AU2005202164B2 - Alpha-amylase variants - Google Patents

Description

S&F Ref: 568571D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Novozymes A/S, of Krogshoejvej 36, DK-2880, Bagsvaerd, Denmark Carsten Andersen Christel Thea Jorgensen Henrik Bisgard-Frantzen Allan Svendsen Soren Kjaerulff Spruson Ferguson St Martins Tower Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Alpha-amylase variants The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c [R:\LIBW]36355.doc:TYB 1 Alpha-AMYLASE VARIANTS FIELD OF THE INVENTION 00 The present invention relates, inter alia, to novel variants of parent Termamyl-like alpha-amylases, notably variants exhibiting altered properties, in particular altered cleavage pattern (relative to the parent) which are advantageous with S respect to applications of the variants in, in particular, l industrial starch processing starch liquefaction or saccharification) BACKGROUND OF THE INVENTION Alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolases,

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3.2.1.1) constitute a group of enzymes which catalyze hydrolysis of starch and other linear and branched 1,4-glucosidic oligoand polysaccharides.

There is a very extensive body of patent and scientific literature relating to this industrially very important class of enzymes. A number of alpha-amylase such as Termamyl-like alpha-amylases variants are known from, WO 90/11352, WO 95/10603, WO 95/26397, WO 96/23873, WO 96/23874 and WO 97/41213.

Among recent disclosure relating to alpha-amylases, WO 96/23874 provides three-dimensional, X-ray crystal structural data for a Termamyl-like alpha-amylase, reffered to as BA2, which consists of the 300 N-terminal amino acid residues of the B. amyloliquefaciens alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 6 herein and amino acids 301-483 of the C-terminal end of the B. licheniformis alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 4 herein 3o (the latter being available commercially under the tradename TermamylTM), and which is thus closely related to the industrially important Bacillus alpha-amylases (which in the present context are embraced within the meaning of the term "Termamyl-like alpha-amylases", and which include, inter alia, the B. licheniformis, B. amyloliquefaciens and B.

stearothermophilus alpha-amylases). WO 96/23874 further describes methodology for designing, on the basis of an analysis r of the structure of a parent Termamyl-like alpha-amylase, variants of the parent Termamyl-like alpha-amylase which exhibit altered properties relative to the parent.

WO 96/23874 and WO 97/41213 (Novo Nordisk) discloses Termamyl-like alphaamylase variants with an altered cleavage pattern containing mutations in the amino acid residues V54, D53, Y56, Q333, G57 and A52 of the sequence shown in SEQ ID NO:4 herein.

Brief Disclosure of the Invention According to a first embodiment of the invention, there is provided an isolated variant of a parent Termamyl-like alpha-amylase, which variant has an improved reduced ability to 0o cleave a substrate close to a branching point, and further has improved substrate specificity and/or improved specific activity, comprising an alteration at one or more positions selected from the group of: G48, T49, G107, wherein the alteration(s) are independently an insertion of an amino acid downstream of the amino acid which occupies the position, (ii) a deletion of the amino acid which occupies the position, or (iii) a substitution of the amino acid which occupies the position with a different amino acid, the variant has alpha-amylase activity, and each position corresponds to a position of the amino acid sequence of the parent Termamyl-like alpha-amylase having the amino acid sequence of SEQ ID NO:4.

According to a second embodiment of the invention, there is provided a DNA construct comprising a DNA sequence encoding an alpha-amylase variant in accordance with the first embodiment of the present invention.

According to a third embodiment of the invention, there is provided a recombinant expression vector which carries a DNA construct in accordance with the second embodiment of the present invention.

According to a fourth embodiment of the invention, there is provided a cell which is transformed with a DNA construct in accordance with the second embodiment of the present invention or a vector in accordance with the third embodiment of the present invention.

810755-1 gcc O 2a According to a fifth embodiment of the invention, there is provided a composition comprising: a mixture of the alpha-amylase from B. licheniformis having the sequence shown in SEQ ID NO:4 with one or more variants in accordance with the first embodiment of the present invention derived from (as the parent Termamyl-like alpha-amylase) the B.

INO stearothermophilus alpha-amylase having the sequence shown in SEQ ID NO:8; or (ii) a mixture of the alpha-amylase from B. stearothermophilus having the sequence 1 shown in SEQ ID NO:8 with one or more variants in accordance with the first embodiment 0 of the present invention derived from one or more other parent Termamyl-like alpha- N 10 amylases; or (iii) a mixture of one or more variants in accordance with the first embodiment of the present invention derived from (as the parent Termamyl-like alpha-amylase) the B.

stearothermophilus alpha-amylase having the sequence shown in SEQ ID NO:8 with one or more variants according to the invention derived from one or more other parent Termamyllike alpha-amylases.

According to a sixth embodiment of the invention, there is provided a composition comprising: a mixture of one or more variants in accordance with the first embodiment of the present invention derived from (as the parent Termamyl-like alpha-amylase) the B.

stearothermophilus alpha-amylase having the sequence shown in SEQ ID NO:8 and a Termamyl-like alpha-amylase derived from the B. licheniformis alpha-amylase having the sequence shown in SEQ ID NO:4.

According to a seventh embodiment of the invention, there is provided a composition comprising: a mixture of one or more variants in accordance with the first embodiment of the present invention derived from (as the parent Termamyl-like alpha-amylase) the B.

stearothermophilus alpha-amylase having the sequence shown in SEQ ID NO:8 and a hybrid alpha-amylase comprising a part of the B. amyloliquefaciens alpha-amylase shown in SEQ ID NO:6 and a part of the B. licheniformis alpha-amylase shown in SEQ ID NO:4.

According to an eighth embodiment of the invention, there is provided a composition comprising: 810755-1 gcc a mixture of one or more variants in accordance with the first embodiment of the present invention derived from (as the parent Termamyl-like alpha-amylase) a hybrid alphaamylase comprising a part of the B. amyloliquefaciens alpha-amylase shown in SEQ ID NO:6 and a part of the B. licheniformis alpha-amylase shown in SEQ ID NO:4.

According to a ninth embodiment of the invention, there is provided the use of an alpha-amylase variant in accordance with the first embodiment of the present invention or a composition in accordance with any one of the fifth to eighth embodiments of the present invention for starch liquefaction; in detergent composition, such as laundry, dish washing and hard surface cleaning compositions; ethanol production, such as fuel, drinking and industrial ethanol production; desizing of textiles, fabrics or garments.

The present invention relates to novel alpha-amylolytic variants (mutants) of a Termamyl-like alpha-amylase, in particular variants exhibiting altered cleavage pattern (relative to the parent), which are advantageous in connection with the industrial processing of starch (starch liquefaction, saccharification and the like).

The inventors have surprisingly found variants with altered properties, in particular altered cleavage pattern which have improved reduced capability of cleaving a substrate close to the branching point, and further have improved substrate specificity and/or improved specific activity, in comparison to the WO 96/23874 and WO 97/41213 (Novo Nordisk) disclosed Termamyl-like alpha-amylase variants with an altered cleavage pattern containing mutations in the amino acid residues V54, D53, Y56, Q333, G57 and A52 of the sequence shown in SEQ ID NO:4 herein.

The invention further relates to DNA constructs encoding variants of the invention, to composition comprising variants of the invention, to methods for preparing variants of the invention, and to the use of variants and compositions of the invention, alone or in combination with other alpha-amylolytic enzymes, in various industrial processes, e.g., starch liquefaction, and in detergent compositions, such as laundry, dish washing and hard surface cleaning compositions; ethanol production, such as fuel, drinking and industrial ethanol production; desizing of textiles, fabrics or garments etc.

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Nomenclature SIn the present description and claims, the conventional one- OO letter and three-letter codes for amino acid residues are used.

For ease of reference, alpha-amylase variants of the invention s are described by use of the following nomenclature: Original amino acid(s) :position(s):substituted amino acid(s) According to this nomenclature, for instance the 0 substitution of alanine for asparagine in position 30 is shown n as: o 0 10 Ala30Asn or A3ON a deletion of alanine in the same position is shown as: or and insertion of an additional amino acid residue, such as lysine, is shown as: *30aLys or A deletion of a consecutive stretch of amino acid residues, such as amino acid residues 30-33, is indicated as (30-33)* or A(A30-N33) or delta(A30-N33).

Where a specific alpha-amylase contains a "deletion" in comparison with other alpha-amylases and an insertion is made in such a position this is indicated as: *36aAsp or *36aD for insertion of an aspartic acid in position 36 Multiple mutations are separated by plus signs, i.e.: Ala30Asp Glu34Ser or A30N+E34S representing mutations in positions 30 and 34 substituting alanine and glutamic acid for asparagine and serine, respectively. Multiple mutations may also be separated as follows, meaning the same as the plus sign: Ala30Asp/Glu34Ser or A30N/E34S When one or more alternative amino acid residues may be inserted in a given position it is indicated as or or Furthermore, when a position suitable for modification is identified herein without any specific modification being suggested, or A30X, it is to be understood that any amino acid residue may be substituted for the amino acid residue present in the position. Thus, for instance, when a modification of an OO alanine in position 30 is mentioned, but not specified, or S specified as it is to be understood that the alanine may be s deleted or substituted for any other amino acid, any one \O of: R,N,D,C,Q,E,G,H, I,L,K,M,F,P,S,T,W,Y,V.

S DETAILED DISCLOSURE OF THE INVENTION tVt The Termamyl-like alpha-amylase 0 10 It is well known that a number of alpha-amylases produced by Bacillus spp. are highly homologous on the amino acid level. For instance, the B. licheniformis alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 4 (commercially available as TermamylTM) has been found to be about 89% homologous with the B. amyloliquefaciens alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 6 and about 79% homologous with the B. stearothermophilus alphaamylase comprising the amino acid sequence shown in SEQ ID NO: 8. Further homologous alpha-amylases include an alpha-amylase derived from a strain of the Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of which are described in detail in WO 95/26397, and the #707 alpha-amylase described by Tsukamoto et al., Biochemical and Biophysical Research Communications, 151 (1988), pp. 25-31.

Still further homologous alpha-amylases include the alphaamylase 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. Other commercial Termamyl-like B.

licheniformis alpha-amylases are OptithermT and TakathermTM (available from Solvay), MaxamylTM (available from Gistbrocades/Genencor), Spezym AATM and Spezyme Delta AAM (available from Genencor), and KeistaseTM (available from Daiwa).

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 alphaamylases".

>Accordingly, in the present context, the term "Termamyl-like alpha-amylase" is intended to indicate an alpha-amylase, which OO at the amino acid level exhibits a substantial homology to TermamylTM, the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 4 herein. In other D words, a Termamyl-like alpha-amylase is an alpha-amylase, which Cl has the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 8 C-i herein, and the amino acid sequence shown in SEQ ID NO: 1 or 2 of WO 95/26397 or in Tsukamoto et al., 1988, or i) which displays at least 60%, preferred at least 70%, more preferred at least 75%, even more preferred at least 80%, especially at least especially preferred at least 90%, even especially more preferred at least 95% homology, more preferred at least 97%, more preferred at least 99% with at least one of said amino acid sequences and/or ii) displays immunological cross-reactivity with an antibody raised against at least one of said alphaamylases, and/or iii) is encoded by a DNA sequence which hybridises to the DNA sequences encoding the above-specified alpha-amylases which are apparent from SEQ ID NOS: 1, 3, 5 and 7 of the present application and SEQ ID NOS: 4 and 5 of WO 95/26397, respectively.

In connection with property the "homology" may be determined by use of any conventional algorithm, preferably by use of the GAP progamme from the GCG package version 7.3 (June 1993) using default values for GAP penalties, which is a GAP creation penalty of 3.0 and GAP extension penalty of 0.1, (Genetic Computer Group (1991) Programme Manual for the GCG Package, version 7, 575 Science Drive, Madison, Wisconsin, USA 53711).

A structural alignment between Termamyl and a Termamyllike alpha-amylase may be used to identify equivalent/corresponding positions in other Termamyl-like alphaamylases. One method of obtaining said structural alignment is to use the Pile Up programme from the GCG package using default values of gap penalties, 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., 6 (1987), FEES LETTERS 224, pp. 149-155) and reverse threading (Huber, T Torda, AE, PROTEIN SCIENCE Vol. 7, No. 1 pp. 142- 00 149 (1998). Property ii) of the alpha-amylase, the immunological cross reactivity, may be assayed using an antibody raised against, or reactive with, at least one epitope of the \O relevant Termamyl-like alpha-amylase. The antibody, which may either be monoclonal or polyclonal, may be produced by methods S known in the art, as described by Hudson et al., Practical V) Immunology, Third edition (1989), Blackwell Scientific C o Publications. The immunological cross-reactivity may be determi- Sned using assays known in the art, examples of which are Western Blotting or radial immunddiffusion assay, as described by Hudson et al., 1989. In this respect, immunological crossreactivity between the alpha-amylases having the amino acid sequences SEQ ID NOS: 2, 4, 6, or 8, respectively, have been found.

The oligonucleotide probe used in the characterization of the Termamyl-like alpha-amylase in accordance with property iii) 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 hybridization involve presoaking in 5xSSC and prehybridizing for 1 hour at -400C 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 hybridization in the same solution supplemented with 100mM ATP for 18 hours at -400C, followed by three times washing of the filter in 2xSSC, 0.2% SDS at 40 0 C for minutes (low stringency), preferred at 50 0 C (medium stringency), more preferably at 650C (high stringency), even more preferably at -750C (very high stringency). More details about the hybridization method can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.

In the present context, "derived from" is intended not only to indicate an alpha-amylase produced or producible by a strain of the organism in question, but also an alpha-amylase encoded by a DNA sequence isolated from such strain and produced in a host organism transformed with said DNA sequence. Finally, the OO term is intended to indicate an alpha-amylase, which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has s the identifying characteristics of the alpha-amylase in S question. The term is also intended to indicate that the parent alpha-amylase may be a variant of a naturally occurring alpha- S amylase, i.e. a variant, which is the result of a modification In (insertion, substitution, deletion) of one or more amino acid C Lo residues of the naturally occurring alpha-amylase.

Parent hybrid alpha-amylases The parent alpha-amylase may be a hybrid alpha-amylase, 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 and/or immunological crossreactivity and/or DNA hybridization (as defined above) can be determined to belong to the Termamyl-like alpha-amylase family.

In this case, 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.

Thus, the parent hybrid alpha-amylase may comprise a combination of partial amino acid sequences deriving from at least two Termamyl-like alpha-amylases, or from at least one Termamyl-like and at least one non-Termamyl-like bacterial alpha-amylase, or from at least one Termamyl-like and at least one fungal alpha-amylase. The Termamyl-like alpha-amylase from which a partial amino acid sequence derives may, be any of those specific Termamyl-like alpha-amylases referred to herein.

For instance, the parent alpha-amylase may comprise a Cterminal part of an alpha-amylase derived from a strain of B.

licheniformis, and a N-terminal part of an alpha-amylase derived from a strain of B. amyloliquefaciens or from a strain of B.

stearothermophilus. For instance, 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, comprise O0 a) an amino acid segment corresponding to the 37 N-terminal amino acid residues of the B. amyloliquefaciens alpha-amylase s having the amino acid sequence shown in SEQ ID NO: 6 and an O amino acid segment corresponding to the 445 C-terminal amino acid residues of the B. licheniformis alpha-amylase having the S amino acid sequence shown in SEQ ID NO: 4, or b) an amino acid in segment corresponding to the 68 N-terminal amino acid residues Ic 0 of the B. stearothermophilus alpha-amylase having the amino acid sequence shown in SEQ ID NO: 8 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: 4.

In a preferred embodiment the parent Termamyl-like alphaamylase is a hybrid Termamyl-like alpha-amylase identical to the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 4, except that the N-terminal 35 amino acid residues (of the mature protein) is replaced with the N-terminal 33 amino acid residues of the mature protein of the Bacillus amyloliquefaciens alphaamylase (BAN) shown in SEQ ID NO: 6. Said hybrid may further have the following mutations: H156Y+A181T+N190F+A209V+Q 264

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(using the numbering in SEQ ID NO: 4) referred to as LE174.

