amylases of the invention as they have an improved performance in alkaline solutions.
  • the ⁇ - amylases may be used alone or in combination with a cellulase, a hemi-cellulase and / or a pectinase, when desizing cellulose-containing fabric or textile.
  • the ⁇ -amylases of the invention may also be very useful in a beer-making process; the ⁇ -amylases will typically be added during the mashing process.
  • the enzyme of the invention may be added to and thus become a component of a detergent composition.
  • the detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.
  • the invention provides a detergent additive comprising the enzyme of the invention.
  • the detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, a cyclydextrin glycosytransferase, a maltogenic alpha-amylase, a xyloglucanase, a pullulanase, an amylopullulanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase.
  • enzymes such as a protease, a lipase, a cutinase, an amylase, a carbo
  • proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included.
  • the protease may be a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease.
  • alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).
  • trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.
  • Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with 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.
  • Preferred commercially available protease enzymes include AlcalaseTM, SavinaseTM, PrimaseTM, DuralaseTM, EsperaseTM, and KannaseTM (Novo Nordisk A/S), MaxataseTM, MaxacalTM, MaxapemTM, ProperaseTM, PurafectTM, Purafect OxPTM, FN2TM, and FN3TM (Genencor International Inc.).
  • Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB
  • lipase variants such as those described in WO 2011/001100600A1
  • WO 2011/001100A1 lipase variants
  • LipolaseTM Preferred commercially available lipase enzymes include LipolaseTM and
  • Amylases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, ⁇ -amylases obtained from Bacillus, e.g. a special strain of ⁇ . licheniformis, described in more detail in GB 1 ,296,839.
  • amylases are the variants described in WO 94/02597,
  • amylases are DuramylTM, TermamylTM, FungamylTM and BANTM (Novo Nordisk A/S), RapidaseTM and PurastarTM (from Genencor Interna- tional Inc.).
  • Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia,
  • Acremonium e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US
  • cellulases are the alkaline or neutral cellulases having colour care benefits.
  • Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940.
  • Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471 , WO 98/12307 and PCT/DK98/00299.
  • Commercially available cellulases include CelluzymeTM, and CarezymeTM
  • 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. from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.
  • the detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes.
  • a detergent additive of the invention i.e. a separate additive or a combined additive, can be formulated e.g. as a granulate, a liquid, a slurry, a tablet etc.
  • Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.
  • Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661 ,452 and may optionally be coated by methods known in the art.
  • waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 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 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
  • Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods.
  • Protected enzymes may be prepared according to the method disclosed in EP 238,216.
  • the detergent composition of the invention may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid.
  • a liquid detergent may be aqueous, typically containing up to 70 % water and 0-30 % organic solvent, or non- aqueous.
  • the detergent composition comprises one or more surfactants, which may be non-ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic.
  • the surfactants are typically present at a level of from 0.1 % to 60% by weight.
  • the detergent When included therein the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha- olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
  • an anionic surfactant such as linear alkylbenzenesulfonate, alpha- olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
  • the detergent When included therein the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides").
  • a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides”).
  • glucamides N-acyl N-alkyl derivatives of glucosamine
  • the detergent may contain 0-65 % of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
  • a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
  • the detergent may comprise one or more polymers.
  • examples are carboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid co- polymers.
  • the detergent may contain a bleaching system which may comprise a H 2 0 2 source such as perborate or percarbonate which may be combined with a peracid- forming bleach activator such as tetraacetylethylenediamine or nonanoyloxyben- zenesulfonate.
  • a bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone type.
  • the enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., 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 borate ester, or a phenyl boronic acid derivative such as 4- formylphenyl boronic acid, and the composition may be formulated as described in, e.g., WO 92/19709 and WO 92/19708.
  • 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 borate ester, or a phenyl boronic acid derivative such as 4- formylphenyl boronic acid
  • the detergent may also contain other conventional detergent ingredients such as, e.g., fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
  • fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
  • any enzyme 0 in particular the enzyme of the invention, may be added in an amount corresponding to 0.01-100 mg of enzyme protein per liter of wash liqour, preferably 0.05-5 mg of enzyme protein per liter of wash liqour, in particular 0.1-1 mg of enzyme protein per liter of wash liqour.
