US20090263881A1 - Alpha-Amylase Mutants - Google Patents

Alpha-Amylase Mutants Download PDF

Info

Publication number
US20090263881A1
US20090263881A1 US11/953,532 US95353207A US2009263881A1 US 20090263881 A1 US20090263881 A1 US 20090263881A1 US 95353207 A US95353207 A US 95353207A US 2009263881 A1 US2009263881 A1 US 2009263881A1
Authority
US
United States
Prior art keywords
amylase
seq
variant
variants
primer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US11/953,532
Inventor
Torben Vedel Borchert
Allan Svendsen
Carsten Andersen
Bjarne Nielsen
Torben Lauesgaard Nissen
Soren Kjaerulff
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novozymes AS
Original Assignee
Novozymes AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=55701615&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20090263881(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Novozymes AS filed Critical Novozymes AS
Priority to US11/953,532 priority Critical patent/US20090263881A1/en
Publication of US20090263881A1 publication Critical patent/US20090263881A1/en
Priority to US13/785,190 priority patent/US20140017715A1/en
Priority to US14/163,585 priority patent/US20140248685A1/en
Priority to US15/012,229 priority patent/US20160222365A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38609—Protease or amylase in solid compositions only
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38636—Preparations containing enzymes, e.g. protease or amylase containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)

Definitions

  • the present invention relates to variants (mutants) of parent Termamyl-like ⁇ -amylases with higher activity at medium temperatures and/or high pH.
  • ⁇ -Amylases ( ⁇ -1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute a group of enzymes which catalyze hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides.
  • ⁇ -amylases such as Termamyl-like ⁇ -amylases variants are known from e.g. WO 90/11352, WO 95/10603, WO 95/26397, WO 96/23873 and WO 96/23874.
  • WO 96/23874 provides three-dimensional, X-ray crystal structural data for a Termamyl-like ⁇ -amylase which consists of the 300 N-terminal amino acid residues of the B. amyloliquefaciens ⁇ -amylase (BANTM) and amino acids 301-483 of the C-terminal end of the B.
  • BANTM amyloliquefaciens ⁇ -amylase
  • licheniformis ⁇ -amylase comprising the amino acid sequence (the latter being available commercially under the tradename TermamylTM), and which is thus closely related to the industrially important Bacillus ⁇ -amylases (which in the present context are embraced within the meaning of the term “Termamyl-like ⁇ -amylases”, and which include, inter alia, the B. licheniformis, B. amyloliquefaciens (BANTM) and B. stearothermophilus (BSGTM) ⁇ -amylases).
  • B. licheniformis B. amyloliquefaciens (BANTM)
  • BSGTM B. stearothermophilus
  • WO 96/23874 further describes methodology for designing, on the basis of an analysis of the structure of a parent Termamyl-like ⁇ -amylase, variants of the parent Termamyl-like ⁇ -amylase which exhibit altered properties relative to the parent.
  • the present invention relates to novel ⁇ -amylolytic variants (mutants) of a Termamyl-like ⁇ -amylase which exhibit improved wash performance (relative to the parent ⁇ -amaylase) at high pH and at a medium temperature.
  • intermediate temperature means in the context of the invention a temperature from 10° C. to 60° C., preferably 20° C. to 50° C., especially 30-40° C.
  • high pH means the alkaline pH which today are used for washing, more specifically from about pH 8 to 10.5.
  • a “low temperature ⁇ -amylase” means an ⁇ -amylase which has an relative optimum activity in the temperature range from 0-30° C.
  • a “medium temperature ⁇ -amylase” means an ⁇ -amylase which has an optimum activity in the temperature range from 30-60° C.
  • SP690 and SP722 ⁇ -amylases are “medium temperature ⁇ -amylases.
  • a “high temperature ⁇ -amylase” is an ⁇ -amylase having the optimum activity in the temperature range from 60-110° C.
  • Termamyl is a “high temperature ⁇ -amylase.
  • the relative temperature optimum often is dependent on the specific pH used.
  • the relative temperature optimum determined at, e.g., pH 8 may be substantially different from the relative temperature optimum determined at, e.g., pH 10.
  • the dynamics in the active site and surroundings are dependent on the temperature and the amino acid composition and of strong importance for the relative temperature optimum of an enzyme.
  • regions of importance for the function of high temperature ⁇ -amylases at medium temperatures can be determined.