Another preferred parent hybrid alpha-amylase is LE429 shown in SEQ ID NO: 2.

The non-Termamyl-like alpha-amylase may, be a fungal alpha-amylase, a mammalian or a plant alpha-amylase or a bacterial alpha-amylase (different from a Termamyl-like alphaamylase) Specific examples of such alpha-amylases include the Aspergillus oryzae TAKA alpha-amylase, the A. niger acid alphaamylase, the Bacillus subtilis alpha-amylase, the porcine pancreatic alpha-amylase and a barley alpha-amylase. All of these alpha-amylases have elucidated structures, which are markedly different from the structure of a typical Termamyl-like alpha-amylase as referred to herein.

The fungal alpha-amylases mentioned above, derived from A. niger and A. oryzae, are highly homologous on the amino 9 acid level and generally considered to belong to the same family of alpha-amylases. The fungal alpha-amylase derived from Aspergillus oryzae is commercially available under the tradename FungamylM.

Furthermore, when a particular variant of a Termamyl-like alpha-amylase (variant of the invention) is referred to in a conventional manner by reference to modification 0 deletion or substitution) of specific amino acid residues in the n amino acid sequence of a specific Termamyl-like alpha-amylase, it is to be understood that variants of another Termamyl-like alpha-amylase modified in the equivalent position(s) (as determined from the best possible amino acid sequence alignment between the respective amino acid sequences) are encompassed thereby.

A preferred embodiment of a variant of the invention is one derived from a B. licheniformis alpha-amylase (as parent Termamyl-like alpha-amylase), one of those referred to above, such as the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 4.

Construction of variants of the invention The construction of the variant of interest may be accomplished by -cultivating a microorganism comprising a DNA sequence encoding the variant under conditions which are conducive for producing the variant. The variant may then subsequently be recovered from the resulting culture broth. This is described in detail further below.

Altered properties The following discusses the relationship between mutations, which may be present in variants of the invention, and desirable alterations in properties (relative to those of a parent Termamyl-like alpha-amylase), which may result there from.

In the first aspect the invention relates to a variant of a parent Termamyl-like alpha-amylase, comprising an alteration at one or more positions selected from the group of: W13, G48, T49, S50, Q51, A52, D53, V54, G57, G107, G108, Alll, o oD SS168, M197, wherein the alteration(s) are independently an insertion of an amino acid downstream of the amino acid which occupies the position, 00 (ii) a deletion of the amino acid which occupies the position, or T (iii) a substitution of the amino acid which occupies the position with a different amino acid, 0 the variant has alpha-amylase activity and each position i corresponds to a position of the amino acid sequence of the parent Termamyl-like alpha-amylase having the amino acid c( sequence of SEQ ID NO: 4.

In a preferred embodiment the above variants of the invention comprise a mutation in a position corresponding to at least one of the following mutations in the amino acid sequence 1i shown in SEQ ID NO: 4: V54N, A52S, A52S+V54N, T49L, T49+G107A, A52S+V54N+T49L+G107A, A52S+V54N+T49L, G107A, Q51R, Q51R+A52S, A52N; or T49F+G107A, T49V+G107A, T49D+G107A, T49Y+G107A, T49S+G107A, T49N+G107A, T49I+G107A, T49L+A52S+G107A, T49L+A52T+G107A, T49L+A52F+G107A, T49L+A52L+G107A, T49L+A52I+G107A, T49L+A52V+G107A; or T49V, T49I, T49D, T49N, T49S, T49Y, T49F, T49W, T49M, T49E, T49Q, T49K, T49R, A52T, A52L, A52I, A52V, A52M, A52F, A52Y, A52W, V54M, G107V, G07I, G107L, G107C.

In a preferred embodiment a variant of the invention comprises at least one mutation in a position corresponding to the following mutations in the amino acid sequence shown in SEQ ID NO: 4: W13F,L,I,V,Y,A; G48A,V,S,T,I,L; *48aD or *48aY insertion of D or Y); T49X; *49aX insertion of any possible amino acid residue) in particular

D,Y,L,T,V,I;

Q51R,K; A52X, in particular A52S,N,T,F,L,I,V; D53E,Q,Y,I,N,S,T,V,L; V54X, in particular V54I,N,W,Y,F,L; G57S,A,V,L,I,F,Y,T; 00 G107X, in particular G107A,V,S,T,I,L,C; G108X, in particular G108A,V,S,T,I,L; A111V,I,L; S S168Y; M197X, in particular Y,F,L,I,T,A,G.

O In a preferred embodiment a variant of the invention V) comprises the following mutations corresponding to the following 0 io mutations in the amino acid sequence shown in SEQ ID NO: 4: T49X+A52X+V54N/I/L/Y/F/W+G107A, and may further comprise G108A.

In a preferred embodiment a variant of the invention comprises at least one mutation corresponding to the following mutations in the amino acid sequence shown in SEQ ID NO: 4: T49L+G107A; T49I+G107A; T49L+G107A+V54I; T49I+G107A+V54I; A52S+V54N+T49L+G107A; A52S+V54I+T49L+G107A; A52S+T49L+G107A; A52T+T49L+G107A; A52S+V54N+T49I+G107A; A52S+V54I+T49I+G107A; A52S+T49I+G107A; T49L+G108A; T49I+G108A; T49L+G108A+V54I; T49I+G108A+V54I.

All of the above-mentioned variants of the invention have altered properties (meaning increased or decreased properties), in particular at least one of the following properties relative to the parent alpha-amylase: reduced ability to cleave a substrate close to the branching point, improved substrate 3s specificity and/or improved specific activity, altered substrate binding, altered thermal stability, altered pH/activity profile, altered pH/stability profile, altered stability towards i 12 oxidation, altered Ca 2 dependency.

00 Stability In the context of the present invention, mutations (including amino acid substitutions and/or deletions) of importance with respect to achieving altered stability, in particular improved stability higher or lower), at especially low pH pH 4-6) include any of the mutations

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listed in the in "Altered properties" section, above and the variants mentioned right below.

The following variants: Q360A,K; N102A, N326A,L, N190G, N190K; Y262A,K,E (using the BAN, SEQ ID N: 6, numbering) were also tested for pH stability. A preferred parent alphaamylase may be BA2 described above. The pH stability was determined as described in the "Materials Methods" section.

Ca 2 stability Altered Ca 2 stability means the stability 'of the enzyme under Ca 2 depletion has been improved, higher or lower stability. In the context of the present invention, mutations (including amino acid substitutions) of importance with respect to achieving altered Ca 2 stability, in particular improved Ca 2 stability, higher or lower stability, at especially low pH pH 4-6) include any of the mutations listed in the in "Altered properties'' section above.

Specific activity In a further aspect of the present invention, important mutations with respect to obtaining variants exhibiting altered specific activity, in particular increased or decreased specific activity, especially at temperatures from 60-100 0 C, preferably 70-95°C, especially 80-90 0 C, include any of the mutations listed in the in "Altered properties" section above.

The specific activity of LE174 and LE429 was determined to 16,000 NU/mg using the Phadebas® assay described in the 'Materials and Methods" section.

00 Altered cleavage pattern In the starch liquefaction process it is desirable to use an alpha-amylase, which is capable of degrading the starch molecules into long, branched oligosaccharides, rather than an 0 alpha-amylase, which gives rise to formation of shorter, l branched oligosaccharides (like conventional Termamyl-like alpha-amylases). Short, branched oligosaccharides (panose precursors) are not hydrolyzed satisfactorily by pullulanases, which are used after alpha-amylase treatment in the liquefaction process, or simultaneously with a saccharifying amyloglucosidase (glucoamylase), or before adding a saccharifying amyloglucosidase (glucoamylase). Thus, in the presence of panose precursors, the product mixture present after the glucoamylase treatment contains a significant proportion of short, branched, so-called limit-dextrin, viz. the trisaccharide panose. The presence of panose lowers the saccharification yield significantly and is thus undesirable.

It has been reported previously (US patent 5,234,823) that, when saccharifying with glucoamylase and pullulanase, the presence of residual alpha-amylase activity arising from the liquefaction process, can lead to lower yields of glucose, if the alpha-amylase is not inactivated before the saccharification stage. This inactivation can be typically carried out by adjusting the pH to below 4.7 at 950C, before lowering the temperature to 600C for saccharification.

The reason for this negative effect on glucose yield is not fully understood, but it is assumed that the liquefying alpha-amylase (for example Termamyl 120 L from B.licheniformis) generates "limit dextrins" (which are poor substrates for pullulanase), by hydrolysing 1,4-alphaglucosidic linkages close to and on both sides of the branching points in amylopectin. Hydrolysis of these limit dextrins by glucoamylase leads to a build up of the trisaccharide panose, which is only slowly hydrolysed by D 14 glucoamylase.

The development of a thermostable alpha-amylase, which 00 does not suffer from this disadvantage, would be a significant improvement, as no separate inactivation step would be required.

Thus, the aim of the present invention is to arrive at a mutant alpha-amylase having appropriately modified starch- O degradation characteristics but retaining the thermostability of tIt the parent Termamyl-like alpha-amylase.

Accordingly, the invention relates to a variant of a Termamyl-like alpha-amylase, which has an improved reduced ability to cleave a substrate close to the branching point, and further has improved substrate specificity and/or improved specific activity.

is Of particular interest is a variant, which cleaves an amylopectin substrate, from the reducing end, more than one glucose unit from the branching point, preferably more than two or three glucose units from the branching point, at a further distance from the branching point than that obtained by use of a wild type B. licheniformis alpha-amylase.

It may be mentioned here that according to WO 96/23874, variants comprising at least one of the following mutations are expected to prevent cleavage close to the branching point: V54L,I,F,Y,W,R,K,H,E,Q; D53L,I,F,Y,W; Y56W; Q333W; G57,all possible amino acid residues; A52, amino acid residues larger than A, A52W,Y,L,F,I.

Mutations of particular interest in relation to obtaining variants according to the invention having an improved reduced ability to cleave a substrate close to the branching point, and further has improved substrate specificity and/or improved specific activity include mutations at the following positions in B. licheniformis alpha-amylase, SEQ ID NO: 4: H156, A181, N190, A209, Q264 and 1201.

It should be emphazised that not only the Termamyl-like 0

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alpha-amylases mentioned specifically below may be used. Also other commercial Termamyl-like alpha-amylases can be used. An 00 unexhaustive list of such alpha-amylases is.the following: Alpha-amylases 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. Other commercial Termamyl-like B.

S licheniformis alpha-amylases are OptithermTM and TakathermTM C-i (available from Solvay), Maxamyl TM (available from Gist- O brocades/Genencor), Spezym AATM Spezyme Delta AA'" (available

C

(i 10 from Genencor), and KeistaseTM (available from Daiwa) All Termamyl-like alpha-amylase may suitably be used as backbone for preparing variants of the invention.

In a preferred embodiment of the invention the parent Termamyl-like alpha-amylase is a hybrid alpha-amylase of SEQ ID NO: 4 and SEQ ID NO: 6. Specifically, the parent hybrid Termamyl-like alpha-amylase may be a hybrid alpha-amylase comprising the 445 C-terminal amino acid residues of the B.

licheniformis alpha-amylase shown in SEQ ID NO: 4 and the 37 Nterminal amino acid residues of the mature alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6, which may suitably further have the following mutations: H156Y+A181T+Nl90F+A209V+Q264S (using the numbering in SEQ ID NO: This hybrid is referred to as LE174. The LE174 hybrid may be combined with a further mutation I201F to form a parent hybrid Termamyl-like alpha-amylase having the following mutations H156Y+A181T+N190F+A209V+Q264S+I201F (using SEQ ID NO: 4 for the numbering). This hybrid variant is shown in SEQ ID NO: 2 and is used in the examples below, and is referred to as LE429.

Also, LE174 or LE429 (SEQ ID NO: 2) or B. licheniformis alpha-amylase shown in SEQ ID NO: 4 comprising one or more of the following mutations may be used as backbone (using SEQ ID NO: 4 for the numbering of the mutations): E119C; S130C; D124C; R127C; 16 A52all possible amino acid residues; possible amino acid residues; 00 N96all possible amino acid residues; 0 V129all possible amino acid residues; s A269all possible amino acid residues; A378all possible amino acid residues; Q AS148all possible amino acid residues, in particular S148N; E211all possible amino acid residues, in particular E211Q; V N188all possible amino acid residues, in particular N188S, N188P 0 10 M197all possible amino acid residues, in particular M197T, M197A, M197G, M197I, M197L, M197Y, M197F, M197I; W138all possible amino acid residues, in particular W138Y; D207all possible amino acid residues, in particular D207Y; H133all possible amino acid residues, in particular H133Y; H205all possible amino acid residues, in particular H205H, H205C, H205R; S187all possible amino acid residues, in particular S187D; A210all possible amino acid residues, in particular A210S, A210T; H405all possible amino acid residues, in particular H405D; K176all possible amino acid residues, in particular K176R; F279all possible amino acid residues, in particular F279Y; Q298all possible amino acid residues, in particular Q298H; G299all possible amino acid residues, in particular G299R; L308all possible amino acid residues, in particular L308F; T412all possible amino acid residues, in particular T412A; Further, B. licheniformis alpha-amylase shown in SEQ ID NO: 4 comprising at least one of the following mutations may be used as backbone: M15all possible amino acid residues; A33all possible amino acid residues; When using LE429 (shown in SEQ ID NO: 2) as the backbone as the parent Termamyl-like alpha-amylase) by combining LE174 with the mutation I201F (SEQ ID NO: 4 numbering), the mutations/alterations, in particular substitutions, deletions and insertions, may according to the invention be made in one or more of the following positions to improve the reduced ability 17 to cleave a substrate close to the branching point, and to S improve substrate specificity and/or improved specific activity: 00 W13, G48, T49, S50, Q51, A52, D53, V54, G57, G107, G108, All1, S168, M197 (using the SEQ ID NO: 4 numbering) s wherein the alteration(s) are independently S(i) an insertion of an amino acid downstream of the amino acid which occupies the position, O (ii) a deletion of the amino acid which occupies the VS position, or (iii) a substitution of the amino acid which occupies the position with a different amino acid, the variant has alpha-amylase activity and each position corresponds to a position of the amino acid sequence of the parent Termamyl-like alpha-amylase having the amino acid sequence of SEQ ID NO: 4.

In a preferred embodiment a variant of the invention comprises at least one mutation in a position corresponding to the following mutations in the amino acid sequence shown in SEQ ID NO: 4: V54N, A52S, A52S+V54N, T49L, T49+G107A, A52S+V54N+T49L+G107A, A52S+V54N+T49L, G107A, Q51R, Q51R+A52S, A52N; or T49F+G107A, T49V+G107A, T49D+G107A, T49Y+G107A, T49S+G107A, T49N+G107A, T49I+G107A, T49L+A52S+G107A, T49L+A52T+G107A, T49L+A52F+G107A, T49L+A52L+G107A, T49L+A52I+G107A, T49L+A52V+G107A; or T49V, T49I, T49D, T49N, T49S, T49Y, T49F, T49W, T49M, T49E, T49Q, T49K, T49R, A52T, A52L, A52I, A52V, A52M, A52F, A52Y, A52W, V54M, G107V, G07I, G107L, G107C.

In a preferred embodiment a variant of the invention comprises at least one mutation in a position corresponding to the following mutations in the amino acid sequence shown in SEQ ID NO: 4: W13F,L,I,V,Y,A; G48A,V,S,T,I,L; *48aD or *48aY insertion of D or Y); T49X; *49aX insertion of any amino acid residue) 18 SBOX, in particular D,Y,L,T,V,I; Q51R,K; 00 A52X, in particular A52S,N,T,F,L,I,V; D53E,Q,Y, I,N,S,T,V,L; V54X, in particular V541,N,W,Y,F,L; G57S,A,V,L, I,F,Y,T; G107X, in particular G1O7A,V,S,T,I,L,C; G1OBX, in particular GlO8A,V,S,T,I,L.; A111V,I,L; io1 S1G8Y; M197X, in particular Y,F,L,I,T,A,G.

