Classifications

• • 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
  • C11D3/38—Products with no well-defined composition, e.g. natural products
  • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• the present invention relates to variants (mutants) of parent Termamyl-like alpha-amylases, which variant has alpha- amylase activity and exhibits an alteration in at least one of the following properties relative to said parent alpha- amylase: stability under, e.g., high temperature and/or low pH conditions, in particular at low calcium concentrations.
• the variant of the invention are suitable for starch conversion, ethanol production, laundry wash, dish wash, hard surface cleaning, textile desizing, and/or sweetner production.
• Alpha-Amylases (alpha-1 , 4-glucan-4-glucanohydrolases, E.C. 3.2.1.1) constitute a group of enzymes, which catalyze hydrolysis of starch and other linear and branched 1, 4-glucosidic oligo- and polysaccharides.
• the object of the present invention is to provide Termamyl- like amylases which variants in comparison to the. corresponding parent alpha-amylase, i.e., un-mutated alpha- amylase, has alpha-amylase activity and exhibits an alteration in at least one of the following properties relative to said parent alpha-amylase: stability under, e.g., high temperature and/or low pH conditions, in particular at low calcium concentrations .
• Ala30Asn or A3 ON a deletion of alanine in the same position is shown as:
• Ala30* or A30* and insertion of an additional amino acid residue, such as lysine, is shown as:
• a deletion of a consecutive stretch of amino acid residues, such as amino acid residues 30-33, is indicated as (30-33)* or ⁇ (A30-N33) .
• Ala30Asp + Glu34Ser or A30N+E34S representing mutations in positions 30 and 34 substituting alanine and glutamic acid for asparagine and serine, respectively.
• A3 ON or A30E when a position suitable for modification is identified herein without any specific modification being suggested, it is to be understood that 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.
• A30X means any one of the following substitutions: A30R, A30N, A30D, A30C, A30Q, A30E, A30G, A30H, A30I, A30L, A30K, A30M, A30F, A30P, A30S, A30T, A30W, A30Y, or A30 V; or in short: A30R,N,D, C, Q, E, G,H, I , L, K,M, F, P, S, T, W, Y, V. If the parent enzyme - used for the numbering - already has the amino acid residue in question suggested for substitution in that position the following nomenclature is used:
• Figure 1 is an alignment of the amino acid sequences of five parent Termamyl-like alpha-amylases. The numbers on the extreme left designate the respective amino acid sequences as follows :
• SEQ ID NO 2 (SP690) 3 3 S SEEQQ I IDD N NOO: 10 (BAN)
• the object of the present invention is to provide Termamyl- like amylases, which variants have alpha-amylase activity and exhibits altered stability at high temperatures and/or at low pH, in particular at low calcium concentrations.
• Termamyl-like alpha-amylases which variants have alpha-amylase activity and exhibits altered stability at high temperatures and/or at low pH, in particular at low calcium concentrations.
• alpha-amylases produced by Bacillus spp. are highly homologous . (identical) on the amino acid level. The identity of a number of known ' Bacillus alpha-amylases can be found in the below Table 1 :
• the B. licheniformis alpha-amylase comprising the amino acid sequence shown in SEQ ID NO : 8 (commercially available as TermamylTM) has been found to be about 81% homologous with the B. amyloliquefaciens alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 10 and about 65% homologous with the B. stearothermophilus alpha- amylase (BSG) comprising the amino acid sequence shown in SEQ ID NO: 6.
• BSG B. stearothermophilus alpha- amylase
• Further homologous alpha-amylases include SP690 and SP722 disclosed in WO 95/26397 and further depicted in SEQ ID NO: 2 and SEQ ID NO : 4, respectively, herein.
• amylases are the AA560 alpha-amylase derived from Bacillus sp. and shown in SEQ ID NO: 12, and the #707 alpha-amylase derived from Bacillus sp., shown in SEQ ID NO: 13 and described by Tsukamoto et al . , Biochemical and Biophysical Research Communications, 151 (1988), pp. 25-31.
• the KSM AP1378 alpha-amylase is disclosed in WO 97/00324 (from KAO Corporation) .