  • the temperature activity profile of the SP722 ⁇ -amaylase (SEQ ID NO: 2) and the B. licheniformis ⁇ -amylase (available from Novo Nordisk as Termamyl®) (SEQ ID NO: 4) are shown in FIG. 2 .
  • the relative temperature optimum of SP722 in absolute activities are shown to be higher at medium range temperatures (30-60° C.) than the homologous B. licheniformis ⁇ -amylase, which have an optimum activity around. 60-100° C.
  • the profiles are mainly dependent on the temperature stability and the dynamics of the active site residues and their surroundings. Further, the activity profiles are dependent on the pH used and the pKa of the active site residues.
  • the invention relates to a variant of a parent Termamyl-like ⁇ -amylase, which variant has ⁇ -amylase activity, said variant comprises one or more mutations corresponding to the following mutations in the amino acid sequence shown in SEQ ID NO: 2:
  • a variant of the invention have one or more of the following substitutions or deletions:
  • variants having a deletion in positions D183 and G184 and further one or more of the following substitutions or deletions;
  • variants of the invention mentioned above exhibits an alteration in at least one of the following properties relative to the parent ⁇ -amylase:
  • the invention further relates to DNA constructs encoding variants of the invention; to methods for preparing variants of the invention; and to the use of variants of the invention, alone or in combination with other enzymes, in various industrial products or processes, e.g., in detergents or for starch liquefaction.
  • the invention relates to a method of providing ⁇ -amylases with altered pH optimum, and/or altered temperature optimum, and/or improved stability,
  • a deletion of a consecutive stretch of amino acid residues, such as amino acid residues 30-33, is indicated as (30-33)* or ⁇ (A30-N33).
  • alanine may be deleted or substituted for any other amino acid, i.e., any one of:
  • FIG. 1 is an alignment of the amino acid sequences of six parent Termamyl-like ⁇ -amylases. The numbers on the extreme left designate the respective amino acid sequences as follows;
  • FIG. 2 shows the temperature activity profile of SP722 (SEQ ID NO: 2) (at pH 9) and B. licheniformis ⁇ -amylase (SEQ ID NO: 4) (at pH 7.3).
  • FIG. 3 shows the temperature profile for SP690 (SEQ ID NO: 1), SP722 (SEQ ID NO: 2), B. licheniformis ⁇ -amylase (SEQ ID NO: 4) at pH 10.
  • FIG. 4 is an alignment of the amino acid sequences of five ⁇ -amylases. The numbers on the extreme left designate the respective amino acid sequences as follows:
  • amyp_mouse 2 amyp_rat 3: amyp_pig porcine pancreatic alpha-amylase (PPA) 4: amyp_human 5: amy_altha
  • PPA pancreatic alpha-amylase
  • AHA haloplanctis alpha-amylase
  • ⁇ -amylases produced by Bacillus spp. are highly homologous on the amino acid level.
  • the B. licheniformis ⁇ -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 ⁇ -amylase comprising the amino acid sequence shown in SEQ ID NO: 5 and about 79% homologous with the B. stearothermophilus ⁇ -amylase comprising the amino acid sequence shown in SEQ ID NO: 3.
  • ⁇ -amylases include an ⁇ -amylase derived from a strain of the Bacillus Bp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of which are described in detail in WO 95/26397, and the ⁇ -amylase described by Tsukamoto et al., Biochemical and Biophysical Research Communications, 151 (1988), pp. 25-31, (see SEQ ID NO: 6).
  • Still further homologous ⁇ -amylases include the ⁇ -amylase produced by the B. licheniformis strain described in BP 0252666 (ATCC 27811), and the ⁇ -amylases identified in WO 91/00353 and WO 94/18314.
  • Other commercial Termamyl-like B. licheniformis ⁇ -amylases are comprised in the products OptithermTM and TakathermTM (available from Solvay), MaxamylTM (available from Gist-brocades/Genencor), Spezyro AATM and Spezyme Delta AATM (available from Genencor), and KeistaseTM (available from Daiwa).
  • ⁇ -amylases Because of the substantial homology found between these ⁇ -amylases, they are considered to belong to the same class of ⁇ -amylases, namely the class of “Termamyl-like ⁇ -amylases”.
  • Termamyl-like a amylase is intended to indicate an ⁇ -amylase which, at the amino acid level, exhibits a substantial homology to TermamylTM, i.e., the B. licheniformis ⁇ -amylase having the amino acid sequence shown in SEQ ID NO:4 herein.