In a preferred embodiment a variant of the invention comprises at least one mutation in a position corresponding to the following mutations in the amino acid sequence shown in SEQ is ID NO: 4: T49X+A52X+V54N/I/L/Y/F/W+GlO7A, and may further comprise GlOBA.

In a preferred embodiment a variant of the invention comprises at least one mutation in a position corresponding to the following mutations in the amino acid sequence shown in SEQ ID NO: 4: T49L+GlO7A; T491+GlO7A; T49L1+GlO7A+V54I; T491+G1O7A+V541; A52S+V54N+T49L+GlO7A; A52S+VS4I+T49L+G1O7A; A52S+T49L+GlO7A; A52T+T4 9L+GlO7A; A52S-iV54N+T4 91+Gl 07A; A52S+V54I+T49I-G1O7A; A52S+T491+GlO7A; T49L+G1O8A; T49I+GlOBA; T49L+G1O8A+V541; T49I-iGlO8A+V54I.

General mutations in variants of the invention It may be preferred that a variant of the invention comprises one or more modifications in addition to those 00 outlined above. Thus, it may be advantageous that one or more proline residues present in the part of the alpha-amylase s 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, 0 glycine, serine, threonine, valine or leucine.

tV Analogously, it may be preferred that one or more cysteine Sio residues present among the amino acid residues with which the parent alpha-amylase is modified is/are replaced with a noncysteine residue such as serine, alanine, threonine, glycine, valine or leucine.

Furthermore, 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. 4 is replaced by an Asn and/or Gln, 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: 4 by an Arg.

It will be understood that the present invention encompasses variants incorporating two or more of the above outlined modifications.

Furthermore, it may be advantageous to introduce pointmutations in any of the variants described herein.

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

D Cloning a DNA sequence encoding an alpha-amylase The DNA sequence encoding a parent alpha-amylase may be 00 isolated from any cell or microorganism producing the alphaamylase in question, using various methods well known in the art. First, 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. Then, if S the amino acid sequence of the alpha-amylase is known, l homologous, labelled oligonucleotide probes may be synthesized and used to identify alpha-amylase-encoding clones from a genomic library prepared from the organism in question. Alternatively, a labelled 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 alphaamylase-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.

Alternatively, 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 (1981) or the method described by Matthes et al.

(1984). In the phosphoroamidite method, oligonucleotides are synthesized, in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, 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 C 21 et al. (1988) 00 Site-directed mutagenesis Once an alpha-amylase-encoding DNA sequence has been isolated, and desirable sites for mutation identified, mutations M\ may be introduced using synthetic oligonucleotides. These oligo- C( nucleotides contain nucleotide sequences flanking the desired c mutation sites; mutant nucleotides are inserted during oligotn nucleotide synthesis. In a specific method, a single-stranded o0 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. The remaining gap is then filled in with DNA polymerase I (Klenow fragment) and the construct is ligated using T4 ligase. A specific example of this method is described in Morinaga et al.

(1984). US 4,760,025 disclose the introduction of oligonucleotides encoding multiple mutations by performing minor alterations of the cassette. However, an even greater variety of mutations can be introduced at any one time by the Morinaga method, because a multitude of oligonucleotides, of various lengths, can be introduced.

Another method for introducing mutations into alpha-amylaseencoding DNA sequences is described in Nelson and Long (1989) It involves the 3-step generation of a PCR fragment containing the desired mutation introduced by using a chemically synthesized DNA strand as one of the primers in the PCR reactions.

From the PCR-generated fragment, a DNA fragment carrying the mutation may be isolated by cleavage with restriction endonucleases and reinserted into an expression plasmid.

Random Mutagenesis Random mutagenesis is suitably performed either as localised or region-specific random mutagenesis in at least three parts of the gene translating to the amino acid sequence shown in question, or within the whole gene.

The random mutagenesis of a DNA sequence encoding a parent alpha-amylase may be conveniently performed by use of any method known in the art.

0 In relation to the above, a further aspect of the present invention relates to a method for generating a variant of a s parent alpha-amylase, wherein the variant exhibits a Sreduced capability of cleaving an oligo-saccharide substrate close to the branching point, and further exhibits improved 0 substrate specificity and/or improved specific activity ln relative to the parent, the method: o(a) subjecting a DNA sequence encoding the parent alphaamylase to random mutagenesis, expressing the mutated DNA sequence obtained in step (a) in a host cell, and screening for host cells expressing an alpha-amylase is variant which has an altered property thermal stability) relative to the parent alpha-amylase.

Step of the above method of the invention is preferably performed using doped primers. For instance, the random mutagenesis may be performed by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the random mutagenesis may be performed by use of any combination of these mutagenizing agents. The mutagenizing agent may, be one, which induces transitions, transversions, inversions, scrambling, deletions, and/or insertions.

Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) ir-radiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 0methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed by incubating the DNA sequence encoding the parent enzyme to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions for the mutagenesis to take place, and selecting for mutated DNA having the desired properties. When the mutagenesis is performed by the use of an oligonucleotide, the oligonucleotide may be doped or spiked with the three non-parent nucleotides during the synthesis of the 00 oligonucleotide at the positions, which are to be changed. The doping or spiking may be done so that codons for unwanted amino s acids are avoided. The doped or spiked oligonucleotide can be 4 incorporated into the DNA encoding the alpha-amylase enzyme by any published technique, using PCR, LCR or any DNA O polymerase and ligase as deemed appropriate. Preferably, the in doping is carried out using "constant random doping", in which O 10 the percentage of wild type and mutation in each position is predefined. Furthermore, the doping may be directed toward a preference for the introduction of certain nucleotides, and thereby a preference for the introduction of one or more specific amino acid residues. The doping may be made, so as to allow for the introduction of 90% wild type and mutations in each position. An additional consideration in the choice of a doping scheme is based on genetic as well as protein-structural constraints. The doping scheme may be made by using the DOPE program, which, inter alia, ensures that introduction of stop codons is avoided. When PCR-generated mutagenesis is used, either a chemically treated or non-treated gene encoding a parent alpha-amylase is subjected to PCR under conditions that increase the mis-incorporation of nucleotides (Deshler 1992; Leung et al., Technique, Vol.1, 1989, pp. 11-15).

A mutator strain of E. coli (Fowler et al., Molec. Gen. Genet., 133, 1974, pp. 179-191), S. cereviseae or any other microbial organism may be used for the random mutagenesis of the DNA encoding the alpha-amylase by, transforming a plasmid containing the parent glycosylase into the mutator strain, growing the mutator strain with the plasmid and isolating the mutated plasmid from the mutator strain. The mutated plasmid may be subsequently transformed into the expression organism.

The DNA sequence to be mutagenized may be conveniently present in a genomic or cDNA library prepared from an organism expressing the parent alpha-amylase. Alternatively, the DNA sequence may be present on a suitable vector such as a plasmid or a bacteriophage, which as such may be incubated with or otherwise exposed to the mutagenising agent. The DNA to be mutagenized may also be present in a host cell either by being 00 integrated in the genome of said cell or by being present on a vector harboured in the cell. Finally, the DNA to be mutagenized may be in isolated form. It will be understood that the DNA \O sequence to be subjected to random mutagenesis is preferably a cDNA or a genomic DNA sequence. In some cases it may be 0 convenient to amplify the mutated DNA sequence prior to performing the expression step b) or the screening step Such S1o amplification may be performed in accordance with methods known S in the art, the presently preferred method being PCR-generated amplification using oligonucleotide primers prepared on the basis of the DNA or amino acid sequence of the parent enzyme.

Subsequent to the incubation with or exposure to the mutagenising agent, the mutated DNA is expressed by culturing a suitable host cell carrying the DNA sequence under conditions allowing expression to take place. The host cell used for this purpose may be one which has been transformed with the mutated DNA sequence, optionally present on a vector, or one which was carried the DNA sequence encoding the parent enzyme during the mutagenesis treatment. Examples of suitable host cells are the following: gram positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, Streptomyces lividans or Streptomyces murinus; and gram-negative bacteria such as E. coli. The mutated DNA sequence may further comprise a DNA sequence encoding functions permitting expression of the mutated DNA sequence.

Localised random mutagenesis 00 The random mutagenesis may be advantageously localised to a part of the parent alpha-amylase in question. This may, be advantageous when certain regions of the enzyme have been k\ identified to be of particular importance for a given property of the enzyme, and when modified are expected to result in a O variant having improved properties. Such regions may normally be Vt identified when the tertiary structure of the parent enzyme has i0 been elucidated and related to the function of the enzyme.

The localised, or region-specific, random mutagenesis is conveniently performed by use of PCR generated mutagenesis techniques as described above or any other suitable technique known in the art. Alternatively, the DNA sequence encoding the part of the DNA sequence to be modified may be isolated, e.g., by insertion into a suitable vector, and said part may be subsequently subjected to mutagenesis by use of any of the mutagenesis methods discussed above.

Alternative methods of providing alpha-amylase variants Alternative methods for providing variants of the invention include gene-shuffling method known in the art including the methods described in WO 95/22625 (from Affymax Technologies and WO 96/00343 (from Novo Nordisk A/S).

Expression of alpha-amylase variants According to the invention, 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 0 26 host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, a vector, which 00 exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or San artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the 0 host cell genome and replicated together with the chromosome(s) ln into which it has been integrated.

In the vector, 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. Examples of as 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, the promoters of the Bacillus licheniformis alphaamylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens alpha-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral alphaamylase, 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, polyadenylation 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 S such sequences are the origins of replication of plasmids pUC19, 00 pACYC177, pUB110, pE194, pAMB1 and pIJ702.

The vector may also comprise a selectable marker, a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B. licheniformis, or one which confers antibiotic resistance such as ampicillin, 0 kanamycin, chloramphenicol or tetracyclin resistance. Fur- Vt thermore, 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, as described in WO 91/17243.

While intracellular expression may be advantageous in some respects, when using certain bacteria as host cells, it is generally preferred that the expression is extracellular. In general, the Bacillus alpha-amylases mentioned herein comprise a pre-region permitting secretion of the expressed protease into the culture medium. If desirable, this pre-region may be replaced by a different preregion or signal sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective preregions.

The procedures used to ligate the DNA construct of the invention encoding an alpha-amylase variant, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989).

The cell of the invention, either comprising a DNA construct or an expression vector of the invention as defined above, is advantageously used as a host cell in the recombinant production of an alpha-amylase variant of the invention. The cell may be transformed with the DNA construct of the invention encoding the variant, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, by homologous or 00 heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.

SThe cell of the invention may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial 0 cell, a bacterial or a fungal (including yeast) cell.

V Examples of suitable bacteria are gram-positive bacteria io 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.favourably be selected from a species of Saccharomyces or Schizosaccharomyces, Saccharomyces cerevisiae. The filamentous fungus may advantageously belong to a species of Aspergillus, 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.

In yet a further aspect, the present invention relates to a method of producing an alpha-amylase variant of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the variant and recovering the variant from the cells and/or culture medium.

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

O0 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 S components of the medium by means of a salt such as ammonium I sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

Industrial applications The alpha-amylase variants of this invention possess valuable properties allowing for a variety of industrial applications. In particular, enzyme variants of the invention are applicable as a component in washing, dishwashing and hard surface cleaning detergent compositions. Numerous variants are particularly useful in the production of sweeteners and ethanol, fuel, drinking or industrial ethanol, from starch, and/or for textile desizing. Conditions for conventional starchconversion processes, including starch liquefaction and/or saccharification processes, are described in, US 3,912,590 and in EP patent publications Nos. 252 730 and 63 909.

Production of sweeteners from starch: A "traditional" process for conversion of starch to fructose syrups normally consists of three consecutive enzymatic processes, viz. a liquefaction process followed by a saccharification process and an isomerization process. During the liquefaction process, starch is degraded to dextrins by an alpha-amylase Termamyl

T

at pH values between 5.5 and 6.2 and at temperatures of 95-160 0 C for a period of approx. 2 hours.

In order to ensure optimal enzyme stability under these conditions, 1 mM of calcium is added (40 ppm free calcium ions).

After the liquefaction process the dextrins are converted into dextrose by addition of a glucoamylase AMG

TM

and a debranching enzyme, such as an isoamylase or a pullulanase Promozyme'). Before this step the pH is reduced to a O value below 4.5, maintaining the high temperature (above 950C) 00 and the liquefying alpha-amylase activity is denatured. The tems perature is lowered to 600C, and glucoamylase and debranching enzyme are added. The saccharification process proceeds for 24-72 hours.

Q After the saccharification process the pH is increased to a C value in the range of 6-8, preferably pH 7.5, and the calcium is removed by ion exchange. The dextrose syrup is then converted cq into high fructose syrup using, an immmobilized glucoseisomerase (such as SweetzymeM).

At least one enzymatic improvement of this process could be envisaged: Reduction of the calcium dependency of the is liquefying alpha-amylase. Addition of free calcium is required to ensure adequately high stability of the alpha-amylase, but free calcium strongly inhibits the activity of the glucoseisomerase and needs to be removed, by means of an expensive unit operation, to an extent, which reduces the level 2c of free calcium to below 3-5 ppm. Cost savings could be obtained if such an operation could be avoided and the liquefaction process could be performed without addition of free calcium ions.

To achieve that, a less calcium-dependent Termamyl-like alpha-amylase which is stable and highly active at low concentrations of free calcium 40 ppm) is required. Such a Termamyl-like alpha-amylase should have a pH optimum at a pH in the range of 4.5-6.5, preferably in the range of 4.5-5.5.

The invention also relates to a composition comprising a 3: mixture of one or more variants of the invention derived from (as the parent Termamyl-like alpha-amylase) the B.

stearothermophilus alpha-amylase having the sequence shown in SEQ ID NO: 8 and a Termamyl-like alpha-amylase derived from the B. licheniformis alpha-amylase having the sequence shown in SEQ ID NO: 4.

Further, the invention also relates to a composition comprising a mixture of one or more variants according the 0 31

(N

invention derived from (as the parent Termamyl-like alphaamylase) the B. stearothermophilus alpha-amylase having the O sequence shown in SEQ ID NO: 8 and a hybrid alpha-amylase comprising a part of the B. amyloliquefaciens alpha-amylase shown in SEQ ID NO: 6 and a part of the B. licheniformis alphaamylase shown in SEQ ID NO: 4. The latter mentioned hybrid Termamyl-like alpha-amylase comprises the 445 C-terminal amino 0 acid residues of the B. licheniformis alpha-amylase shown in SEQ ln ID NO: 4 and the 37 N-terminal amino acid residues of the alphao0 amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6.

Said latter mentioned hybrid alpha-amylase may suitably comprise the following mutations: H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 4) Preferably, said latter mentioned hybrid alpha-amylase may suitably comprise the following s1 mutations: H156Y+Al81T+N190F+A209V+Q264S+I201F (using the SEQ ID NO: 4 numbering). In the examples below said last-mentioned parent hybrid Termamyl-like alpha-amylase referred to as LE429 (shown in SEQ ID NO: 2) is used for preparing variants of the invention, which variants may be used in compositions of the invention.

An alpha-amylase variant of the invention or a composition of the invention may in an aspect of the invention be used for starch liquefaction, in detergent composition, such as laundry, dish wash compositions and hard surface cleaning, ethanol production, such as fuel, drinking and industrial ethanol production, desizing of textile, fabric and garments.

MATERIALS AND METHODS Enzymes: LE174: hybrid alpha-amylase variant: LE174 is a hybrid Termamyl-like alpha-amylase being identical to the Termamyl sequence, the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 4, except that the Nterminal 35 amino acid residues (of the mature protein) has been replaced by the N-terminal 33 residues of BAN (mature protein), the Bacillus amyloliquefaciens alpha-amylase shown in SEQ ID NO: 6, which further have following mutations: D 32 SH156Y+A181T+N190F+A209V+Q264S (SEQ ID NO: 4).