  • the enzyme of the invention may additionally be incorporated in the 5 detergent formulations disclosed in WO 97/07202 which is hereby incorporated as reference.
  • the enzyme of the invention may also be used in dish wash detergent o compositions, including the following:
  • NTA Nitrilotrisodium acetate
  • Liquid nonionic surfactant e.g. alcohol ethoxylates 2.0 - 10.0%
  • Liquid carrier selected from higher glycols, polyglycols, polyoxides, glycolethers 25.0 - 45.0%
  • Stabilizer e.g. a partial ester of phosphoric acid and a C16-C18 alkanol
  • Liquid nonionic surfactant e.g. alcohol ethoxylates 2.0 - 10.0%
  • Stabilizing system e.g. mixtures of finely divided silicone and low molecular weight dialkyl polyglycol ethers
  • Clay gel thickener e.g. bentonite 0.0 - 10.0%
  • Liquid carrier selected from higher lycols, polyglycols, polyoxides and glycol ethers Balance
  • Oleic acid 0 - 10%
  • the manganese catalyst may, e.g., be one of the compounds described in "Efficient manganese catalysts for low-temperature bleaching", Nature 369, 1994, pp. 637-639.
  • Chemicals used as buffers and substrates were commercial products of at least reagent grade.
  • SP690 is the NCIB 12512 ⁇ -amylase disclosed in SEQ ID NO: 1 of US patent no.
  • SP722 is the NCIB 12513 ⁇ -amylase disclosed in SEQ ID NO: 2 of US patent no.
  • JE1 ⁇ -amylase variant of SP722 having a two amino acid deletion, delta(D183,G184). JE1 is thus also termed "NCIB 12513 + deletion of (D183.G184)".
  • Termamyl ⁇ -amylase from Bacillus licheniformis disclosed in SEQ ID NO:2 of US patent no. 5,830,837 (SEQ ID NO:11 of the present specification).
  • Duramyl ® ⁇ -amylase variant from Bacillus licheniformis stable towards oxidation.
  • BAN ® ⁇ -amylase from Bacillus amyloliquefacience disclosed in SEQ ID NO:12.
  • AA180 ⁇ -amylase of the invention shown in SEQ ID NO: 2.
  • AAG20 ⁇ -amylase of the invention shown in SEQ ID NO: 4.
  • Amrk385 ⁇ -amylase of the invention shown in SEQ ID NO: 6.
  • E. coli strain SJ2 (Diderichsen et al., 1990; Cloning of aldB, which encodes alpha- acetolactate decarboxylase, an exoenzyme from Bacillus brevis; J. Bacteriol., 1990, vol. 172, pp. 4315-4321 ). Plasmids:
  • the gene bank vector was pSJ1678 which is further disclosed in WO 94/19454 which is hereby incorporated by reference.
  • Phadebas assay for ⁇ -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-coloured starch polymer which has been mixed with bovine serum albumin and a buffer substance and tabletted.
  • the measured 620 nm absorbance after 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. Under a specified set of conditions (temp., pH, reaction time, buffer conditions) - 1 mg of a given ⁇ -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 ⁇ -amylase protein) of the ⁇ -amylase in question under the given set of conditions.
  • the specific conditions for determine the specific activity was 37°C, pH 7.3, 50 mM Britton-Robinson buffer, 15 minutes of incubation. From the absorption measured the specific activity is calculated using the Termamyl standard curve.
  • a standard curve was made by using a Termamyl sample (commercially available from Novo Nordisk A/S) with a known activity. The Termamyl standard is diluted to 0.3 - 2 NU/ml and the absorbance is measured using the Phadebas assay. A standard curve can then be made.
  • the Phadebas Assay is useful for preparation of temperature profiles for which the temperature is varied (e.g. in the range 20°C to 70°C) while the pH and other conditions are fixed (e.g. pH 8.5, 50 mM Britton-Robinson buffer, 15 min incubation).