  • ⁇ -amylases which has the amino acid sequences shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 or 8 herein, or the amino acid sequence shown in SEQ ID NO: 1 of WO 95/26397 (the same as the amino acid sequence shown as SEQ ID NO: 7 herein) or in SEQ ID NO: 2 of WO 95/26397 (the same as the amino acid sequence shown as SEQ ID NO: 8 herein) or in Tsukamoto et al., 1988, (which amino acid sequence is shown in SEQ ID NO: 6 herein) are considered to be “Termamyl-like ⁇ -amylase”.
  • Termamyl-like ⁇ -amylases are ⁇ -amylases i) which displays at least 60%, such as at least 70%, e.g., at least 75%, or at least 80%, e.g., at least 85%, at least 90% or at least 95% homology with at least one of said amino acid sequences shown in SEQ ID NOS: 1-8 and/or ii) displays immunological cross-reactivity with an antibody raised against at least one of said ⁇ -amylases, and/or iii) is encoded by a DMA sequence which hybridizes to the DNA sequences encoding the above-specified a amylases which are apparent from SEQ ID NOS: 9, 10, 11, or 12 of the present application (which encoding sequences encode the amino acid sequences shown in SEQ ID NOS: 1, 2, 3, 4 and 5 herein, respectively), from SEQ ID NO: 4 of WO 95/26397 (which DNA sequence, together with the stop codon TAA, is shown in SEQ ID NO:
  • 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, Wis., USA 53711).
  • a structural alignment between Termamyl (SEQ ID NO: 4) and a Termamyl-like ⁇ -amylase may be used to identify equivalent/corresponding positions in other Termamyl-like ⁇ -amylases.
  • One method of obtaining said structural alignment is to use the Pile Up programme from the GCG package using default values of gap penalties, i.e., a gap creation penalty of 3.0 and gap extension penalty of 0.1.
  • Other structural alignment methods include the hydrophobic cluster analysis (Gaboriaud et al., (1987), FSBS LETTERS 224, pp. 149-155) and reverse threading (Huber, T; Torda, A E, PROTEIN SCIENCE Vol. 7, No. 1 pp. 142-149 (1998).
  • Property ii) of the ⁇ -amylase i.e., the immunological cross reactivity
  • the antibody which may either be monoclonal or polyclonal, may be produced by methods known in the art, e.g., as described by Hudson et al., Practical Immunology, Third edition (1989), Blackwell Scientific Publications.
  • the immunological cross-reactivity may be determined using assays known in the art, examples of which are Western Blotting or radial immunodiffusion assay, e.g., as described by Hudson et al., 1989. In this respect, immunological cross-reactivity between the ⁇ -amylases having the amino acid sequences SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, or 8, respectively, has been found.
  • the oligonucleotide probe used in the characterisation of the Termamyl-like ⁇ -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 ⁇ -amylase in question.
  • Suitable conditions for testing hybridisation involve pre-soaking in 5 ⁇ SSC and prehybridising for 1 hour at ⁇ 40° C. in a solution of 20% formamide, 5 ⁇ Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 mg of denatured sonicated calf thymus DNA, followed by hybridisation in the same solution supplemented with 100 mM ATP for 18 hours at ⁇ 40° C., followed by three times washing of the filter in 2 ⁇ SSC, 0.2% SDS at 40° C. for 30 minutes (low stringency), preferred at 50° C. (medium stringency), more preferably at 65° C. (high stringency), even more preferably at ⁇ 75° C. (very high stringency). More details about the hybridisation method can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.
  • derived from is intended not only to indicate an ⁇ -amylase produced or producible by a strain of the organism in question, but also an ⁇ -amylase encoded by a DNA sequence isolated from such strain and produced in a host organism transformed with said DNA sequence.
  • the term is intended to indicate an ⁇ -amylase which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the ⁇ -amylase in question.
  • the parent ⁇ -amylase may be a variant of a naturally occurring ⁇ -amylase, i.e. a variant which is the result of a modification (insertion, substitution, deletion) of one or more amino acid residues of the naturally occurring ⁇ -amylase.
  • the parent ⁇ -amylase (i.e., backbone ⁇ -amylase) may be a hybrid ⁇ -amylase, i.e., an ⁇ -amylase which comprises a combination of partial amino acid sequences derived from at least two ⁇ -amylases.