00 LE429 hybrid alpha-amylase variant: LE429 is a hybrid Termamyl-like alpha-amylase being identical to the Termamyl sequence, the Bacillus licheniformis Salpha-amylase shown in SEQ ID NO: 4, except that the Nterminal 35 amino acid residues (of the mature protein) has 0 been replaced by the 'N-terminal 33 residues of BAN (mature -i protein), the Bacillus amyloliquefaciens alpha-amylase shown in SEQ ID NO: 6, which further have following mutations: (C H156Y+A181T+N190F+A209V+Q264S+I201F (SEQ ID NO: LE429 is shown as SEQ ID NO: 2 and was constructed by SOE-PCR (Higuchi et al. 1988, Nucleic Acids Research 16:7351).

Dextrozyme T E: a balanced mixture of glucoamylase (AMG) and pullulanase obtainable from selected strains of Aspergillus niger and Bacillus deramificans (available from Novo Nordisk

A/S)

Fermentation and purification of alpha-amylase variants A B. subtilis strain harbouring the relevant expression plasmid is streaked on an LB-agar plate with 10 micro g/ml kanamycin from -80 0 C stock, and grown overnight at 37 0

C.

The colonies are transferred to 100 ml BPX media supplemented with 10 micro g/ml kanamycin in a 500 ml shaking flask.

Composition of BPX medium: Potato starch 100 g/l Barley flour 50 g/l BAN 5000 SKB 0.1 g/l Sodium caseinate 10 g/l Soy Bean Meal 20 g/l NaHPO 4 12 H0O 9 g/l Pluronicm 0.1 g/l The culture is shaken at 37 0 C at 270 rpm for 5 days.

Cells and cell debris are removed from the fermentation broth by centrifugation at 4500 rpm in 20-25 minutes. Afterwards the supernatant is filtered to obtain a completely clear i33 >solution. The filtrate is concentrated and washed on an UFfilter (10000 cut off membrane) and the buffer is changed to OO 20mM Acetate pH 5.5. The UF-filtrate is applied on a S-sepharose F.F. and elution is carried out by step elution with 0.2M NaCl in the same buffer. The eluate is dialysed against 10mM Tris, pH 9.0 and applied on a Q-sepharose F.F. and eluted with a linear gradient from 0-0.3M NaCl over 6 column volumes. The fractions 0 that contain the activity (measured by the Phadebas assay) are In pooled, pH was adjusted to pH 7.5 and remaining color was Sac removed by a treatment with 0.5% W/vol. active coal in minutes.

Activity determination

(KNU)

One Kilo alpha-amylase Unit (1 KNU) is the amount of enzyme which breaks down 5.26 g starch (Merck, Amylum Solubile, Erg. B 6, Batch 9947275) per hour in Novo Nordisk's standard method for determination of alpha-amylase based upon the following condition: Substrate soluble starch Calcium content in solvent 0.0043 M Reaction time 7-20 minutes Temperature 37 0

C

pH 5.6 Detailed description of Novo Nordisk's analytical method (AF 9) is available on request.

Assay for Alpha-Amylase Activity Alpha-Amylase activity is determined by a method employing Phadebas® tablets as substrate. Phadebas tablets (Phadebas® Amylase Test, supplied by Pharmacia Diagnostic) contain a crosslinked insoluble blue-coloured starch polymer, which has been mixed with bovine serum albumin and a buffer substance and tabletted.

For every single measurement one tablet is suspended in a tube containing 5 ml 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, 0.1 mM CaC1,, pH adjusted to the value of interest with NaOH). The test is 0 34 performed in a water bath at the temperature of interest. The alpha-amylase to be tested is diluted in x ml of 50 mM Britton- 0O Robinson buffer. 1 ml of this alpha-amylase solution is added to the 5 ml 50 mM Britton-Robinson buffer. The starch is hydrolysed by the alpha-amylase giving soluble blue fragments. The absorbance of the resulting blue solution, measured F- spectrophotometrically at 620 nm, is a function of the alpha- 0 amylase activity.

I It is important that the measured 620 nm absorbance after 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 alphaamylase will hydrolyse 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.

Determining Specific Activity The specific activity is determined using the Phadebas assay (Pharmacia) as activity/mg enzyme.

Measuring the pH activity profile (pH stability) The variant is stored in 20 mM TRIS ph 7.5, 0.1 mM, CaC1 2 and tested at 300C, 50 mM Britton-Robinson, 0.1 mM CaC1 2 The pH activity is measured at pH 4.0, 4.5, 5.0, 5.5, 6.0, 8.0, 9.5, 9.5, 10, and 10.5, using the Phadebas assay described above.

Determination Of AGU Activity and As AGU/mg One Novo Amyloglucosidase Unit (AGU) is defined as the amount of enzyme, which hydrolyzes 1 micromole maltose per minute at 37 0 C and pH 4.3. A detailed description of the analytical method (AEL-SM-0131) is available on request from Novo Nordisk.

00 The activity is determined as AGU/ml by a method modified after (AEL-SM-0131) using the Glucose GOD-Perid kit from Boehringer Mannheim, 124036. Standard: AMG-standard, batch 7- ND 1195, 195 AGU/ml.

C-i 375 microL substrate maltose in 50 mM Sodium acetate, C( pH 4.3) is incubated 5 minutes at 370C. 25 microL enzyme diluted in sodium acetate is added. The reaction is stopped Sio after 10 minutes by adding 100 microL 0.25 M NaOH. 20 microL is transferred to a 96 well microtitre plate and 200 microL GOD-Perid solution is added. After 30 minutes at room temperature, the absorbance is measured at 650 nm and the activity calculated in AGU/ml from the AMG-standard.

The specific activity in AGU/mg is then calculated from the activity (AGU/ml) divided with the protein concentration (mg/ml).

EXAMPLES

EXAMPLE 1 Construction of Termamyl variants in accordance with the invention Termamyl licheniformis alpha-amylase SEQ ID NO: 4) is expressed in B. subtilis from a plasmid denoted pDN1528. This plasmid 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 pUB110 and the cat gene from plasmid pC194 conferring resistance towards chloramphenicol. pDN1528 is shown in Fig. 9 of WO 96/23874. A specific mutagenesis vector containing a major part of the coding region of SEQ ID NO: 3 was prepared.

The important features of this vector, denoted pJeEN1, include an origin of replication derived from the pUC plasmids, the cat 3E gene conferring resistance towards chloramphenicol, and a frameshift-containing version of the bla gene, the wild type of which normally confers resistance towards ampicillin (amp' 0 36 phenotype). This mutated version results in an amps phenotype.

SThe plasmid pJeEN1 is shown in Fig. 10 of WO 96/23874, and the E. coli origin of replication, ori, bla, cat, the 00 version of the Termamyl amylase gene, and selected restriction s sites are indicated on the plasmid.

SMutations are introduced in amyL by the method described by S Deng and Nickoloff (1992, Anal. Biochem. 200, pp. 81-88) except 0 that plasmids with the "selection primer" (primer #6616; see S below) incorporated are selected based on the amp' phenotype of transformed E. coli cells harboring a plasmid with a repaired CI( bla gene, instead of employing the selection by restriction enzyme digestion- outlined by Deng and Nickoloff. Chemicals and enzymes used for the mutagenesis were obtained from the ChameleonO mutagenesis kit from Stratagene (catalogue number 200509).

After verification of the DNA sequence in variant plasmids, the truncated gene, containing the desired alteration, is subcloned into pDN1528 as a PstI-EcoRI fragment and transformed into the protease- and amylase-depleted Bacillus subtilis strain SHA273 (described in W092/11357 and W095/10603) in order to express the variant enzyme.

The Termamyl variant V54W was constructed by the use of the following mutagenesis primer (written 5' to left to right): PG GTC GTA GGC ACC GTA GCC CCA ATC CGC TTG (SEQ ID NO: 9) The Termamyl variant A52W V54W was constructed by the use of the following mutagenesis primer (written 5' to left to right): PG GTC GTA GGC ACC GTA GCC CCA ATC CCA TTG GCT CG (SEQ ID NO: Primer #6616 (written 5' to left to right; P denotes a phosphate): P CTG TGA CTG GTG AGT ACT CAA CCA AGT C (SEQ ID NO: 11) The Termamyl variant V54E was constructed by the use of the following mutagenesis primer (written left to right): PGG TCG TAG GCA CCG TAG CCC TCA TCC GCT TG (SEQ ID NO: 12) 37 SThe Termamyl variant V54M was constructed by the use of the following mutagenesis primer (written left to 00 right): PGG TCG TAG GCA CCG TAG CCC ATA TCC GCT TG (SEQ ID NO: 13) The Termamyl variant V54I was constructed by the use of S the following mutagenesis primer (written left to right): PGG TCG TAG GCA CCG TAG CCA ATA TCC GCT TG (SEQ ID NO: 14) V) The Termamyl variants Y290E and Y290K were constructed by io the use of the following mutagenesis primer (written left to right): PGC AGC ATG GAA CTG CTY ATG AAG AGG CAC GTC AAA C (SEQ ID Y represents an equal mixture of C and T. The presence of a is codon encoding either Glutamate or Lysine in position 290 was verified by DNA sequencing.

The Termamyl variant N190F was constructed by the use of the following mutagenesis primer (written left to right): PCA TAG TTG CCG AAT TCA TTG GAA ACT TCC C (SEQ ID NO: 16) The Termamyl variant N188P+N190F was constructed by the use of the following mutagenesis primer (written left to right): PCA TAG TTG CCG AAT TCA GGG GAA ACT TCC CAA TC (SEQ ID NO: 17) The Termamyl variant H140K+H142D was constructed by the use of the following mutagenesis primer (written left to right): PCC GCG CCC CGG GAA ATC AAA TTT TGT CCA GGC TTT AAT TAG (SEQ ID NO: 18) The Termamyl variant H156Y was constructed by the use of the following mutagenesis primer (written left to right): PCA AAA TGG TAC CAA TAC CAC TTA AAA TCG CTG (SEQ ID NO: 19) The Termamyl variant A181T was constructed by the use of the following mutagenesis primer (written left to right): PCT TCC CAA TCC CAA GTC TTC CCT TGA AAC (SEQ ID NO: The Termamyl variant A209V was constructed by the use of the following mutagenesis primer (written left to 00 right): PCTT AAT TTC TGC TAC GAC GTC AGG ATG GTC ATA ATC (SEQ ID NO: 21) SThe Termamyl variant Q264S was constructed by the use of the following mutagenesis primer (written left to 0 right) SPCG CCC AAG TCA TTC GAC CAG TAC TCA GCT ACC GTA AAC (SEQ ID NO: 22) C The Termamyl variant S187D was constructed by the use of the following mutagenesis primer (written left to right): PGC CGT TTT CAT TGT CGA CTT CCC AAT CCC (SEQ ID NO: 23) The Termamyl variant DELTA(K370-G371-D372) deleted of amino acid residues nos. 370, 371 and 372) was constructed by the use of the following mutagenesis primer (written left to right): PGG AAT TTC GCG CTG ACT AGT CCC GTA CAT ATC CCC (SEQ ID NO: 2C 24) The Termamyl variant DELTA(D372-S373-Q374) was constructed by the use of the following mutagenesis primer (written left to-right): PGG CAG GAA TTT CGC GAC CTT TCG TCC CGT ACA TAT C (SEQ ID NO: The Termamyl variants A181T and A209V were combined to A181T+A209V by digesting the A181T containing pDN1528-like plasmid pDN1528 containing within amyL the mutation resulting in the A181T alteration) and the A209V-containing pDN1528-like plasmid pDN1528 containing within amyL the mutation resulting in the A209V alteration) with restriction enzyme Clal which cuts the pDN1528-like plasmids twice resulting in a fragment of 1116 bp and the vector-part (i.e.

contains the plasmid origin of replication) of 3850 bp. The 3E fragment containing the A209V mutation and the vector part containing the A181T mutation were purified by QIAquick gel extraction kit (purchased from QIAGEN) after separation on an 0 39 agarose gel. The fragment and the vector were ligated and transformed into the protease and amylase depleted Bacillus O0 subtilis strain referred to above. Plasmid from amy+ (clearing zones on starch containing agar-plates) and chloramphenicol resistant transformants were analysed for the presence of both mutations on the plasmid.

In a similar way as described above, H156Y and A209V were 0 combined utilizing restriction endonucleases Acc65I and EcoRI, l giving H156Y+A209V.

H156Y +A209V and A181T+A209V were combined into H156Y+ A181T+A209V by the use of restriction endonucleases Acc65I and HindIII.

The 35 N-terminal residues of the mature part of Termamyl variant H156Y+ A181T+A209V were substituted by the 33 Nterminal residues of the B. amyloliquefaciens alpha-amylase (SEQ ID NO: 4) (which in the present context is termed BAN) by a SOE-PCR approach (Higuchi et al. 1988, Nucleic Acids Research 16:7351) as follows: Primer 19364 (sequence CCT CAT TCT GCA GCA GCA GCC GTA AAT GGC ACG CTG (SEQ ID NO: 26) Primer 19362: CCA GAC GGC AGT AAT ACC GAT ATC CGA TAA ATG TTC CG (SEQ ID NO: 27) Primer 19363: CGG ATA TCG GTA TTA CTG CCG TCT GGA TTC (SEQ ID NO: 28) Primer 1C: CTC GTC CCA ATC GGT TCC GTC (SEQ ID NO: 29) A standard PCR, polymerase chain reaction, was carried out using the Pwo thermostable polymerase from Boehringer Mannheim according to the manufacturer's instructions and the temperature cyclus: 5 minutes at 94°C, 25 cycles of (940C for 30 seconds, 500C for 45 seconds, 720C for 1 minute), 720C for minutes.

An approximately 130 bp fragment was amplified in a first PCR denoted PCR1 with primers 19364 and 19362 on a DNA fragment containing the gene encoding the B. amyloliquefaciens alpha-amylase.

An approximately 400 bp fragment was amplified in another PCR denoted PCR2 with primers 19363 and 1C on template pDN1528.

PCR1 and PCR2 were purified from an agarose gel and used as templates in PCR3 with primers 19364 and 1C, which resulted in a fragment of approximately 520 bp. This fragment thus contains one part of DNA encoding the N-terminus from BAN fused to a part of DNA encoding Termamyl from the 35th amino acid.

0 The 520 bp fragment was subcloned into a pDN1528-like In plasmid (containing the gene encoding Termamyl variant H156Y+ A181T+A209V) by digestion with restriction endonucleases PstI Cl and SacII, ligation and transformation of the B. subtilis strain as previously described. The DNA sequence between restriction sites PstI and SacII was verified by DNA sequencing in extracted plasmids from amy+ and chloramphenicol resistant transformants.

The final construct containing the correct N-terminus from BAN and H156Y+ A181T+A209V was denoted BAN(1-35)+ H156Y+ A181T+A209V.

N190F was combined with BAN(1-35)+ H156Y+ A181T+A209V giving BAN(1-35)+ H156Y+ A181T+N190F+A209V by carrying out mutagenesis as described above except that the sequence of amyL in pJeEN1 was substituted by the DNA sequence encoding Termamyl variant BAN(1-35)+ H156Y+ A181T+A209V Q264S was combined with BAN(1-35)+ H156Y+ A181T+A209V giving BAN(1-35)+ H156Y+ A181T+A209V+Q264S by carrying out mutagenesis as described above except that the sequence of amyL in pJeEN was substituted by the the DNA sequence encoding Termamyl variant BAN(1-35)+ H156Y+ A181T+A209V BAN(1-35)+ H156Y+ A181T+A209V+Q264S and BAN(1-35)+ H156Y+ A181T+N190F+A209V were combined into BAN(1-35)+ H156Y+ A181T+N190F+A209V+Q264S utilizing restriction endonucleases BsaHI (BsaHI site was introduced close to the A209V mutation) and PstI.