• alpha-amylases include the alpha- amylase produced by the B. licheniformis strain described in EP 0252666 (ATCC 27811) , and the alpha-amylases identified in WO 91/00353 and WO 94/18314.
• Termamyl-like alpha-amylases are comprised in the products sold under the following tradenames : OptithermTM and TakathermTM (Solvay) ; MaxamylTM (available from Gist-brocades/Genencor) , Spezym AATM and Spezyme Delta AATM (available from Genencor) , and KeistaseTM (available from Daiwa) , Dex lo, GC 521 (available from Genencor) and Ultraphlow (from Enzyme Biosystems) .
• alpha-amylases Because of the substantial homology found between these alpha-amylases, they are considered to belong to the same class of alpha-amylases, namely the class of "Termamyl-like alpha-amylases" .
• Termamyl- like alpha-amylase is intended to indicate an alpha-amylase, in particular Bacillus alpha-amylase, which, at the amino acid level, exhibits a substantial identity to TermamylTM, i.e., the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 8, herein.
• alpha-amylases which has the amino acid sequences shown in SEQ ID NOS : 2, 4, 6, 8, 10, 12 and 13 herein are considered to be "Termamyl-like alpha- amylase".
• Other Termamyl-like alpha-amylases are alpha- amylases i) which displays at least 60%, such as at least 70%, e.g., at least 75%, or at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% homology (identity) with at least one of said amino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, and 13, and/or is encoded ' by a DNA sequence which hybridizes to the DNA sequences encoding the above-specified alpha-amylases which are apparent from SEQ ID NOS: 1, 3, 5, 7, 9, and of the present specification (which encoding sequences encode the amino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10 and 12 here
• the homology may be determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second.
• the homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (described above) .
• GAP provided in the GCG program package (described above) .
• Gap GCGv ⁇ may be used with the default scoring matrix for identity and the following default parameters: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, respectively for nucleic acidic sequence comparison, and GAP creation penalty of 3.0 and GAP extension penalty of 0.1, respectively, for protein sequence comparison.
• GAP uses the method of Needleman and Wunsch, (1970), ' J.Mol. Biol. 48, p. 43 -453, to make alignments and to calculate the identity.
• a structural alignment between Termamyl (SEQ ID NO: 8) and, e.g., another alpha-amylase may be used to identify equivalent/corresponding positions in other Termamyl-like alpha- amylases.
• One method of obtaining said structural alignment is to use the Pile Up programme from the GCG package using default values of gap penalties, i.e., a gap creation penalty of 3.0 and gap extension penalty of 0.1.
• Other structural alignment methods include the hydrophobic cluster analysis (Gaboriaud et al . , (1987), FEBS LETTERS 224, pp. 149-155) and reverse threading (Huber, T; Torda, AE, PROTEIN SCIENCE Vol. 7, No. 1 pp. 142-149 (1998) .
• oligonucleotide probe used in the characterisation of the Termamyl-like alpha-amylase above may suitably be prepared on the basis of the full or partial nucleotide or amino acid sequence of the alpha-amylase in question.
• Suitable conditions for testing hybridisation involve pre- soaking in 5xSSC and prehybridizing for 1 hour at 40 °C in a solution of 20% formamide, 5xDenhardt ' s solution, 50mM sodium phosphate, pH 6.8, and 50mg of denatured sonicated calf thymus DNA, followed by hybridisation in the same solution supplemented with 100 mM ATP for 18 hours at 40°C, followed by three times washing of the filter in 2xSSC, 0.2% SDS at . 40°C for 30 minutes (low stringency) , preferred at 50°C (medium stringency) , more preferably at 65°C (high stringency) , even more preferably at 75°C (very high stringency) . More details about the hybridisation method can be found in Sambrook et al . , Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.
• derived from is intended not only to indicate an alpha-amylase produced or producible by a strai of the organism in question, but also an alpha-amylase encoded by a DNA sequence isolated from such strain and pro- . **d in a host organism transformed with said DNA sequence.
• the term is intended to indicate an alpha-amylase, which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics • of the alpha-amylase in question.
• parent alpha-amylase may be a variant of a naturally occurring alpha-amylase, i.e., a variant, which is the result of a modification (insertion, substitution, deletion) of one or more amino acid residues of the naturally occurring alpha-amylase.