  • the parent hybrid ⁇ -amylase may be one which on the basis of amino acid homology and/or immunological cross-reactivity and/or DNA hybridisation (as defined above) can be determined to belong to the Termamyl-like ⁇ -amylase family.
  • the hybrid ⁇ -amylase is typically composed of at least one part of a Termamyl-like ⁇ -amylase and part(s) of one or more other ⁇ -amylases selected from Termamyl-like ⁇ -amylases or non-Termamyl-like ⁇ -amylases of microbial (bacterial or fungal) and/or mammalian origin.
  • the parent hybrid ⁇ -amylase may comprise a combination of partial amino acid sequences deriving from at least two Termamyl-like ⁇ -amylases, or from at least one Termamyl-like and at least one non-Termamyl-like bacterial ⁇ -amylase, or from at least one Termamyl-like and at least one fungal ⁇ -amylase.
  • the Termamyl-like ⁇ -amylase from which a partial amino acid sequence derives may, e.g., be any of those specific Termamyl-like ⁇ -amylase referred to herein.
  • the parent ⁇ -amylase may comprise a C-terminal part: of an ⁇ -amylase derived from a strain of B. licheniformis , and a N-terminal part of an ⁇ -amylase derived from a strain of B. amyloliquefaciens or from a strain of B. stearothermophilus .
  • the parent ⁇ -amylase may comprise at least 430 amino acid residues of the C-terminal part of the B. licheniformis ⁇ -amylase, and may, e.g., comprise a) an amino acid segment corresponding to the 37 N-terminal amino acid residues of the B.
  • amyloliquefaciens ⁇ -amylase having the amino acid sequence shown in SEQ ID NO: 5 and an amino acid segment corresponding to the 445 C-terminal amino acid residues of the B.
  • licheniformis ⁇ -amylase having the amino acid sequence shown in SEQ ID NO: 4, or a hybrid Termamyl-like ⁇ -amylase being identical to the Termamyl sequence, i.e., the Bacillus licheniformis ⁇ -amylase shown in SEQ ID NO: 4, except that the N-terminal 35 amino acid residues (of the mature protein) has been replaced by the N-terminal 33 residues of BAN (mature protein), i.e., the Bacillus amyloliquefaciens ⁇ -amylase shown in SEQ ID NO: 5; or b) an amino acid, segment corresponding to the 68 N-terminal amino acid residues of the B.
  • stearothermophilus ⁇ -amylase having the amino acid sequence shown in SEQ ID NO: 3 and an amino acid segment corresponding to the 415 C-terminal amino acid residues of the B. licheniformis ⁇ -amylase having the amino acid sequence shown in SEQ ID NO: 4.
  • Another suitable parent hybrid ⁇ -amylase is the one previously described in WO 96/23374 (from Novo Nordisk) constituting the N-terminus of BAN, Bacillus amyloliquefaciens ⁇ -amylase (amino acids 1-300 of the mature protein) and the C-terminus from Termamyl (amino acids 301-483 of the mature protein). Increased activity was achieved by substituting one or more of the following positions of the above hybrid ⁇ -amylase (BAN:1-300/Termamyl:301-483); Q360, F290, and N 102 .
  • Particularly interesting substitutions are one or more of the following substitutions: Q360E,D; F290A, C, D, E, G, H, I, K, L, M, N, P,Q, R,S,T; N 102 D, E;
  • the corresponding positions in the SP722 ⁇ -amylase shown in SEQ ID NO: 2 are one or more of: S365, Y2 95, N106.
  • Corresponding substitutions of particular interest in said ⁇ -amylase shown in SEQ ID NO: 2 are one or more of: S365D,E; Y295A,C,D,E,G,H,I,K,L,M,N,P,Q,R,S,T; and N106D,E.
  • the corresponding positions in the SP690 ⁇ -amylase shown in SEQ ID NO: 1 are one or more of: S365, Y295, N 106 .
  • the corresponding substitutions of particular interest are one or more of: S365D,E; Y295 A,C,D,E,G,H,I,K,L,M,N,P,Q,R,S,T; N106D.E.
  • non-Termamyl-like ⁇ -amylase may, e.g., be a fungal ⁇ -amylase, a mammalian or a plant ⁇ -amylase or a bacterial ⁇ -amylase (different from a Termamyl-like ⁇ -amylase).