1201F was combined with BAN(1-35)+ H156Y+ A181T+N190F+A209V+Q264S giving BAN(1-35)+ H156Y+ A181T+N190F+A209V+Q264S+I201F (SEQ ID NO: 2) by carrying out mutagenesis as described above. The mutagenesis primer AM100 was used, introduced the I201F substitution and removed simultaneously a Cla I restriction site, which facilitates easy pin-pointing of mutants.

primer AM100: 3' (SEQ ID NO: EXAMPLE 2 Construction of Termamyl-like alpha-amylase variants with an altered cleavage pattern according to the invention The variant of the thermostable B. licheniformis alphaamylase consisting comprising the 445 C-terminal amino acid residues of the B. licheniformis alpha-amylase shown in SEQ ID NO: 4 and the 37 N-terminal amino acid residues of the alphais amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6, and further comprising the following mutations: H156Y+A181T+N190F+A209V+Q264S+I201F (the construction of this variant is described in Example 1, and the amino acid sequence shown in SEQ ID NO: 2) has a reduced capability of cleaving an substrate close to the branching point.

In an attempt to further improve the reduced capability of cleaving an substrate close to the branching point of said alpha-amylase variant site directed mutagenesis was carried out using the Mega-primer method as described by Sarkar and Sommer, 1990 (BioTechniques 8: 404-407): Construction of LE313: BAN/Termamyl hybrid H156Y+A181T+N190F+ A209V+Q264S+V54N: Gene specific primer 27274 and mutagenic primer AM115 are used to amplify by PCR an approximately 440 bp DNA fragment from a pDN1528-like plasmid (harbouring the BAN(1- 35)+H156Y+A181T+N190F+I201F+A209V+Q264S mutations in the gene encoding the amylase from SEQ ID NO: 4).

The 440 bp fragment is purified from an agarose gel and used as a Mega-primer together with primer 113711 in a second PCR carried out on the same template.

The resulting approximately 630 bp fragment is digested 0 42

(N

with restriction enzymes EcoR V and Acc65 I and the resulting approximately 370 bp DNA fragment is purified and ligated with 00 the pDN1528-like plasmid digested with the same enzymes.

Competent Bacillus subtilis SHA273 (amylase and protease low) cells are transformed with the ligation and Chlorampenicol Sresistant transformants are checked by DNA sequencing to verify the presence of the correct mutations on the plasmid.

(N

In Primer 27274: 5' CATAGTTGCCGAATTCATTGGAAACTTCCC 3' (SEQ ID NO: 31) Primer 1B: CCGATTGCTGACGCTGTTATTTGC 3' (SEQ ID NO: 32) primer AM115: GCCAAGCGGATAACGGCTACGGTGC 3' (SEQ ID NO:33) Construction of LE314: BAN/Termamyl hybrid H156Y+A181T+N190F+ A209V+Q264S A52S is carried our in a similar way, except that mutagenic primer AM116 is used.

AM116: GAACGAGCCAATCGGACGTGGGCTACGG 3' (SEQ ID NO: 34) Construction of LE315: BAN/Termamyl hybrid H156Y+A181T+N190F+ A209V+Q264S A52S+V54N is carried our in a similar way, except that mutagenic primer AM117 is used.

AM117: 5' GGAACGAGCCAATCGGATAACGGCTACGGTGC 3' (SEQ ID NO: Construction of LE316: BAN/Termamyl hybrid H156Y+A181T+N190F+ A209V+Q264S T49L is carried our in a similar way, except that mutagenic primer AM118 is used.

AM118: GCATATAAGGGACTGAGCCAAGCGG 3' (SEQ ID NO: 36) 0 43 Construction LE317: BAN/Termamyl hybrid H156Y+A181T+N190F+ OO A209V+Q264S T49L+G107A is carried our in a similar way, except that mutagenic primer AM118 and mutagenic primer AM119 s are used simultaneously.

\O

AM119: 0 5' CAACCACAAAGCCGGCGCTGATGCG 3' (SEQ ID NO: 37) In S1o Construction of LE318: BAN/Termamyl hybrid H156Y+A181T+N190F+ A209V+Q264S A52S+V54N+T49L+G107A is carried our in a similar way, except that mutagenic primer AM120 and mutagenic primer AM119 are used simultaneously.

AM120: GCATATAAGGGACTGAGCCAATCGGATAACGGCTACGGTGC 3' (SEQ ID NO: 38) Construction of LE 319: BAN/Termamyl hybrid H156Y+A181T+N190F+ A209V+Q264S A52S+V54N+T49L is carried our in a similar way, except that mutagenic primer AM120 is used.

Construction of LE320: BAN/Termamyl hybrid H156Y+A181T+N190F+ A209V+Q264S G107A is carried our in a similar way, except that mutagenic primer AM119 is used.

Construction of LE322: BAN/Termamyl hybrid H156Y+A181T+N190F+A209V+Q264S Q51R+A52S is carried our in a similar way, except that mutagenic primer AM121 is used.

AM121: GAACGAGCCGATCGGACGTGGGCTACGG 3' (SEQ ID NO:39) Construction of LE323: BAN/Termamyl hybrid H156Y+A181T+N190F+ A209V+Q264S A52N is carried our in a similar way, except that mutagenic primer AM122 is used.

AM122: GAACGAGCCAAAACGACGTGGGCTACGG 3' (SEQ ID NO: EXAMPLE 3 Testing of LE429 variants (saccharification) o 44 The standard reaction conditions were: Substrate concentration 30 w/w 00 Temperature 600C Initial pH (at 60 0 C) IN Enzyme dosage Glucoamylase 0.18 AGU/g DS Pullulanase 0.06 PUN/g DS Alpha-amylase 10 micro g enzyme/g DS Dextrozyme" E was used to provide glucoamylase and pullulanase activities Substrates for saccharification were prepared by dissolving common corn starch in deionized water and adjusting the dry substance to approximately 30% w/w. The pH was adjusted to 5.5 (measured at 600C), and aliquots of substrate corresponding to 10 g dry weight were transferred to blue cap glass flasks.

The flasks were then placed in a shaking water bath equilibrated at 60 0 C, and the enzymes added. The pH was readjusted to 5.5 where necessary. The samples were taken after 48 hours of saccharification; the pH was adjusted to about 3.0, and then heated in a boiling water bath for s1 minutes to inactivate the enzymes. After cooling, the samples were treated with approximately 0.1 g mixed bed ion exchange resin (BIO-RAD 501 X8 for 30 minutes on a rotary mixer to remove salts and soluble N. After filtration, the carbohydrate composition was determined by HPLC. The following results were obtained: The parent alpha-amylase for the variants is LE429.

UP

1

LP

2

DP

3

SPEC.

Added

ACT.

Alpha-amylase (NU/mg) Variants 4N I 1.7b 1.17- 8200 r7571+Vb4-N 69.3 1.T4 1.9 IU0000 00

O

oo 14 9 L+ Gl /A 7 1.8 UT72 I3UU Ab26+ Vb4N+14.L+Gu 0A EU5 2T5 .43 A52S+Vb4N+149L -9.8 16 0.b4 84UU GL U/A 4.4 1.89 f .U4 19600 QbiR+Ab 29b9 ITTT 1.2T Ab2N 51.89 1bb6 17 bUU LEi /4 (CONTROL) 9.W/ 1TT77 1600 95.8 1.83 1.35 Compared with the control, the presence of an active alpha-amylase variant of the invention during liquefaction results in decreased panose levels (DP3).

Especially the T49L+GlO7A variant of LE429 and the A52S+V54N+T49L variant of LE429, respectively, result in a drastically decreased panose level (DP 3 If these alphaamylase variants are used for starch liquefaction, it will not be necessary to inactivate the enzyme before the commencement ao of saccharification.

Example 4 Liquefaction and saccharification of LE429 variants The experiment in Example 3 was repeated for a number of as other LE429 variants under the same conditions.

The result is shown below: Variant/sugar profile DP1 DP2 DP3 DP4+ T49V+G107A 95.9% 1.72% 1.27% 1.11% T49Y+G1O7A 95.3% 1.73% 1.29% 1.65% T49N+G107A 95.7% 1.64% 1.51% 1.18% T49L+A52S+G107A 95.7% 1.73% 0.95% 1.67% T49L+A2T+G07A 95.8% 1.66% 1.03% 1.48% T49L+A52F+G07A 95.7% 1.69% 1.16% 1.42% T49L+A52L+G107A 95.5% 1.70% 1.40% 1.38% T49L+A521+G107A 95.9% 1.72% 1.31% 1.07% T49L+A52V+G07A 94.7% 1.69% 1.16% 2.44% 46 T49L+A52V+GIO7A+A11lV 94.5% 1.75% 0.72% 2.99% LE429 94.9% 1.71% 1.85% 1.51% 00 Example The experiment in Example 3 was repeated for a number of LE429 variants, except that the liquefaction was carried out at pH 6.0 and the saccharification at 60 0 C, pH 4.5, 40 ppm CaCd 2 followed by inactivation. The variant referred to below are LE429 variant. The results found are as follows: Variant/sugar profile DP4+ DP3 DP2 DPi T4 9F 1.15 0.92 1.83 96.12 T49D+GlO7A 0.84 1.03 1.82 96.3 T491+G1O7A 0.97 0.64 1.84 96.55 T49L+GlO7A 0.96 0.81 1.82 96.42 T4 9L+A52S+G1O7A 1.37 0.75 1.88 96.01 T49L+A52T+G1O7A 0.87 0.81 1.8 96.52 T49L+A52F+Gl07A 0.98 0.83 1.87 96.31 T49V+G107A 0.65 0.8 2.13 96.43 T49Y+G1O7A 0.83 0.94 1.89 96.35 LE429 1.16 1.21 1.77 95.87 0) 47 REFERENCES CITED OO Klein, et al., Biochemistry 1992, 31, 8740-8746, Mizuno, et al., J. Mol. Biol. (1993) 234, 1282-1283, Chang, et al, J. Mol. Biol. (1993) 229, 235-238, Larson, J. Mol. Biol. (1994) 235, 1560-1584, Lawson, J. Mol. Biol. (1994) 236, 590-600, 0 Qian, et al., J. Mol. Biol. (1993) 231, 785-799, l Brady, et al., Acta Crystallogr. sect. B, 47, 527-535, Swift, et al., Acta Crystallogr. sect. B, 47, 535-544 A. Kadziola, Ph.D. Thesis: "An alpha-amylase from Barley and its Complex with a Substrate Analogue Inhibitor Studied by X-ray Crystallography", Department of Chemistry University of Copenhagen 1993 is MacGregor, Food Hydrocolloids, 1987, Vol.1, No. 5-6, p.

B. Diderichsen and L. Christiansen, Cloning of a maltogenic amylase from Bacillus stearothermophilus, FEMS Microbiol. letters: 56: pp. 53-60 (1988) Hudson et al., Practical Immunology, Third edition (1989), Blackwell Scientific Publications, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989 S.L. Beaucage and M.H. Caruthers, Tetrahedron Letters 22, 1981, pp. 1859-1869 Matthes et al., The EMBO J. 3, 1984, pp. 801-805.

R.K. Saiki et al., Science 239, 1988, pp. 487-491.

Morinaga et al., (1984, Biotechnology 2:646-639) Nelson and Long, Analytical Biochemistry 180, 1989, pp. 147-151 Hunkapiller et al., 1984, Nature 310:105-111 3C R. Higuchi, B. Krummel, and R.K. Saiki (1988). A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucl. Acids Res.

16:7351-7367.

Dubnau et al., 1971, J. Mol. Biol. 56, pp. 209-221.

Gryczan et al., 1978, J. Bacteriol. 134, pp. 318-329.

S.D. Erlich, 1977, Proc. Natl. Acad. Sci. 74, pp. 1680-1682.

Boel et al., 1990, Biochemistry 29, pp. 6244-6249.

b 48 Sarkar and Sommer, 1990, BioTechnigues 8, pp. 404-407.

00

Claims (10)

1. An isolated variant of a parent Termamyl-like alpha-amylase, which variant has O an improved reduced ability to cleave a substrate close to a branching point, and further has improved substrate specificity and/or improved specific activity, comprising an alteration at one or more positions selected from the group of: G48, T49, G107, wherein C the alteration(s) are independently C an insertion of an amino acid downstream of the amino acid which Soccupies the position, C (ii) a deletion of the amino acid which occupies the position, or (iii) a substitution of the amino acid which occupies the position with a different amino acid, the variant has alpha-amylase activity, and each position corresponds to a position of the amino acid sequence of the parent Termamyl-like alpha-amylase having the amino acid sequence of SEQ ID NO:4. Is

2. The variant of claim 1, comprising a mutation in a position corresponding to at least one of the following mutations in the amino acid sequence shown in SEQ ID NO:4: T49L, T49+G107A, A52S+V54N+T49L+G107A, A52S+V54N+T49L, G107A; or T49F+G107A, T49V+G107A, T49D+G107A, T49Y+G107A, T49S+G107A, T49N+G107A, T49I+G107A, T49L+A52S+G107A, T49L+A52T+G107A, T49L+A52F+G107A, T49L+A52L+G107A, T49L+A52I+G107A, T49L+A52V+G107A; or T49V, T49I, T49D, T49N, T49S, T49Y, T49F, T49W, T49M, T49E, T49Q, T49K, T49R, G107V, G07I, G107L, G107C.

3. The variant of claims 1 or 2, comprising a mutation in a position corresponding to at least one of the following mutations in the amino acid sequence shown in SEQ ID NO:4: G48A,V,S,T,I,L; *48aD or *48aY insertion of D or Y); T49X; *49aX insertion of any amino acid residue) G107X, in particular G107A,V,S,T,I,L,C.

810755-1 gcc

4. The variant of any one of claims 1-3, comprising the following mutations con esponding to at least one of the following mutations in the amino acid sequence shown in SEQ ID NO:4: T49X+A52X+V54N/I/UY/F/W+G 107A.

5. The variant of claims 1-4, further comprising G108A.

6. The variant of claims 1-5, comprising the following mutations corresponding to at least one of the following mutations in the amino acid sequence shown in SEQ ID NO:4: T49L+G107A; T49I+G107A; o0 T49L+G107A+V54I; T49I+G107A+V54I; A52S+V54N+T49L+G107A; A52S+V54I+T49L+G 107A; A52S+T49L+G107A; A52T+T49L+G107A; A52S+V54N+T49I+G 107A; A52S+V541+T49I+G107A; A52S+T49I+G 07A; T49L+G108A; T49I+G 08A; T49L+G108A+V54I; T49I+G 108A+V54I.

7. A variant of any one of claims 1-6, wherein said variant has a reduced capability of cleaving an oligosaccharide substrate close to the branching point as compared to the parent alpha-amylase.

8. A variant of any one of claims 1-7, which further exhibits improved substrate specificity and/or improved specific activity relative to the parent Termamyl-like alpha- amylase.