• parent Termamyl-like Alpha-amylases are also intended to indicate that the parent alpha-amylase may be a variant of a naturally occurring alpha-amylase, i.e., a variant, which is the result of a modification (insertion, substitution, deletion) of one or more amino acid residues of the naturally occurring alpha-amylase.
• Termamy-like alpha-amylases may be used as the parent (i.e., backbone) alpha-amylase.
• the parent alpha-amylase is derived from B. licheniformis, e.g., one of those referred to above, such as the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 8.
• the parent alpha-amylase (i.e., backbone alpha-amylase) may also be a hybrid alpha-amylase, i.e., an alpha-amylase, which comprises a combination of partial amino acid sequences derived from at least two alpha-amylases.
• the parent hybrid alpha-amylase may be one, which on the basis of amino acid homology (identity) and/or DNA hybridization (as defined above) can be determined to belong to the Termamyl-like alpha-amylase family.
• the hybrid alpha-amylase is typically composed of at least one part of a Termamyl-like alpha-amylase and part (s) of one or more other alpha-amylases selected from Termamyl-like alpha- amylases or non-Termamyl-like alpha-amylases of microbial (bacterial or fungal) and/or mammalian origin.
• the parent hybrid alpha-amylase may comprise a combination of partial amino acid sequences deriving from at least two Termamyl-like alpha-amylases, or from at least one Termamyl-like and at least one non-Termamyl-like bacterial alpha-amylase, or from at least one Termamyl-like and at least one fungal alpha-amylase.
• the Termamyl-like alpha-amylase from which a partial amino acid sequence derives may be any of the specific Termamyl-like alpha-amylase referred to herein.
• the parent alpha-amylase may comprise a C- terminal part of an alpha-amylase derived from a strain of B.
• the parent alpha- amylase may comprise at least 430 amino acid residues of the C-terminal part of the B. licheniformis alpha-amylase, and may, e.g., comprise a) an amino acid segment corresponding to the 37 N-terminal amino acid residues of the B. amyloliquefa- ciens alpha-amylase having the amino acid sequence shown in SEQ ID NO: 10 and an amino acid segment corresponding to the 445 C-terminal amino acid residues of the B.
• licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 8, or a hybrid Termamyl-like alpha-amylase being identical to the Termamyl sequence, i.e., the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8, except that the N- terminal 35 amino acid residues (of the mature protein) has been replaced by the N-terminal 33 residues of BAN (mature protein), i.e., the Bacillus amyloliquefaciens alpha-amylase shown in SEQ ID NO: 10; or b) an amino acid segment corresponding to the 68 N-terminal amino acid residues of the B.
• BAN mature protein
• stearothermophilus alpha-amylase having the amino acid sequence shown in SEQ ID NO: 6 and an amino acid segment corresponding to the 415 C-terminal amino acid residues of the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO : 8.
• Another suitable parent hybrid alpha-amylase is the one previously described in WO 96/23874 (from Novo Nordisk) constituting the N-terminus of BAN, Bacillus amyloliquefaciens alpha-amylase (amino acids 1-300 of the mature protein) and the C-terminus from Termamyl (amino acids 301-483 of the mature protein) .
• the parent Termamyl-like alpha-amylase is a hybrid alpha-amylase of SEQ ID NO: 8 and SEQ ID NO: 10.
• the parent hybrid Termamyl-like alpha-amylase may be a hybrid alpha-amylase comprising the 445 C-terminal amino acid residues of the B.
• the latter mentioned hybrid is used in the examples below and is referred to as LE174.
• parent alpha-amylase include LE174 with fewer mutations, i.e., the right above mentioned hydrid having the following mutations A181T+N190F+A209V+Q264S; N190F+A209V+Q264S ; A209V+Q264S Q264S; H156Y+N190F+A209V+Q264S ; H156Y+A209V+Q264S H156Y+Q264S; H156Y+A181T+A209V+Q264S ; ' H156Y+A181T+Q264S H156Y+Q264S; . H156Y+A181T+N190F+Q264S; H156Y+A181T+N190F H156Y+A181T+N190F+A209V.
• These hybrids ⁇ are also considered to be part of the invention.