  • the Phadebas assay is also very useful for the preparation of pH profiles for which the temperature is varied (e.g. in the range pH 4 to pH 11 ) while the temperature and other conditions are fixed (e.g. 37°C, 50 mM Britton-Robinson buffer adjusted to the actual pH with NaOH, 15 min incubation).
  • Alternative assay for ⁇ -Amylase Activity is determined by a method employing the PNP-G7 substrate.
  • PNP-G7 which is a abbreviation for para-nitrophenyl- ⁇ ,D-maltoheptaoside is a blocked oligosaccharide which can be cleaved by an endo-amylase.
  • Kits containing PNP-G7 substrate and ⁇ - Glucosidase is manufactured by Boehringer-Mannheim (cat.No. 1054635).
  • BM 1442309 To prepare the substrate one bottle of substrate (BM 1442309) is added to 5 ml buffer (BM 1442309).
  • ⁇ -Glucosidase To prepare the ⁇ -Glucosidase one bottle of ⁇ -Glucosidase (BM 1462309) is added to 45 ml buffer (BM 1442309).
  • the working solution is made by mixing 5 ml ⁇ -Glucosidase solution with 0.5 ml substrate.
  • the assay is performed by transforming 20 ⁇ l enzyme solution to a 96 well microtitre plate and incubating at 25°C 200 ⁇ l working solution, 25°C is added. The solution is mixed and pre-incubated 1 minute and absorption is measured every 15 sec. over 3 minutes at OD 405 nm.
  • the specific activity (activity per mg enzyme) of the ⁇ -amylase under the given conditions can be calculated using the Termamyl standard curve.
  • a standard curve was made by using a Termamyl ® sample (commercially available from Novo Nordisk A/S) with a known activity.
  • the Termamyl standard is diluted to 10 - 100 NU/ml and the absorbance is measured using the PNP-G7 assay. A standard curve can then be made.
  • EXAMPLE 1 Isolation of genomic DNA of the Amrk385 strain The strain Bacillus sp. (obtained from a soil sample of California, USA)
  • Amrk385 (the Amrk385 ⁇ -amylase) was propagated in liquid TY medium (as described in Ausubel et al.(1995)) pH adjusted by addition of Na 2 HCO 3 to pH 9. After 20 hours incubation at 37°C, the cells were harvested, and genomic DNA isolated by using the Genom purification kit from Machery-Nagel of purchased from AH Diagnostic, Aarhus, Denmark. The kit was used according to the manufactures manual.
  • the isolated genomic DNA was used as template for PCR amplification using degenerate primers directed towards the conserved regions in known Bacillus ⁇ - amylases.
  • the degenerate primers were directed towards the following regions/amino acid sequences:
  • Amy-1 N H H N G T N G T (SEQ ID NO. 7)
  • Amy-1C G G (A/S) V S (V/l) W V (K/N) (Q/K/R) (SEQ ID NO. 8)
  • SEQ ID NO:5 which includes the N- and the C-terminal of the coding region, the signal sequence and the non- coding flanking sequence
  • Inverse PCR was used. 5 ul of genomic DNA was 5 digested with 10U of BamH I restriction enzyme in 50 ul 1x restriction buffer at 37°C for 16 hours. The digested DNA was purified by QIAquick spin-columns (QUIGEN) and 10% of the digested DNA was ligated at 16°C for 16 hours. Finally, PCR amplification was carried out using the specific primers DAX385-5 and -7.
  • the resulting PRC fragment was sequenced using the same o primers, and totally about 2.3 kb DNA, coding the Amrk385 alpha-amylase was obtained by use of degenerated primers and inverse PCR.
  • Dax385-5 GGTACAGTAGTATCAAAACATCCAATACATGC (SEQ ID NO. 9)
  • Dax385-7 GTAATTGACTCCGCGTACCATATTTCGTTCG (SEQ ID NO. 10)
  • the sequence of Amrk385 is shown in SEQ ID NO:5-6. (amino acids 1-486 of SEQ s ID NO:6 is the amino acid sequence of the mature polypeptide Amrk385).