9. A variant of any one of claims 1-8, wherein the parent alpha-amylase is a hybrid alpha-amylase of SEQ ID NO:4 and SEQ ID NO:6. The variant of any one of claims 1-9, wherein the parent hybrid alpha-amylase is a hybrid alpha-amylase comprising the 445 C-terminal amino acid residues of the B. licheniformis alpha-amylase shown in SEQ ID NO:4 and the 37 N-terminal amino acid residues of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO:6. 810 7 55-1 gcc 11. The variant of any one of claims 1-10, wherein the parent hybrid Termamyl-like alpha-amylase further has the following mutations: H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO:4) or LEI74. 12. The variant of any one of claims 1-11, wherein the parent hybrid Termamyl-like alpha-amylase further has the following mutations: H156Y+A181T+N190F+A209V+Q264S+I201F (using the numbering of SEQ ID NO:4) or LE429. 13. A DNA construct comprising a DNA sequence encoding an alpha-amylase 0o variant according to any one of claims 1-12. 14. A recombinant expression vector which carries a DNA construct according to claim 13. A cell which is transformed with a DNA construct according to claim 13 or a vector according to claim 14. is 16. The cell of claim 15, which is a microorganism, in particular a bacterium or a fungus, such as a gram positive bacterium such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus or Bacillus thuringiensis. 17. A composition comprising: a mixture of the alpha-amylase from B. licheniformis having the sequence shown in SEQ ID NO:4 with one or more variants of claims 1-12 derived from (as the parent Termamyl-like alpha-amylase) the B. stearothermophilus alpha-amylase having the sequence shown in SEQ ID NO:8; or (ii) a mixture of the alpha-amylase from B. stearothermophilus having the sequence shown in SEQ ID NO:8 with one or more variants of claims 1-12 derived from one or more other parent Termamyl-like alpha-amylases; or (iii) a mixture of one or more variants of claim 1-12 derived from (as the parent Termamyl-like alpha-amylase) the B. stearothermophilus alpha-amylase having the sequence shown in SEQ ID NO:8 with one or more variants according to the invention derived from one or more other parent Termamyl-like alpha-amylases. 18. A composition comprising: a mixture of one or more variants of claims 1-12 derived from (as the parent Termamyl-like alpha-amylase) the B. stearothermophilus alpha-amylase having the 810755-1 gcc C sequence shown in SEQ ID NO:8 and a Termamyl-like alpha-amylase derived from the SB. licheniformis alpha-amylase having the sequence shown in SEQ ID NO:4. _19. A composition comprising: a mixture of one or more variants of claims 1-12 derived from (as the parent Termamyl-like alpha-amylase) the B. stearothermophilus alpha-amylase having the IN sequence shown in SEQ ID NO:8 and a hybrid alpha-amylase comprising a part of the B. amyloliquefaciens alpha-amylase shown in SEQ ID NO:6 and a part of the C B. licheniformis alpha-amylase shown in SEQ ID NO:4. A composition comprising: S 10 a mixture of one or more variants of claims 1-12 derived from (as the parent Termamyl-like alpha-amylase) a hybrid alpha-amylase comprising a part of the B. amyloliquefaciens alpha-amylase shown in SEQ ID NO:6 and a part of the B. licheniformis alpha-amylase shown in SEQ ID NO:4. 21. A composition of claim 20, wherein the hybrid alpha-amylase is a hybrid alpha- is amylase comprising the 445 C-terminal amino acid residues of the B. licheniformis alpha- amylase shown in SEQ ID NO:4 and the 37 N-terminal amino acid residues of the alpha- amylase derived from B. amyloliquefaciens shown in SEQ ID NO:6. 22. A composition of claim 21, wherein the hybrid alpha-amylase further has the following mutations: H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO:4) or LE174. 23. A composition of claim 21, wherein the hybrid alpha-amylase further has the following mutations: H156Y+A181T+N190F+A209V+Q264S+I201F as shown in SEQ ID NO:2 or LE429. 24. Use of an alpha-amylase variant of any one of claims 1-12 or a composition of any one of claims 17-23 for starch liquefaction; in detergent composition, such as laundry, dish washing and hard surface cleaning compositions; ethanol production, such as fuel, drinking and industrial ethanol production; desizing of textiles, fabrics or garments. 25. An isolated variant of a parent Termamyl-like alpha-amylase, which variant has an improved reduced ability to cleave a substrate close to a branching point, and further has improved substrate specificity and/or improved specific activity, comprising an alteration at one or more positions selected from the group of: G48, T49, G107, substantially as hereinbefore described with reference to any one of the examples. 810755.1 gcc O 53 26. A DNA construct comprising a DNA sequence encoding an alpha-amylase Svariant according to claim 27. A recombinant expression vector which carries a DNA construct according to claim 26. 28. A cell which is transformed with a DNA construct according to claim 26 or a IND vector according to claim 27. (N 29. The composition of any one of claims 17-20, substantially as hereinbefore Sdescribed with reference to any one of the examples. Use of an alpha-amylase variant of claim 25 or a composition of claim 29 for to starch liquefaction; in detergent composition, such as laundry, dish washing and hard surface cleaning compositions; ethanol production, such as fuel, drinking and industrial ethanol production; desizing of textiles, fabrics or garments. Dated 31 May, 2007 Novozymes A/S Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 810755-1:gcc 00 1 SEQUENCE LISTING <110> Novo Nordisk A/S <120> <130> <160> <170> Patentln Ver. 2.1 <210> 1 <211> 1. <212> DJ <213> B <220> <221> Cl <222>( <400> 1 gta aat Val Asn 1 443 NA acillus amylol iquefaciens DS 1) (1443) ggc acg ctg Gly Thr Leu 5 atg cag tat ttt Met Gin Tyr Phe gaa Giu 10 tgg tat acg ccg Trp Tyr Thr Pro aac gac Asn Asp ggc cag cat Gly Gin His atc ggt att Ile Gly Ile tg Trp aaa cga ttg cag Lys Arg Leu Gin gat gcg gaa cat Asp Ala Giu His tta tog gat Leu Ser Asp gga acg agc Gly Thr Ser act gcc gtc tgg Thr Ala Val Trp att Ile ccc ocg gca tat Pro Pro Ala Tyr caa gcg Gin Ala gat gtg ggc tac Asp Val Gly Tyr gct tac gac ctt Ala Tyr Asp Leu tat Tyr gat tta ggg gag Asp Leu Gly Giu ttt Phe cat caa aaa ggg His Gin Lys Gly gtt cgg aca aag Val Arg Thr Lys ggc aca aaa gga Gly Thr Lys Gly ctg caa tct gcg Leu Gin Ser Ala atc Ile aaa agt ctt cat Lys Ser Leu His tc Ser 90 cgc gao att aac Arg Asp Ile Asn gtt tac Val Tyr ggg gat gtg Gly Asp Val gta acc gog Val Thr Ala 115 gt c Val1 100 ato aao cac aaa Ile Asn His Lys ggo ggc got gat gog acc gaa gat Gly Gly .Ala Asp Ala Thr Glu Asp 105 110 gtt gaa gto gat Vai Giu Vai Asp ccc Pro 120 got gao cgc aao Ala Asp Arg Asn gta att tca Val Ile Ser gga gaa Gly Glu 130 cac ota att aaa His Leu Ile Lys goc tgg aca cat ttt cat ttt cog ggg ogo Ala Trp Thr His Phe His Phe Pro Gly Arg 135 140 ggC Gly 145 ago aca tao ago Ser Thr Tyr Ser ttt aag tgg tat Phe Lys Trp Tyr tao cat ttt gao Tyr His Phe Asp gg9a Gly 160 aco gat tgg gao Thr Asp Trp Asp too oga aag ctg Ser Arg Lys Leu aac ogo ato tat aag ttt oaa Asn Arg Ile Tyr Lys Phe Gin 170 175 00 ggg aag act Gly Lys Thr tg Trp 180 gat tgg gaa gtt Asp Trp Glu Val tc Ser 185 aat gaa ttc ggc Asn Glu Phe Gly aac tat gat Asn Tyr Asp 190 gtc gta gca Val Val Ala tat ttg atg Tyr Leu Met 195 tat gee gae ttt Tyr Ala Asp Phe gat Asp 200 tat gac cat cet Tyr Asp His Pro gag att Glu Ile 210 aag aga tgg gge Lys Arg Trp Gly tgg tat gee aat Trp Tyr Ala Asn gaa Glu 220 etg caa ttg gac Leu Gin Leu Asp ggt Gly 225 tte egt ett gat Phe Arg Leu Asp get Al a 230 gte aaa cac Val Lys His att aaa Ile Lys .235 Phe Ser Phe Leu egg Arg 240 gat tyg gtt aat Asp Trp Val Asn cat His 245 gte agg gaa aaa Val Arg Giu Lys acq Thr 250 gg9 aag gaa atg Gly Lys Giu Met ttt acg Phe Thr 255 gta get gag Val Ala Giu tac Tyr 260 tgg teg aat gae Trp Ser Asn Asp gge gcg etg gaa Gly Ala Leu Glu aac tat ttg Asn Tyr Leu 270 ett eat tat Leu His Tyr aae aaa aea Asn Lys Thr 275 aat ttt aat eat Asn Phe Asn His tea Ser 280 gtg ttt gac gtg Val Phe Asp Val eeg Pro 285 cag tte Gin Phe 290 eat get gea teg His Ala Ala Ser aca Thr 295 eag gga gge gge Gin Gly Gly Gly tat Tyr 300 gat atg agg aaa Asp Met Arg Lys ttg Leu 305 etg aae ggt aeg Leu Asn Gly Thr gtt tee aag eat Val Ser Lys His ttg aaa teg gtt Leu Lys Ser Val ttt gte gat aac Phe Val Asp Asn eat His 325 gat aea eag eeg Asp Thr Gin Pro ggg Gly 330 eaa teg ett gag Gin Ser Leu Giu teg act Ser Thr 335 gte eaa aca Val Gin Thr ttt aag ceg ett Phe Lys Pro Leu get Ala 345 tac get ttt att Tyr Ala Phe Ile etc aca agg Leu Thr Arg 350 ggg aeg aaa Gly Thr Lys gaa tet gga Glu Ser Gly 355 tac eet cag gtt Tyr Pro Gin Val ttc Phe 360 tac ggg gat atg Tyr Gly Asp Met tac Tyr 365 gga gae Gly Asp 370 tee eag ege gaa Ser Gin Arg Glu att Ile 375 ect gee ttg aaa Pro Ala Leu Lys aaa att gaa eeg Lys Ile Glu Pro 1008 1056 1104 1152 1200 1248 1296 1344 ate Ile 385 tta aaa geg aga Leu Lys Ala Arg eag tat geg tac Gin Tyr Ala Tyr gga Gly 395 gca cag eat gat Ala Gin His Asp tat Tyr 400 tte gac cac cat Phe Asp His His gac Asp 405 att gte ggc tgg Ile Val Gly Trp aca Thr 410 agg gaa gge gac Arg Giu Gly Asp age teg Ser Ser 415 gtt gea aat Val Ala Asn ggt ttg geg gca Gly Leu Ala Ala tta Leu 425 ata aea gac gga Ile Thr Asp Gly ecC ggt ggg Pro Gly Gly 430 aea tgg eat Thr Trp His gea aag ega Ala Lys Arg 435 atg tat gte ggc Met Tyr Val Gly eg Arg 440 eaa aae gee ggt Gin Asn Ala Gly gag Glu 445 00 gac att acc gga aac cgt, tcg gag cog gtt gtc atc aat tc9 gaa ggc Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser Giu Gly 450 455 460 tgg gga gag ttt cac gia aac ggc ggg tcg gtt tca att tat gtt caa Trp Gly Giu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gin 465 470 475 480 aga. Arg <210> 2 <211> 48i <212> PRT <213> Bacillus amyioiiquefacieis 1392 1440 1443 <400> 2 Val Asn Giy Thr Leu Met Gin Tyr Phe 1 Giy Ilie Gin Phe Leu Gly Vai Gly Giy 145 s0 Thr Gly Tvr Glu G ly 225 Gin Gly Al a s0 His Gin Asp Thr Giu 130 Ser Asp Lys Leu Ile 210 Phe His Ile 35 Asp Gin Scr Val1 Al a 115 His Thr Trp Thr Met 195 Lys Arg 5 Trp Lys Thr Ala Vai Gly Lys Gly Ala Ile Val Ile 100 Val Giu Leu Ile Tyr Ser Asp Giu 165 Trp Asp 180 Tyr Ala Arg Trp Leu Asp Arg Val1 Tyr Thr 70 Lys Asn Val1 Lys Asp 150 Ser Trp Asp Gly Al a 230 Leu Trp Gly 55 Val1 Ser His Asp Al a 135 Phe Arg Giu Phe Thr 215 Val Gin Ile 40 Al a Arg Leu Lys Pro 120 Trp Lys Lys Val Asp 200 Trp, Lys As n 25 Pro Tyr Thr His Gly 105 Al a Thr Trp Leu Ser 185 Tyr Tyr His Glu