  • ⁇ -amylases include the Aspergillus oryzae TAKA ⁇ -amylase, the A. niger acid ⁇ -amylase, the Bacillus subtilis ⁇ -amylase, the porcine pancreatic ⁇ -amylase and a barley ⁇ -amylase. All of these ⁇ -amylases have elucidated structures which are markedly different from the structure of a typical Termamyl-like ⁇ -amylase as referred to herein.
  • the fungal ⁇ -amylases mentioned above i.e., derived from A. niger and A. oryzae , are highly homologous on the amino acid level and generally considered to belong to the same family of ⁇ -amylases.
  • the fungal ⁇ -amylase derived from Aspergillus oryzae is commercially available under the tradename FungamylTM.
  • variants of a Termamyl-like ⁇ -amylase variant of the invention
  • modification e.g., deletion or substitution
  • variants of another Termamyl-like ⁇ -amylase modified in the equivalent position(s) are encompassed thereby.
  • the ⁇ -amylase backbone is derived from B. licheniformis (as the parent Termamyl-like ⁇ -amylase), e.g., one of those referred to above, such as the B. licheniformis ⁇ -amylase having the amino acid sequence shown in SEQ ID NO: 4.
  • mutations include mutations corresponding to mutations in one or more of the following positions in SP722 ⁇ -amylase (having the amino acid sequence shown in SEQ ID NO: 2): T141, K142, F143, D144, F145, P146, G147, R148, G149, R181, A186, S193, N195, K269, N270, K311, K458, P459, T461.
  • the variant of the invention have one or more of the following substitutions (using the SEQ ID NO: 2 numbering):
  • Preferred, high pH stability variants include one or more of the following substitutions in the SP722 ⁇ -amylase (having the amino acid sequence shown in SEQ ID NO: 2):
  • Bacillus strain NCIB 12512 ⁇ -amylase having the sequence shown in SEQ ID NO: 1, or the B. stearothermophilus ⁇ -amylase having the sequence shown in SEQ ID NO: 3, or the B. licheniformis ⁇ -amylase having the sequence shown in SEQ ID NO: 4, or the S, amyloliquefaciens ⁇ -amylase having the sequence shown in SEQ ID NO: 5 is used as the backbone, i.e., parent Termamyl-like ⁇ -amylase, for these mutations.
  • the B. stearothermophilus ⁇ -amylase already has a Tyrosine at position corresponding to N270 in SP722.
  • the Bacillus strain NCIB 12512 ⁇ -amylase, the B. stearothermophilus ⁇ -amylase, the B. licheniformis ⁇ -amylase and the B. amyloliquefaciens ⁇ -amylase already have Arginine at position corresponding to K458 in SP722.
  • the B. licheniformis ⁇ -amylase already has a Proline at position corresponding to T461 in SP722. Therefore, for said ⁇ -amylases these substitutions are not relevant.
  • ⁇ -amylase variants with improved stability at high pH can be constructed by making substitutions in the regions found using the molecular dynamics simulation mentioned in Example 2.
  • the simulation depicts the region(s) that has a higher flexibility or mobility at high pH (i.e., pH 8-10.5) when compared to medium pH.
  • the homology of said bacterial ⁇ -amylase may be at least 60%, preferably be more than 70%, more preferably more than 80%, most preferably more than 90% homologous to the above mentioned Termamyl-like ⁇ -amylase (BA2), measured using the UWGCG GAP program from the GCG package version 7.3 (June 1993) using default values for GAP penalties [Genetic Computer Group (1991) Programme Manual for the GCG Package, version 7, 575 Science Drive, Madison, Wis., USA 53711], Substitution of the unfavorable residue for another would be applicable.
  • BA2 Termamyl-like ⁇ -amylase
  • Improved Ca stability means the stability of the enzyme under Ca + depletion has been improved.
  • mutations (including amino acid substitutions) of importance with respect to achieving improved Ca 2+ stability at high pH include mutation or deletion in one or more positions corresponding to the following positions in the SP722 ⁇ -amylase having the amino acid sequence shown in SEQ ID NO; 2: R181, G182, D183, G184, K185, A18S, W189, M195, N270, E346, K385, K458, P459.
  • a variant of the invention have one or more of the following substitutions or deletions:
  • Bacillus strain NCIB 12512 ⁇ -amylase having the sequence shown in SEQ ID NO: 1, or the B. amyloliquefaciens ⁇ -amylase having the sequence shown in SEQ ID NO: 5, or the B. licheniformis ⁇ -amylase having the sequence shown in SEQ ID NO: 4 are used as the backbone for these mutations.