  • the wild type Bacillus strain (obtained from a soil sample of California, USA) was grown under vigorous shaking for 5 days at 37°C in a rich media adjusted to pH 9.0.
  • the culture broth was flocculated by adding 0.01 ml 50%(w/w) CaCI 2 ,2H 2 0, 0.03 ml 20% (w/w) Sodium aluminate, 0.025 ml 10% C521 and 0.075 ml 0.1 % A130 pr. 5 ml culture broth. A clear solution was obtained after centrifugation.
  • the enzyme solution was added ammonium sulphate to a final concentration of 1.2 M and applied on a Butyl Toyo Pearl column (100 ml) previously equilibrated in 1.2 M ammonium sulphate, 10 mM Tris-HCl, pH 7.0.
  • the amylase was eluted using 5 mM Tris-HCl, pH 7.0 and the eluted pool was dialysed against 5 mM Tris-HCl over night.
  • the fraction 0 was then subjected to ion exchange chromatography using a Q-Sepharose column
  • EXAMPLE 4 Characterisation of AA180 pH profile The pH profile shown in Fig. 2 was prepared at 37°C by using the Phadebas Assay described above using Britton-Robinson buffer (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, 0.1 mM CaCI 2 ) adjusted to pH 4 to 1 1 with NaOH.
  • Temperature profile The temperature profile shown in Fig.3 was prepared at pH 8.5 by using the Phadebas Assay described above at temperatures from 20 to 70°C
  • Specific activity The specific activity is determined using the Phadebas assay described below in the "Materials and Methods" section with the assay conditions pH 7.3 and at 37°C
  • AA180 has a specific activity of 56000 (NU/mg) and as indicated in Table 1 this is much higher than the specific activities of SP690 (i.e. SEQ ID NO: 1 of US patent no. 5,856,164), SP722 (SP722 is SEQ ID NO: 2 of US patent no. 5,856,164) and Termamyl ® (SEQ ID NO:2 of US patent no. 5,830,837).
  • SP690 is SEQ ID NO: 1 of US patent no. 5,856,164
  • Genomic DNA of strains above were partially digested with restriction enzyme Isolated DNA fragments were ligated to digested pSJ1678 plasmid DNA, and the ligation mixture was used to transform E. coli SJ2. Identification of positive transformants:
  • the sequence of AA180 is shown in SEQ ID NO:1-2 (amino acids 1-486 of SEQ ID NO:2 is the amino acid sequence of the mature polypeptide AA180).
  • the sequence of AAG20 is shown in SEQ ID NO:3-4 (amino acids 1-486 of SEQ ID NO:4 is the amino acid sequence of the mature polypeptide AAG20).
  • the amino acid sequence of the mature polypeptide of AA180 and AAG20 is identical.
  • AA180 and AAG20 differs from the amino acid sequence of the mature Amrk385 in position 90 and position 420.
  • Wash performance was evaluated by washing soiled test swatches in a detergent solution with the ⁇ -amylase of the invention.
  • the detergent and wash conditions were:
  • test swatches used were orange rice starch swatches, CS-28, available from CFT, Center for Test Material, Holland.
  • the detergents were commercially available batches in which the included amylases were inactivated by heating up to
  • the ⁇ -amylase of the invention was evaluated for wash performance in respectively the European Ariel Futur powder and Tide powder from USA ( Figure 4). The results demonstrate that the ⁇ -amylase of the invention is effective in starch removal in powdered detergents from both Europe and USA.

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Abstract

L'invention concerne des polypeptides possédant une activité de l'alpha-amylase et des séquences nucléotidiques codant pour ces polypeptides. L'invention concerne également des produits de recombinaison d'acides nucléiques, des vecteurs et des cellules hôtes ainsi que des plantes transgéniques contenant ces séquences nucléotidiques ainsi que des méthodes de production et d'utilisation de ces polypeptides.
PCT/DK2001/000133 2000-03-03 2001-02-28 Polypeptides possedant une activite de l'alpha-amylase et acides nucleiques codant pour ces polypeptides Ceased WO2001064852A1 (fr)

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