10 Asp Pro Asp Lys Ser 90 Gly Asp His Tyr Asn 170 Asn Asp Ala Ile Trp Al a Ala Leu Tyr 75 Arg Ala Arg Phe Trp 155 Arg Glu His As n Lys 235 Tyr Giu Tyr Tyr Gly Asp Asp Asn His 140 Tyr Ile Phe Pro Giu 220 Phe Thr His Lys Asp Thr Ile Al a Arg 125 Phe His Tyr Gly Asp 205 Leu Ser Pro Leu Gly Leu Lys Asn Thr 110 Val Pro Phe Lys Asn 190 Val Gin Phe Asn Ser Thr Gly Gly Val Glu Ile Gly Asp Phe 175 Tyr V.7a 1 Leu Leu Asp Asp Ser Giu Glu Tyr Asp Se r Arg Gly 160 Gin Asp Al a Asp Arg 240 Asp Trp, Val Asn His Val Arg Giu Lys Thr Gly Lys Glu Met 245 250 Phe Thr 255 Val Ala Glu Tyr Trp Ser Asn Asp Leu Gly Ala Leu Giu Asn Tyr Leu 260 265 270 Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu His Tyr 27S 280 285 Gin Phe His Ala Ala Ser Thr Gin Gly Gly Gly Tyr Asp Met Arg Lys 290 295 300 Leu Leu Asn Gly Thr Val Vai Ser Lys His Pro Leu Lys Ser Val Thr 305 310 315 320 Phe Val Asp Asn His Asp Thr Gin Pro Gly Gin Ser Leu Giu Ser Thr 325 330 335 Val Gin Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg 340 345 350 Giu Ser Gly Tyr Pro Gin Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys 355 360 365 Gly Asp Ser Gin Arg Glu Ile Pro Ala Leu Lys His Lys Ile Giu Pro 370 375 380 Ilie Leu Lys Ala Arg Lys Gin Tyr Ala Tyr Gly Ala Gin His Asp Tyr 385 390 395 400 Phe Asp His His Asp Ile Val Gly Trp Thr Arg Giu Gay Asp Ser Ser 405 410 415 Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly 420 425 430 Ala Lys Arg Met Tyr Val Gly Arg Gin Asn Ala Gly Giu Thr Trp, His 435 440 445 Asp Ilie Thr Gly Asn Arg Ser Glu Pro Val Vai Ile Asn Ser Glu Gly 450 455 460 Trp Gly Giu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gin 465 '470 475 480 Arg <210> 3 <211> 1920 <212> DNA <213> Bacillus licheniformis <220> <221> CDS <222> (421)..(1872) <400> 3 cggaagattg gaagtacaaa aataagcaaa agattgtcaa tcatgtcatg agccatgcgg gagacggaaa aatcgtctta atgcacgata tttatgcaac gttcgcagat gctgctgaag 120 agattattaa. aaagctgaaa gcaaaaggct atcaattggt aactgtatct cagcttgaag 180 aagtgaagaa gcagagaggc tattgaataa atgagtagaa gcgccatatc ggcgcttttc 240 ttttggaaga aaatataggg aaaatggtac ttgttaaaaa ttcggaatat ttatacaaca. 300 tcatatgttt cacattgaaa ggggaggaga atcatgaaac aacaaaaacg gctttacgcc 360 cgattgctga cgctgttatt tgcgctcatc ttcttgctgc ctcattctgc agcagcggcg 420 gca aat ctt aat Ala Asn Leu Asn 1 aat gac ggc caa Asn Asp Gly Gin ggg Gly 5 acg ctg atg cag Thr Leu Met Gin tat Tyr 10 ttt gaa tgg tac Phe Glu Trp Tyr atg ccc Met Pro cat tgg agg cgt His Trp Arg Arg ttg Leu caa aac gac tcg Gin Asn Asp Ser gca tat ttg Ala Tyr Leu 516 gct gaa cac ggt att act gcc gtc tgg att ccc ccg gca tat aag gga Ala Giu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 acg agc Thr Ser caa gcg gat gtg Gin Ala Asp Val ggc Gly 55 tac ggt gct tac Tyr Gly Ala Tyr gac Asp ctt tat gat tta Leu Tyr Asp Leu ggg Gly gag ttt cat caa Giu Phe His Gin ggg acg gtt cgg Gly Thr Val Arg aag tac ggc aca Lys Tyr Gly Thr gga gag ctg caa Gly Glu Leu Gin gtt tac ggg gat Val Tyr Gly Asp t ct Ser gcg atc aaa agt Ala Ile Lys Ser ctt Leu 90 cat tcc cgc gac His Ser Arg Asp att. aac Ile Asn gtg gtc atc aac Val Val Ilie Asn cac His 105 aaa ggc ggc gct Lys Giy Gly Ala gat gcg acc Asp Ala Thr 110 aac cgc gta Asn Arg Val gaa gat gta Giu Asp Val 115 acc gcg gtt gaa Thr Ala Val Giu gat ccc gct gac Asp Pro Ala Asp att tca Ile Ser 130 gga gaa cac cta Gly Glu His Leu at t Ile 135 aaa gcc tgg aca Lys Ala Trp, Thr cat His 140 ttt cat ttt. ccg Phe His Phe Pro ggg Giy 145 cgc ggc agc aca Arg Gly Ser Thr t ac Tyr 150 agc gat ttt aaa Ser Asp Phe Lys cat tgg tac cat His Trp Tyr His 900 948 gac gga acc gat Asp Gly Thr Asp gac gag tcc cga Asp Giu Ser Arg aag Lys 170 ctg aac cgc atc Leu Asn Arg Ile tat aag Tyr Lys 175 ttt caa gga Phe Gin Gly aag Lys 180 gct tgg gat tgg Ala Trp Asp Trp gtt tcc aat gaa Val Ser Asn Glu aac ggc aac Asn Giy Asn 190 cct gat gtc Pro Asp Val tat gat tat Tyr Asp Tyr 195 ttg atg tat gcc Leu Met Tyr Ala gac Asp 200 atc gat tat gac Ile Asp Tyr Asp gca gca Ala Ala 210 gaa att aag aga Giu Ile Lys Arg ggc act tgg tat Gly Thr Trp Tyr aat gaa ctg caa Asn Giu Leu Gin 1044 1092 1140 1188 ttg Leu 225 gac ggt ttc cgt Asp Gly Phe Arg ctt Leu 230 gat gct gtc aaa Asp Ala Val Lys cac His 235 att aaa ttt tct Ile Lys Phe Ser 6S ttg cgg gat tgg Leu jLrg Asp Trp gtt Val1 245 aat cat gtc agg Asn His Val Arg aaa acg ggg aag Lys Thr Gly Lys gaa atg Giu Met 255 00 ttt acg gta Phe Thr Val tat ttg aac Tyr Leu Asn 275 gct Ala 260 gaa tat tgg cag Giu Tyr Trp Gin gac ttg ggc gcg Asp Leu Gly Ala ctg gaa aac Leu Glu Asn 270 gtg cog ctt Val Pro Leu aaa aca aat ttt Lys Thr Asn Phe aat Asn 280 cat tca gtg ttt His Ser Val Phe 1c cat tat His Tyr 290 cag ttc cat gct Gin Phe His Ala gca Al a 295 tcg aca cag gga Ser Thr Gin Gly ggc Gly 300 ggC tat gat atg Gly Tyr Asp Met agg Arg 305 aaa ttg ctg aac Lys Leu Leu Asn ggt Gly 310 acg gtc gtt tcc Thr Val Val Ser aag Lys 315 cat cog ttg aaa His Pro Leu Lys gtt aca ttt gtc Val Thr Phe Val gat Asp 325 aac cat gat aca Asn His Asp Thr ccg ggg caa tog Pro Gly Gin Ser ctt gag Leu Glu 335 tcg act gtc Ser Thr Val aca agg gaa Thr Arg Giu 355 aca tgg ttt aag Thr Trp Phe Lys ccg Pro 345 Ott gct tao got Leu Ala Tyr Ala ttt att ctc Phe Ile Leu 350 atg tac ggg Met Tyr Gly tct gga tac oct Ser Gly Tyr Pro cag Gin 360 gtt ttc tac ggg Val Phe Tyr Gly gat Asp 365 acg aaa Thr Lys 370 gga gac tcc cag Gly Asp Ser Gin gaa att cot gcO Giu Ile Pro Ala ttg Leu 380 aaa cac aaa att Lys His Lys Ile 1236 1284 1332 1380 1428 1476 1524 1572 1620 1668 1716 1764 1812 1860 1912 1920 ga a Giu 385 cog atc tta aaa Pro Ilie Leu Lys aga aaa cag tat Arg Lys Gin Tyr tao gga gca cag Tyr Gly Ala Gin gat tat ttc gao Asp Tyr Phe Asp cat gao att gtc His Asp Ile Vai ggc Gly 410 tgg aca agg gaa Trp Thr Arg Giu ggc gao Gly Asp 415 ago tog gtt Ser Ser Val goa Al a 420 aat toa ggt ttg Asn Ser Gly Leu goa tta ata aoa Ala Leu Ile Thr gao gga 000 Asp Gly Pro 430 ggt gag aca Gly Giu Thr ggt ggg gca aag oga atg tat gtc Gly Gly Ala Lys Arg Met Tyr Val 435 440 ggo cgg caa aao Gly Arg Gin Asn tgg cat Trp His 450 gao att aco gga Asp Ile Thr Gly aa 0 Asn 455 cgt tog gag cog Arg Ser Giu Pro gto ato aat tog Val Ile Asn Ser gaa S Giu 465 g9c tgg gga gag Gly Trp Gly Glu cac gta aao ggc His Val Asn Gly tog gtt toa att Ser Val Ser Ile gtt caa aga Val Gin Arg tttatttt tag aagagcagag aggaoggatt tootgaagga aatoogtttt <210> 4 <211> 483 <212> PRT <213> Bacillus lijheniformis 7 <400> 4 Ala Asn Leu Asn Gly Thr Leu Met Gin Tyr Phe Glu Trp Tyr Met Pro 1 5 10 00 __Asn Asp Gly Gin His Trp Arg Ary Leu Gin Asn Asp Ser Ala Tyr Leu 25 Ala Ciu His Gly 1ie Thr Ala Val Trp Ie Pro Pro Ala Tyr Lys Gly 35 40 Thr Ser Gin Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 55 Gly Giu Phe His Gin Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys 70 75 Gly Glu Leu Gin Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn C185 90 Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr 100 105 110 Glu Asp Val Thr Ala Val Giu Val Asp Pro Ala Asp Arg Asn Arg Val 25115 120 1.25 Ile Ser Gly Giu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro 130 135 140 Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe 145 150 155 160 Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170 175 Phe Gin Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185 190 Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val 195 200 205 Ala Ala Glu Ilie Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gin 210 215 220 Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp, Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met 245 250 255 Phe Thr Val Ala Giu Tyr Trp Gin Asn Asp Leu Gly Ala Leu Glu Asn 260 265 270 Tyr Leu Asn.Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu 275 280 285 His Tvr Gin Phe His Ala Ala Ser Thr Gin Gly Gly Gly Tyr Asp Met 290 295 300 Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser 305 310 315 320 Val Thr Phe Val Asp Asn His Asp Thr Gin Pro Gly Gin Ser Leu Giu 325 330 335 Ser Thr Val Gin Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 00 Thr 5 Thr Glu 385 Asp Ser G ly Trp Glu 465 Val1 Arg Lys 370 Pro Tyr Ser Gly His 450 Gly Gin Glu 355 Gly Ile Phe Val Al a 435 Asp Trp Arg Ser Asp Leu Asp Ala 420 Lys Ile Gly Gly Ser Lys His 405 Asn Arg Thr Glu Tyr Gin Al a 390 His Ser Met Giy Phe 470 Pro Arg 375 Arg Asp Gly Tyr Asn 455 His Gin 360 Giu Lys Ile Leu Val 440 Arg Val Val Ile Gin Val Ala 425 Gly Ser As n Phe Pro Tyr Gly 410 Ala Arg Giu Gly Tyr Al a Ala 395 Trp Leu Gin Pro Gly 475 Gly Leu 380 Tyr Thr Ile Asn Val 460 Ser Asp 365 Ly s Gly Arg Thr Al a 445 Val1 Val1 Met His Ala Giu Asp 430 Gly Ile Ser Tyr Lys Gin Gly 415 Gly Giu Asn Ile Gly Ile His 400 Asp Pro Thr Ser Tyr 480 <210> <211> 2604 <212> DNA <213> Bacillus amyloliquefaciens <220> <221> <222> (707)..(712) <220> <221> <222> (729)..(734) <220> <221> RBS <222> (759)..(762) <220> <221> sigpeptide <222> (770)..(862) <220> <221> mat peptide <222> (863)..(2314) <220> <221> terminator <222> (2321)..(2376) <220> <221> CDS <222> (863)..(2314) <400> aagcttcaag cggtcaatcg gaatgtgcat ctcgcttcat acttaggttt tcacccgcat attaagcagg cgtttttgaa ccgtgtgaca gaagctgttc gaaaccccgg cgggcggttt 120 00 gattttaagg cattgctggc 5 gatattaccg atccgctgga gccgcgacaa 6tggtcaact aaa gggccCa ctggctgaaa tttgagaaaa tgctgtccag ctgatatgta acgaaagcgg gccgattaca ggggacagta gggggcaatg tgatcagtct ttccccggag atgaccgagg gtgtaagcaa ttcaagtatc a cat tgag cc gaagaagacc actgtccgct aaatataatt acagtttcgt aaaacatcag tgctgcctct ttgcattaag gagcctgcct aattgaaatg cgtgaatcag ggctgaacaa agtatcaaca tttgatgact ataaaaatac gtgtaaaaaa tgtataagaa tcagacttgt cc gta aat Val Asn 1 tcacattaat aaggctgaaa gaaattaaaa aaagatctca gagatagccg ggcagcgtat agcggggcaa gatgatttgg cttgtctgtc taggaataaa aatgagaggg ctcagcggaa cggtgtttcc agctggcgga agcccgcttt caaacgcttc atatgccgaa gccccgcaca ctgaagaagt atcagacagg ggggggttgt agaggaaaca aaagaatcat ggaaggcgct tgaaggacgc tttcattatt tgaaacgcag gatcatccgc tacgaaaaga ggatcgattg gtatttttta tattatttta tgattcaaaa 180 240 300 360 420 480 540 600 660 720 780 gcttatgtgc acgctgttat ttgtcagttt 840 ggc acg ctg atg cag tat ttt gaa Gly Thr Leu Met Gin Tyr Phe Glu tgg tat acg ccg Trp Tyr Thr Pro aac Asn tcg Ser gac ggc cag cat Asp Gly Gin His tgg Trp, 20 act Thr aaa cga ttg cag Lys Arg Leu Gin aat gat Asn Asp cct ccc Pro Pro gcg gaa cat Ala Giu His gca tac aaa Ala Tyr Lys gat atc gga Asp Ile Gly atc Ile gat Asp gcc gtc tgg Ala Val Trp ttg agc caa Leu Ser Gin aac 9ga tac gga Asn Giy Tyr Gly aaa ggg acg gtc Lys Gly Thr Val cct tat gat Pro Tyr Asp aga acg aaa Arg Thr Lys ttg tat Leu Tyr gat tta gga gaa Asp Leu Gly Glu ttc Phe 65 ctt Leu cag caa Gin Gin tac Tyr 7 5 s0 Arg ggc aca aaa tca Gly Thr Lys Ser gag Giu 80 caa gat gcg Gin Asp Ala atc Ile ggc tca ctg cat Gly'Ser Leu His 940 988 1036 1084 1132 1180 1228 1276 1324 1372 aac gtc caa Asn Val Gin gta Vali gaa Giu tac gga gat gtg Tyr Gly Asp Val gtt Vai 100 gtc Val ttg aat cat aag Leu Asn His Lys gct ggt Ala Gly 105 gct gat gca Ala Asp Ala aga aat cag Arg Asn Gin 125 ttt cgt ttt Phe Arg Phe gat gta act Asp Val Thr gcc Al a 115 tat Tyr gaa gtc aat Clu Val Asn act tcg gag Thr Ser Giu gaa Glu 130 aac Asn caa atc aaa Gln Ile Lys gcg Al a 135 ttt Phe ccg gcc aat Pro Ala Asn 120 tgg acg gat Trp Thr Asp aaa tgg cat Lys Trp His ccg ggc cgt Pro Gly Arg 140 tgg tat Trp Tyr 155 gga Gly 145 gcg Ala acg tac agt Thr Tyr Ser gat Asp tcc Ser cat ttc gac His Phe Asp g9a Gly 160 gac tgg gat Asp Trp Asp gaa Glu 165 cgg aag atc Arg Lys Ile 00 cgc atc ttt aag Arg Ile Phe Lys cgt ggg gaa gga Arg Gly Glu Gly gcg tgg gat tg Ala Trp Asp Trp gaa gta Glu Val 185 tca agt gaa Ser Ser Giu ggc aac tat gac Gly Asn Tyr Asp tta atg tat got Leu Met Tyr Ala gat gtt gao Asp Val Asp 200 ggt atc tgg Gly Ile Trp iC tao gao cac Tyr Asp His 205 oct gat gto gtg Pro Asp Val Val gca Al a 210 gag aca aaa aaa Giu Thr Lys Lys tgg Trp 215 tat gog Tyr Ala 220 aat gaa ctg toa Asn Giu Leu Ser tta Leu 225 gac ggc tto cgt Asp Gly Phe Arg gat gcc gco aaa Asp Ala Ala Lys cat His 2C 235 att aaa ttt tca Ile Lys Phe Ser ctg cgt gat tgg Leu Arg Asp Trp cag gcg gtc aga Gin Ala Val Arg cag Gin 250 qog aeg gga aaa Ala Thr Gly Lys gaa Glu 255 atg ttt acg gtt Met Phe Thr Val gcg Al a 260 gag tat tgg cag Glu Tyr Trp Gin aat aat Asn Asn 265 goc qgg aaa Ala Gly Lys gaa aao tao ttg Glu Asn Tyr Leu aat Asn 275 aaa aoa ago ttt Lys Thr Ser Phe aat oaa too Asn Gin Ser 280 too toa oaa Ser Ser Gin gtg ttt gat Val Phe Asp 285 gtt oog ott oat Val Pro Leu His t to Phe 290 aat tta cag gog Asn Leu Gin Ala got Al a 295 gga ggc Gly Gly 300 gga tat gat atg Gly Tyr Asp Met ogt ttg otg gao Arg Leu Leu Asp ggt Gly 310 aoo gtt gtg too Thr Val Val Ser 1420 1468 1516 1564 1612 1660 1708 1756 1804 1852 1900 1948 1996 2044 2092 2140 2188 agg Arg 315 cat oog gaa aag His Pro Glu Lys gog Al a 320 gtt aoa ttt gtt Val Thr Phe Val gaa Giu 325 aat oat gao aoa Asn His Asp Thr cag Gin 330 ocg gga oag toa Pro Gly Gin Ser gaa tog aca gto Glu Ser Thr Val act tgg ttt aaa Thr Trp Phe Lys cog ott Pro Leu 345 gca tao gc Ala Tvr Ala tt t Phe 350 att ttg aca aga Ile Leu Thr Arg gaa Giu 355 too ggt tat cot Ser Gly Tyr Pro cag gtg tto Gin Val Phe 360 gaa att 000 Giu Ile Pro tat ggg gat Tyr Gly Asp 365 atg tao ggg aca Met Tyr Gly Thr ggg aca tog oca Gly Thr Ser Pro toa ctg Ser Leu 380 aaa gat aat ata Lys Asp Asn Ile gag Glu 385 cog att tta aaa Pro Ile Leu Lys gog Al a 390 cgt aag gag tao Arg Lys Glu Tyr gca Ala 6C 395 tao ggg ccc cag Tyr Gly Pro Gin cac gat tat att gac cac cog gat gtg ato 99a His Asp Tyr Ile Asp His Pro Asp Val Ile Gly 400 405 410 tgg acg agg gaa Trp Thr Arg Giu ggt Gly 415 gao ago too gc Asp Ser Ser Ala goc Al a 420 aaa tca ggt ttg Lys Ser Gly Leu goc got Ala Ala 425 tta ato acg gao gga coo ggo gga toa aag cgg atg tat goc ggc otg Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu 00 430 435 