• the parent Termamyl-like alpha amylase is LE174, SP722, or AA560 including any of LE174+G48A+T49I+G107A+I201F; LE174+M197L; LE174+G48A+T49I+G107A+M197L+I201F, or SP722+D183*+G184* ; SP722+D183*+G184*+N195F; SP722+D183*+G184*+M202L; SP722+D183*+G184*+N195F+M202L; BSG+I181*+G182* ; BSG+I181*-t-G182*+N193F; BSG+I181*+G182*+M200L; BSG+I181*+G182*+N193F+M200L; AA560+D183*+G184*; AA560+D183*+G184*+N195F;
• LE429 which is LE174 with an additional substitution in I201F.
• LE335 is the alpha-amylase, which in comparison to LE429 has additional substitutions in T49I+G107A;
• LE399 is LE335+G48A, i.e., LE174, with G48A+T49I+G107A+I201F.
• the invention relates to Termamyl-like alpha-amylases with altered properties (as mentioned above) , in particular at high temperatures and/or at low pH, in particular at low calcium concentrations.
• high temperature means temperatures from 70-120°C, preferably 80-100°C, especially 85-95°C.
• low pH means from a pH in the range from 4-6, preferably 4.2-5.5, especially 4.5-5.
• high pH means from a pH in the range from 8-11, especially 8.5-10.6.
• low calcium concentration means free calcium levels lower than 60 ppm, preferably 40 ppm, more preferably 25 ppm, especially 5 ppm calcium.
• Termamyl-like alpha-amylase and parent hydrid Termamyl-like alpha-amylases .
• the Termamyl ® alpha-amylase is used as the starting point, but corresponding positions in, e.g., the SP722, BSG, BAN, AA560, SP690, KSM AP1378, and #707 should be understood as disclosed and specifically comtemplated too.
• the variant of the invention has in. particular at high temperatures and/or at low pH.
• the invention relates to variant with altered properties as mentioned above.
• a variant of a parent Termamyl-like alpha-amylase comprising an alteration at one or more positions (using SEQ ID NO: 8 for the amino acid numbering) selected from the group of:
• the variant has alpha-amylase activity and (c) each position corresponds to a position of the amino acid sequence of the parent Termamyl-like alpha-amylase having the amino acid sequence shown in SEQ ID NO: 8.
• Termamyl ® (SEQ ID NO: 8) such corresponding positions are : T49; D60; N104; E132; D161; K170; K176; G179; K180; A181; D183; D200; Y203; D204; D207; 1212; K237; S239; E250; N280; Q298; L318; Q374; E385; Q393; Y402; H406; L427 D430; V440; N444 ; E447; Q482.
• SP722 (SEQ ID NO : 4) the corresponding positions are: T51; D62; N106; D134; D163; Q172; K179; G184; K185; A186; D188; D205; M208; D209; X212; L217, K242, S244, N255, N285, S303, M323; D387, N395; Y404; H408; 1429; D432; V442; K446; Q449; K484.
• the variant of the invention (using SEQ ID NO: 8 (TermamylTM) for the numbering) has one or more of the following substitutions :
• the ' variant of the invention (using SEQ ID NO : 4 (SP722) for the numbering) has one or more of the following substitutions:
• Preferred double, triple and multi -mutations - using SEQ ID NO: 8 as the basis for the numbering - are selected from the group consisting of :
• K170Q+K176R K170Q+K176R+D207V; K170Q+K176R+D207E;
• V440A E250G+N280S; E250G+N280S+L318M;
• the variant has the following substitutions: K170Q+D207V+N280S;. E132A+D207V;
• T49I+E132V+V440A T49I+K176R+D207V+Y402F; Q374R+E385V+Q393R;
• G48A+T49I+I201F G48A+T49I+G107A; T49I+I201F; T49I+G107A; G48A+T49I;
• LE174+G107A+I201F; LE174+I201F, are specifically contemplated variants of the invention.
• altered stability in particular improved stability (i.e., higher or lower), at especially high temperatures (i.e., 70-120°C) and/or extreme pH (i.e. low or high pH, i.e, pH 4-6 or pH 8-11, respectively), in particular at free (i.e., unbound, therefore in solution) calcium concentrations below 60 ppm, include any of the mutations listed in the "Altered properties" section.
• the stability may be determined as described in the' "Materials & Methods" section below.
• a variant of the invention may in one embodiment comprise one or more modifications in addition to those outlined above.
• one or more Proline (Pro) residues present in the part of the alpha-amylase variant which is modified is/are replaced with a non-Proline residue which may be any of the possible, naturally occurring non- Proline residues, and which preferably is an Alanine, Glycine, Serine, Threonine, Valine or Leucine.
• one or more Cysteine residues present in the parent alpha-amylase may be replaced with a non-Cysteine residue such as Serine, Alanine, Threonine, Glycine, Valine or Leucine.
• a variant of the invention may - either as the only modification or in combination with any of the above outlined modifications - be modified so that one or more Asp and/or Glu present in an amino acid fragment corresponding to the amino acid fragment 185-209 of SEQ ID NO: 10 is replaced by an Asn and/or Gin, respectively. Also of interest is the replacement, in the Termamyl-like alpha-amylase, of one or more of the Lys residues present in an amino acid fragment corresponding to the amino acid fragment 185-209 of SEQ ID NO: 10 by an Arg. It is to be understood that the present invention encompasses variants incorporating two or more of the above outlined modifications.
• H405, T412, in particular the following single, double or triple or multi mutations M15X, in particular M15T,L;
• V128X in particular V128E;
• H133X in particular H133Y;
• N188X in particular N188S,T,P;
• M197X in particular M197T,L
• A209X in particular A209V
• the DNA sequence encoding a parent alpha-amylase may be isolated from any cell or microorganism producing the alpha- amylase in question, using various methods well known in the art.
• a genomic DNA and/or cDNA library should be constructed . using chromosomal DNA or messenger RNA from the organism that produces the alpha-amylase to be studied.
• homologous, labeled oligonucleotide probes may be synthesized and used to identify alpha-amylase-encoding clones from a genomic library prepared from the organism in question.
• oligonucleotide probe containing sequences homologous to a known alpha-amylase gene could be used as a probe to identify . alpha-amylase-encoding clones, using hybridization and washing conditions of lower stringency.
• Yet another method for identifying alpha-amylase-encoding clones would involve inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming alpha- amylase-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for alpha-amylase, thereby allowing clones expressing the alpha-amylase to be identified.
• an expression vector such as a plasmid
• transforming alpha- amylase-negative bacteria with the resulting genomic DNA library
• the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g., the phosphoroamidite method described by S.L. Beaucage and M.H. Caruthers, Tetrahedron Letters 22, 1981, pp. 1859-1869, or the method described by Matthes et al . , The EMBO J. 3, 1984, pp. 801-805.
• the phosphoroamidite method oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors .
• the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, the fragments corresponding to various parts of the entire DNA sequence), in accordance with standard techniques.
• the DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers-, for instance as described in US 4,683,202 or R.K. Saiki et al . , Science 239, 1988, pp. 487- 491.
• muta- tions may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligonucleotide synthesis.
• a single-stranded gap of DNA, bridging the alpha-amylase- encoding sequence is created in a vector carrying the alpha- amylase gene. Then the synthetic nucleotide, bearing the desired mutation, is annealed to a homologous portion of the single-stranded DNA.
• Alternative methods for providing variants of the invention include gene shuffling, e.g., as described in WO 95/22625 (from Affymax Technologies N.V.) or in WO 96/00343 (from Novo Nordisk A/S) , or other corresponding techniques resulting in a hybrid enzyme comprising the mutation(s), e.g., substitution (s) and/or deletion(s), in question.
• parent alpha-amylases which suitably may be used for providing a hybrid with the desired mutations (s) according to the invention include the KSM-K36 and KSM-K38 alpha-amylases disclosed in EP 1,022,334 (hereby incorporated by reference) .
• a DNA sequence encoding the variant produced by methods described above, or by any alternative methods known in the art can be expressed, in enzyme form, using an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.
• the recombinant expression vector carrying the DNA sequence encoding an alpha-amylase variant of the invention may be any vector, which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
• the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or an artificial chromosome.
• the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome (s) into which it has been integrated.
• the DNA sequence should be operably connected to a suitable promoter sequence.