440 aaa aat gcc ggc gag aca tgg tat gac ata acq ggc aac cgt tca gat Lys Asn Ala Gly Giu Thr Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp 5 445 450 455 act gta aaa atc gga tct gac ggc tgg gga gag ttt cat gta aac gat Thr Val Lys Ile Gly Ser Asp Giy Trp Gly Giu Phe His Vai Asn Asp 460 465 470 ggg tcc gtc tcc att tat gtt cag aaa taa ggtaataaaa aaacacctcc Giy Ser Val Ser Ile Tyr Vai Gin Lys 475 480 aagctgagtg cgggtatcag cttggaggtg cgtttatttt ttcagccgta tgacaaggtc ggcatcaggt gtgacaaata cggtatgctg gctgtcatag gtgacaaatc cgggttttgc gccgtttggc tttttcacat gtctgatttt tgtataatca acaggcacgg agccggaatc tttcgccttg gaaaaataag cggcgatcgt agctgcttcc aatatggatt gttcatcggg atcgctgctt ttaatcacaa cgtgggatcc <210> 6 <211> 483 <212> PRT <213> Bacillus amyloliquefaciens 2236 2284 2334 2394 2454 2514 2574 2604 <400 Val 6 Asn Giy Thr Leu Met Gin Tyr Phe Giu Trp Tyr Thr Pro Asn Asp 1 Gly Ilie Gin Phe 65 Leu Giy Val1 Giu Giv 145 a Gin Gly Ser Gin Gin Asp Thr Giu 130 As n Asp His Ile Asp Gin Asp Vai Ala 115 Tyr Thr Trp Trp, Thr Asn Lys Al a Val 100 Val Gin Tyr Asp Lys 180 Lys Al a Gly Gly Ile Leu Giu Ile Ser Giu Arg Vai !ryr Thr 70 Gly As n Val1 Lys Asp 150 Ser Leu Trp Giy 5 Val Ser His Asn Al a 135 Phe Arg Gin Ile 40 Pro Arg Leu Lys Pro 120 Trp Lys Lys AS n 25 Pro Tyr Thr His Al a 105 Al a Thr Trp Ile Asp Pro Asp Lys Ser 90 Gly As n Asp His Ser 170 Al a Ala Leu Tyr 75 Arg Ala Arg Phe Trp Arg Giu Tyr Tyr Gly As n Asp Asn Arg 140 Tyr Ile His Lys Asp Thr Val Ala Gin 125 Phe His Phe Leu Gly Leu Lys Gin Thr 110 Giu Pro Phe Lys Ser Leu Gly Ser Val Giu Thr Gly Asp Phe 175 Asp Ser Glu Giu Tyr Asp Ser Arg Gly 160 Arg Gly Giu Gly Ala Trp Asp Trp Glu Val Ser Ser Giu Asn Gly Asn 0 12 Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val 195 200 205 Val Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser 00 5 210 215 220 Leu Asp Gly Phe Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp Val Gin Ala Val Arg Gin Ala Thr Gly Lys Glu Met C0 245 250 255 C Phe Thr Val Ala Glu Tyr Trp Gin Asn Asn Ala Gly Lys Leu Glu Asn 260 265 270 STyr Leu Asn Lys Thr Ser Phe Asn Gin Ser Val Phe Asp Val Pro Leu 0 275 280 285 O C' His Phe Asn Leu Gin Ala Ala Ser Ser Gin Gly Gly Gly Tyr Asp Met 290 295 300 Arg Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala 305 310 315 320 Val Thr Phe Val Glu Asn His Asp Thr Gin Pro Gly Gin Ser Leu Glu 325 330 335 Ser Thr Val Gin Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 Thr Arg Glu Ser Gly Tyr Pro Gin Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile 370 375 380 Glu Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gin His 385 390 395 400 Asp Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410 415 Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr 435 440 445 Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser 450 455 460 Asp Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr 465 470 475 480 Val Gin Lys <210> 7 <211> 1548 <212> DNA <213> Bacillus stearothermophilus <220> <221> CDS <222> (1)..(1548) <400> 7 9CC 9Ca ccg ttt aac ggc acc atg atg cag tat ttt gaa tgg Ala Ala Pro Phe Asn Gly Thr Met Met Gin Tyr Phe Glu Trp 1 5 10 tac tt9 Tyr Leu ccg gat gat Pro Asp Asp tta tcc agc Leu Ser Ser gga aca agc 1s Gly Thr Ser acg tta tgg acc Thr Leu Trp, Thr aaa Lys gtg 9CC aat gaa Val Ala Asn Glu gcc aac aac Ala Asn Asn gct tac aaa Ala Tyr Lys ctt ggc atc acc Leu Gly Ile Thr ctt tgg ctg ccg Leu Trp Leu Pro cgc agc gac Arg Ser Asp gta Val 55 ggg tac gga gta Gly Tyr Gly Val tac Tyr gac ttg tat gac Asp Leu Tyr Asp ctc Leu ggc gaa ttc aat Gly Glu Phe Asn aaa g99 acc gtc Lys Gly Thr Val cgc Arg "75 aca aaa tac gga Thr Lys Tyr Gly aca Thr aaa qCt caa tat Lys Ala Gin Tyr ctt Leu caa 9CC att caa Gin Ala Ile Gin 9CC cac 9CC gct Ala His Ala Ala 99a atg Gly Met caa gtg tac Gin Val Tyr gcc Ala 100 gat gtc gtg ttc Asp Val Val Phe gac Asp 105 cat aaa ggC ggC His Lys Gly Gly gct gac ggc Ala Asp Gly 110 cgc aac caa Arg Asn Gin acg gaa tgg Thr Giu Trp 115 gaa atc tC9 Giu Ile Ser 130 gtg gac gcc gtc Val Asp Ala Val gaa Giu 120 gtc aat ccg tcc Val Asn Pro Ser gac Asp 125 ggc acc tat Gly Thr Tyr caa Gin 135 atc caa gca tgg Ile Gin Ala Trp aaa ttt gat ttt Lys Phe Asp Phe ccc Pro 4C 145 999 cgg 99c aac Gly Arg Gly Asn acc Thr 150 tac tcc agc ttt Tyr Ser Ser Phe aag Lys tgg cgc tgg tac Trp Arg Trp Tyr ttt gac 99C gtt Phe Asp Gly Val gat Asp 165 tgg gac gaa agc Trp Asp Glu Ser cga Arg 170 aaa ttg agc C9c Lys Leu Ser Arg att taC Ile Tyr 175 aaa ttC cgC Lys Phe Arg ggc Gly 180 atc ggc aaa gCg Ile Gly Lys Ala tgg Trp 185 gat tgg gaa gta Asp Trp, Giu Val gac acg gaa Asp Thr Glu 190 atg gat cat Met Asp His so aaC 99a aac Asn Gly Asn 195 CCC gaa gtC Pro Giu Val 210 tat gac tac tta Tyr Asp Tyr Leu atg Met 200 tat gCC gac ctt Tyr Ala Asp Leu gat Asp 205 gtg acc gag Val Thr Glu aaa aaC t.9g ggg Lys Asn Trp Gly tgg tat gtc aac Trp Tyr Val Asn aCa Thr 225 acg aac att gat Thr Asn Ile Asp 999 Gly 230 ttC cgq Ctt gat Phe Arg Leu Asp gCC Al a 235 gtc aag cat att Val Lys His Ile aag Lys 240 ttc agt ttt ttt Phe Ser Phe Phe cct Pro 245 gat tgg ttg tcg Asp Trp Leu Ser tat Tyr 250 gtg cgt tct cag Val Arg Ser Gin act ggc Thr Gly 255 aag CC9 cta ttt acc gtc ggg gaa tat tgg agc tat gac atc aac aag Lys ?ro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys 00 260 270 ttg ttt gat Leu Phe Asp ttg cac aat tac att acg aaa aca gac gga acg atg tct Leu His Asn Tyr Ile Thr Lys Thr Asp Gly Thr Met Ser 275 280 285 gcc cog Ala Pro 290 tta cac aac aaa Leu His Asn Lys tat acc gct tcc aaa tca ggg ggc gca Tyr Thr Ala Ser Lys Ser Gly Gly Ala ttt gat atg cyc acg tta atg acc aat act ctc Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu 305 310 315 aty aaa gat caa Met Lys Asp Gin ccg Pro 320 is aca ttg 9cc gtc Thr Leu Ala Val ttc gtt gat aat Phe Val Asp Asn gac acc gaa ccc Asp Thr Giu Pro ggc caa Gly Gin 335 gcg ctg cag 2C Ala Leu Gin ttt att cta Phe Ilie Leu 2 C 355 tca Ser 340 tgg gtc gac cca Trp Val Asp Pro tgg Trp, 345 ttc aaa ccg ttg Phe Lys Pro Leu got tac gcc Ala Tyr Ala 350 tat ggt gac Tyr Gly Asp act cgg cag gaa Thr Arg Gin Giu tao ccg tgc gtc Tyr Pro Cys Val tat tat Tyr Tyr 370 ggc att cca caa Gly Ile Pro Gin tat Tyr 375 aac att Oct tog Asn Ile Pro Ser aaa ago aaa atc Lys Ser Lys Ile gat Asp 385 cog oto ctc atc Pro Leu Leu Ile ogo agg gat tat Arg Arg Asp Tyr got Ala 395 tao gga aog caa Tyr Gly Thr Gin gat tat ctt gat Asp Tyr Leu Asp cac His 405 too gao ato ato Ser Asp Ile Ile ggg tgg aca agg gaa ggg ggo Gly Trp Thr Arg Giu Gly Gly 410 415 2008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 1536 act gaa aaa 4C Thr Glu Lys gga too gga ctg Gly Ser Giy Leu gos otg ato cc Ala Leu Ile Thr gat ggg cog Asp Gly Pro 430 gga aaa gtg Gly Lys Val gga ggs ago Gly Gly Ser 435 as tgg atg tao Lys Trp Met Tyr gtt Val1 440 990 aaa cas cac Gly Lys Gin His tto tat Phe Tyr 450 gao ctt soc ggc Asp Leu Thr Gly aac cgg agt gao aoc gtc aco ato aac agt Asn Arg Ser Asp Thr Vai Thr Ile Asn Ser 455 460 gat Asp 465 gga tgg 999 gas Gly Trp Giy Glu tto Phe 470 aaa gtc sat 990 Lys Vai Asn Gly ggt Giy 475 tog gtt tog gtt Ser Val Ser Val ES gtt Oct aga aaa Val Pro Arg Lys cga 009 tgg act 6C Arg Pro Trp Thr acg Thr 485 soc gtt tot cc Thr Val Ser Thr atc Ile 490 got 099 009 stc Ala Arg Pro Ile aca aco Thr Thr 495 ggt gsa tto gto Gly Giu Phe Val cgt tgg soc gas cca 099 ttg gtg Arg Trp Thr Giu Pro Arg Leu Val 505 510 gos tog cot tga 1548 Ala Trpo Pro 515 <210> 8 <211> 515 <212> PRT <213> Bacillus stearothermophilus 00 _I <400> 8 Ala Ala Pro Phe Asn Gly Thr Met Met Gin Tyr Phe Glu Trp Tyr Leu 1 5 10 Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn IN 20 25 Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys 40 S n Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp 55 1 Leu Gly Glu Phe Asn Gin Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr 65 70 75 Lys Ala Gin Tyr Leu Gin Ala Ile Gin Ala Ala His Ala Ala Gly Met 90 Gin Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly 100 105 110 Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gin 115 120 125 Glu Ile Ser Gly Thr Tyr Gin Ile Gin Ala Trp Thr Lys Phe Asp Phe 130 135 140 Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His 145 150 155 160 Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr 165 170 175 Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu 180 185 190 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His 195 200 205 Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn 210 215 220 Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys 225 230 235 240 Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gin Thr Gly 245 250 255 Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys 260 265 270 Leu His Asn Tyr Ile Thr Lys Thr Asp Gly Thr Met Ser Leu Phe Asp 275 280 285 Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala 290 295 300 Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gin Pro 305 310 315 320 Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gin CD 16 325 330 335 Ala Leu Gin Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350 00 SPhe Ile Leu Thr Arg Gin Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp 355 360 365 Tyr Tyr Gly Ile Pro Gin Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile 370 375 380 Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gin His C1 385 390 395 400 C 15 Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly In 405 410 415 SThr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro C1 420 425 430 S Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gin His Ala Gly Lys Val 435 440 445 Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser 450 455 460 Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp 465 470 475 480 Val Pro Arg Lys Thr Thr Val Ser Thr Ile Ala Arg Pro Ile Thr Thr 485 490 495 Arg Pro Trp Thr Gly Glu Phe Val Arg Trp Thr Glu Pro Arg Leu Val 500 505 510 Ala Trp Pro 515 <210> 9 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 9 ggtcgtaggc accgtagccc caatccgctt g 31 <210> <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> ggtcgtaggc accgtagccc caatcccatt ggctcg 36 <210> 11 <211> 28 17 <212> DNA <213> Artificial Sequence <220> 00 <223> Description of Artificial Sequence: Primer <400> 11 ctgtgactgg tgagtactca accaagtc 28 0 <210> 12 <211> 31 C<212> DNA O <213> Artificial Sequence Cq (f <220> O <223> Description of Artificial Sequence: Primer S<400> 12 ggtcgtaggc accgtagccc tcatccgctt g 31 <210> 13 <211> 31 2E <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 13 ggtcgtaggc accgtagccc atatccgctt g 31 <210> 14 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 14 ggtcgtaggc accgtagcca atatccgctt g 31 <210> <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> gcagcatgga actgctyatg aagaggcacg tcaaac 36 <210> 16 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 16 oC 18 catagttgcc gaattcattg gaaacttccc <210> 17 5 <211> 34 00 <212> DNA <213> Artificial Sequence <220> S 1 <223> Description of Artificial Sequence: Primer NO <400> 17 catagttgcc gaattcaggg gaaacttccc aatc 34 S f <210> 18 0 <211> 41 S<212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 18 ccgcgccccg ggaaatcaaa ttttgtccag gctttaatta g 41 <210> 19 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 19 caaaatggta ccaataccac ttaaaatcgc tg 32 <210> <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> cttcccaatc ccaagtcttc ccttgaaac 29 <210> 21 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer G0 <400> 21 cttaatttct gctacgacgt caggatggtc ataatc 36 <210> 22 E£ <211> 38 <212> DNA <213> Artificial Sequence 19 <220> <223> Description of Artificial Sequence: Primer 00 5 <400> 22 cgcccaagtc attcgaccag tactcagcta ccgtaaac 38 <210> 23 <211> 29 \D <212> DNA <213> Artificial Sequence 0 <220> S 15 <223> Description of Artificial Sequence: Primer <400> 23 gccgttttca ttgtcgactt cccaatccc 29 <210> 24 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 24 ggaatttcgc gctgactagt cccgtacata tcccc <210> <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> ggcaggaatt tcgcgacctt tcgtcccgta catatc 36 <210> 26 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 26 cctcattctg cagcagcagc cgtaaatggc acgctg 36 <210> 27 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 27 ccagacggca gtaataccga tatccgataa atgttccg 38 0 <210> 28 <211> <212> DNA 00 5 <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 28 cggatatcgg tattactgcc gtctggattc 0 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence N <220> 20 <223> Description of Artificial Sequence: Primer <400> 29 ctcgtcccaa tcggttccgt c 21 <210> <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> gatgtatgcc gacttcgatt atgacc 26 <210> 31 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 31 catagttgcc gaattcattg gaaacttccc <210> 32 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 32 ccgattgctg acgctgttat ttgc 24 <210> 33 <211> <212> DNA <213> Artificial Sequence <220> 0 21 ><223> Description of Artificial Sequence: Primer <400> 33 gccaagcgga taacggctac ggtgc 00 <210> 34 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer CK 15 <400> 34 gaacgagcca atcggacgtg ggctacgg 28 r <210> 20 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> ggaacgagcc aatcggataa cggctacggt gc 32 <210> 36 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 36 4C gcatataagg gactgagcca agcgg <210> 37 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 37 caaccacaaa gccggcgctg atgcg <210> 38 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 38 gcatataagg gactgagcca atcggataac ggctacggtg c 41 210 39 <210> 39 0 22 <211> 28 <212> DNA <213> Artificial Sequence 00 5 <220> <223> Description of Artificial Sequence: Primer <400> 39 gaacgagccg atcggacgtg ggctacgg 28 NO <210> N <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> gaacgagcca aaacgacgtg ggctacgg 28