• the promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
• suitable promoters for directing the transcription of the DNA sequence encoding an alpha-amylase variant of the invention, especially in a bacterial host are the promoter of the lac operon of E.coli, the Streptomyces .
• coelicolor agarase gene dagA promoters examples include those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, ⁇ A.
• niger neutral alpha-amylase A. niger acid stable alpha- amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.
• the expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, poly- adenylation sequences operably connected to the DNA sequence encoding the alpha-amylase variant of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
• the vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBHO, pE194, pAMBl and pIJ702.
• the vector may also comprise a selectable marker, e.g.
• the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, e.g., as described in WO 91/17243. While intracellular expression may be advantageous in some respects, e.g., when using certain bacteria as host cells, it is generally preferred that the expression is extracellular.
• Bacillus alpha-amylases mentioned herein comprise a preregion permitting secretion ' of the expressed protease into the culture medium. If desirable, this preregion may be replaced by a different preregion or signal sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective preregions.
• the cell of the invention is advantageously used as a host cell in the recombinant production of an alpha-amylase variant of the invention.
• the cell may be transformed with the DNA construct of the invention encoding the variant, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.
• the cell of the invention may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g., a bacterial or a fungal (including yeast) cell.
• a microbial cell e.g., a bacterial or a fungal (including yeast) cell.
• suitable bacteria are Gram-positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus , thuringiensis, or Streptomyces lividans or Streptomyces murinus, or gramnegative bacteria such as E.coli.
• the transformation of the bacteria may, for instance, be effected by protoplast .transformation or by using competent cells in a manner known per se .
• the yeast organism may favorably be selected from a species of Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae.
• the filamentous fungus may advantageously belong ' to a species of Aspergillus, e.g., Aspergillus oryzae or Aspergillus niger.
• Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se . A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023.
• the present invention relates to a method of producing an alpha-amylase variant of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the variant and recovering the variant from the cells and/or culture medium.
• the medium used to cultivate the cells may be any conven- tional medium suitable for growing the host cell in question and obtaining expression of the alpha-amylase variant of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection) .
• the alpha-amylase • variant secreted from the host cells may conveniently be recovered from the culture medium by well- known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or. the like.
• alpha-amylase variants of this invention possess valuable properties allowing for a variety of industrial applications.
• enzyme variants of the invention are applicable as a component in washing, dishwashing, and hard surface cleaning detergent compositions.
• Variant of the invention with altered properties may be used for starch processes, in particular starch conversion, especially liquefaction of starch (see, e.g., US 3,912,590, EP patent publications Nos . 252 730 and 63 909, WO 99/19467, and WO 96/28567 all references hereby incorporated by reference) .
• compositions for starch conversion purposes which may beside the variant of the invention also comprise a AMG, pullulanase, and other alpha-amylases.
• variants of the invention are also particularly useful in the production of sweeteners and ethanol (see, e.g.,
• a variant of the invention may also be used for textile desizing (see, e.g., WO 95/21247, US patent 4,643,736, EP 119,920 hereby in corporate by reference) .
• variants of the invention may suitably be incorporated in detergent compositions.
• Detergent compositions comprising a variant of the invention may additionally comprise one or more other enzymes, such as a protease, a lipase, a peroxidase, another amylolytic enzyme, glucoamylase, maltogenic amylase, CGTase and/or a cellulase, mannanase (such as MannawayTM from Novozymes, Denmark)), pectinase, pectine lyase, cutinase, laccase, and/or another alpha-amylase.
• enzymes such as a protease, a lipase, a peroxidase, another amylolytic enzyme, glucoamylase, maltogenic amylase, CGTase and/or a cellulase, mannanase (such as MannawayTM from Novozymes, Denmark)), pectinase, pectine lyase, cutinase, lac
• Alpha-amylase variants of the invention may be incorporated in detergents at conventionally employed concentrations. It is at present contemplated that a variant of the invention may be incorporated in an amount corresponding to 0.00001-10 mg (calculated as pure, active enzyme protein) of alpha-amylase per liter of wash/dishwash liquor using conventional dosing levels of detergent .
• the invention also related to composition comprising a variant of the invention, and in a preferred embodiment also a B. stearothermophilus alpha-amylase (BSG), in particular a variant thereof.
• BSG B. stearothermophilus alpha-amylase
• the composition comprises beside a variant of the invention a glucoamylase, in particular a glucoamylase originating from Aspergillus niger (e.g., the GI or G2 A. niger AMG disclosed in Boel et al . (1984), "Glucoamylases GI and G2 from Aspergillus niger are synthesized from two different but closely related mRNAs", EMBO J. 3 (5), p. 1097-1102, or a variant therefore, in particular a variant disclosed in WO 00/04136 or WO 01/04273 or the Talaromyces emersonii AMG disclosed in WO 99/28448.
• a specific combination is LE399 and a variant disclosed in WO 00/04136 or Wo 01/04273, in particular a variant with oe or more of the following substitutions :
• composition of the invention also comprises a pullulanase, in particular a Bacillus pullulanase.
• AA560 SEQ ID NO: 12; disclosed in WO 00/60060; deposited on 25th January 1999 at DSMZ and assigned the DSMZ no. 12649. AA560 were deposited by the inventors under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at Deutshe Sammmlung von Microorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg lb, D-38124 Braunschweig DE.
• DSMZ Sammmlung von Microorganismen und Zellkulturen GmbH
• LB medium In 1 liter H20: 10 g bacto-tryptone, 5 g bacto- yeast extract, 10 g NaCl, pH adjusted to 7.0 w. NaOH, autoclaved
• TY agar plates In 1 liter H20: 16 g bacto-tryptone, 10 g bacto-yeast extract, 5 g NaCl, pH adjusted to -7.0 w. NaOH, and 15 g bacto-agar is added prior to autoclaving
• Plasmids pDN1528 contains the complete gene encoding Termamyl, amyL, the expression of which is directed by its own promoter. Further, the plasmid contains the origin of replication, ori, from plasmid pUBHO and the cat gene from plasmid pC194 conferring resistance towards chloramphenicol. pDN1528 is shown in Fig. 9 of WO 96/23874.
• Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67, Schleicher & Schuell, Dassel, Germany) - and nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plates with 10 micro g/ml chloramphenicol at 37°C for at least 21 hours.
• the cellulose acetate layer is located on the TY agar plate.
• Each filter sandwich is specifically marked with a needle after plating, but before incubation in order to be able to localize positive variants on the filter, and the nitrocellulose filter with bound variants is transferred to a container with citrate buffer, pH 4.5 and incubated at 80°C for 20 minutes (when screening for variants in the wild type backbone) or 85oC for 60 minutes (when screening for variants in the LE399 backbone) .
• the cellulose acetate filters with colonies are stored on the TY-plates at room temperature until use. After incubation, residual activity is detected on assay plates containing 1% agarose, 0.2% starch in citrate buffer, pH 6.0.
• the assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for 2 hours at 50°C. After removal of the filters the assay plates are stained with 10% Lugol solution. Starch degrading variants are detected as white spots on dark blue background and then identified on the -storage plates. Positive variants are rescreened twice under the same conditions as the first screen.
• Positive transformants after rescreening are picked from the storage plate and tested in a secondary plate assay. Positive transformants are grown for 22 hours at 37°C in 5 ml LB + chloramphenicol. The Bacillus culture of each positive transformant and as a control a clone expressing the corresponding backbone are incubated in citrate buffer, pH 4.5 at 90°C and samples are taken at 0, 10, 20, 30, 40, 60 and 80 minutes. A 3 micro liter sample is spotted on an assay plate. The assay plate is stained with 10% Lugol solution. Improved variants are seen as variants with higher residual activity (detected as halos on the assay plate) than the backbone. The improved variants are determined by nucleotide sequencing.
• Bacillus cultures expressing the variants to be analysed are grown for 21 hours at 37°C in 10 ml LB+chloramphenicol .
• 800 micro liter culture is mixed with 200 micro 1 citrate buffer, pH 4.5.
• a number of 70 micro 1 aliquots corresponding to the number of sample time points are made in PCR tubes and incubated at 70°C (for variants in the wt backbone) or 90°C (for variants in LE399) for various time points (typically 5, 10, 15, 20, 25 and 30 minutes) in a PCR machine.
• the 0 min sample is not incubated at high temperature.
• Activity in the sample is measured by transferring 20 micro 1 to 200 micro 1 of the alpha-amylase PNP-G7 substrate MPR3 ( (Boehringer Mannheim Cat. no. 1660730) as described below under "Assays for Alpha-Amylase Activity” . Results are plotted as percentage activity (relative to the 0 time point) versus time, or stated as percentage residual activity after incubation for a certain period of time.
• the culture is shaken at 37°C at 270 rpm for 5 days.
• the enzyme is incubated under the relevant conditions (1-4) .
• Samples are taken at various time points, e.g., after 0, 5,
• the activity measured before incubation (0 minutes) is used as reference (100%) .
• the decline in percent is calculated as a function of the incubation time.
• the table shows the residual activity after, e.g., 30 minutes of incubation.
• Specific activity determination The specific activity is determined using the Phadebas assay (Pharmacia) as activity/mg enzyme. The manufactures instructions are followed (see also below under "Assay for ⁇ - amylase activity) .
• Phadebas ® tablets contain a cross-linked insoluble blue-colored starch polymer, which has been mixed with bovine serum albumin and a buffer substance and tabletted.
• the absorbance of the resulting blue solution is a function of the alpha-amylase activity. It is important that the measured 620 nm absorbance after 10 or 15 minutes of incubation (testing time) 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 alpha-amylase will hydrolyze a certain amount of substrate and a blue colour will be produced. The colour intensity is measured at 620 nm. The measured absorbance is directly proportional to the specific activity (activity/mg of pure alpha-amylase protein) of the alpha-amylase in question under the given set of conditions.
• Alpha-amylase activity is determined by a method employing the PNP-G7 substrate.
• PNP-G7 which 'is a abbreviation for p- nitrophenyl-alpha, D-maltoheptaoside is a blocked oligosaccharide which can be cleaved by an endo-amylase .
• Kits containing PNP-G7 substrate and alpha-Glucosidase is manufactured by Boehringer- Mannheim (cat. No.1054635).
• PCR conditions were: 10 mM Tris-HCl, pH 8.3 , 50 mM KCl, 4 mM MgCl2, 0.3 mM MnCl2 , 0. ImM dGTP/dATP, 0.5 mM dTTP/dCTP, and 2.5 units Taq polymerase per 100 micro 1 reaction.
• the resultant PCR fragment was purified on gel and used in a PCR-based multimerization step with a gel purified vector fragment created by PCR amplification of pDN1528 with primers #24: 5' -GAA TGT ATG TCG GCC GGC AAA ACG CCG GTG A-3' (SEQ ID NO: 16) and #27: 5"-GCC GCC GCT GCT GCA GAA TGA GGC AGC AAG-3'
• a "+" indicates significant increase in stability relative to wt .
• LE493 Mutations from LE493 (K176R+D207V+Y402F) were transferred to LE399 yielding LE495. This was performed by the overlap PCR method (Kirchhoff and Desrosiers, PCR Methods and Applications, 2 (1993) , 301-304) .
• Fragment A #312 Mutl76 5'-CCC GAA AGC TGA ACC GCA TCT ATA GGT TTC AAG GGA AGA CTT GGG ATT-3' (SEQ ID NO: 18) (mutated codon indicated in bold) and #290 ' D207overlap 5'- AGG ATG GTC ATA ATC AAA GTC GG-3' (SEQ ID NO : 19); Fragment B: #313 Mut207 5' -CCG ACT TTG ATT ATG ACC ATC CTG TTG TCG TAG CAG AGA TTA AGA GAT GGG G-3' (SEQ ID NO: 20) and #314 Mut402 5'- CGA CAA TGT CAT GGT GGT CGA AAA AAT CAT GCT GTG CTC CGT ACG-3'
• LE 497 was obtained in a similar manner by amplifying the LE399 encoding template with primers #312 Mutl76 and #314 Mut402 and using the resulting PCR fragment in a multimerization reaction with a vector fragment obtained by PCR amplification of the LE399 template with the primers #296 Y402multi and #305 399Multil76.
• a "+" indicates significant increase in stability, relative to backbone .

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