  • the variant is the Bacillus strain NCTB 12512 ⁇ -amylase with deletions in D183 and G184 and further one of the following substitutions: R181Q,N and/or G182T,S,N and/or D183*; G184* and/or
  • important mutations with respect to obtaining variants exhibiting increased specific activity at temperatures from 10-60° C., preferably 20-50° C., especially 30-40° C. include mutations corresponding to one or more of the following positions in the SP722 ⁇ -amylase having the amino acid sequence shown in SEQ ID NO: 2:
  • the variant, of the invention have one or more of the following substitutions:
  • Preferred variants has one or more of the following substitutions or deletions: ⁇ 174*, D183*, G184*, K269S.
  • the B. licheniformis ⁇ -amylase having the sequence shown in SEQ ID NO: 4 is used as the backbone for these mutations.
  • the particularly interesting amino acid substitution are those that increase the mobility around the active site of the enzyme. This is accomplished by changes that disrupt stabilizing interaction in the vicinity of the active site, i.e., within preferably 10 ⁇ or 8 ⁇ or 6 ⁇ or 4 ⁇ from any of the residues constituting the active site.
  • Such mutations are expected to cause increased flexibility in the active site region either by the introduction of cavities or by the structural rearrangements that fill the space left by the mutation.
  • a variant of the invention comprises one or more modifications in addition to those outlined above.
  • one or more Proline residues present in the part of the ⁇ -amylase variant which is modified is/are replaced with a non-Proline residue which may be any of the possible, naturally occurring non-Proline residues, and which preferably is an Alanine, Glycine, Serine, Threonine, Valine or Leucine.
  • Cysteine residues present among the amino acid residues with which the parent ⁇ -amylase is modified is/are replaced with a non-Cysteine residue such as Serine, Alanine, Threonine, Glycine, Valine or Leucine.
  • a variant of the invention may—either as the only modification or in combination with any of the above outlined modifications—be modified so that one or more Asp and/or Glu present in an amino acid fragment corresponding to the amino acid fragment 185-209 of SEQ ID NO: 4 is replaced by an Asn and/or Gln, respectively. Also of interest is the replacement, in the Termamyl-like ⁇ -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.
  • the mobility of ⁇ -amylase variants of the invention may be increased by replacing one or more amino acid residue at one or more positions close to the substrate site. These positions are (using the SP722 ⁇ -amylase (SEQ ID NO: 2) numbering): V56, K108, D168, Q169, Q172, L201, K269, L272, L275, K446, P45S.
  • the invention relates to variants being mutated in one or more of the above mentioned positions.
  • Preferred substitutions are one or more of the following:
  • the Bacillus strain NCIB 12512 ⁇ -amylase having the sequence shown in SEQ ID NO: 1, or the B. stearothermophilus ex-amylase having the sequence shown in SEQ ID NO: 3, or the B. licheniformis ⁇ -amylase having the sequence shown in SEQ ID NO; 4, or the B. amyloliquefaciens ⁇ -amylase having the sequence shown in SEQ ID NO: 5 are used as the backbone for these mutations.
  • the B. licheniformis ⁇ -amylase and the B. amyloliquefaciens ⁇ -amylase have a Glut amine at position corresponding to K269 in SP722.
  • the B. stearothermophilus ⁇ -amylase has a Serine at position corresponding to K269 in SP722Therefore, for said ⁇ -amylases these substitutions are not relevant.
  • the B. amyloliquefaciens ⁇ -amylase has an Alanine at position corresponding to L272 in SP722, and the B. stearothermophilus ⁇ -amylase has a Isoleucine at the position corresponding to L272 in SP722. Therefore, for said ⁇ -amylases these substitutions are not relevant.
  • the Bacillus strain 12512 ⁇ -amylase has a Isoleucine at position corresponding to L275 in SP722. Therefore for said ⁇ -amylase this substitution is not relevant.
  • the B. amyloliquefaciens ⁇ -amylase has a Phenylalanine at position corresponding to 295 in SP722. Further, the B. stearothermophilus ⁇ -amylase has an Asparagine at position corresponding to Y295 in SP722. Therefore, for said ⁇ -amylases these substitutions are not relevant.
  • the B. licheniformis ⁇ -amylase and the B. amyloliquefaciens ⁇ -amylase have a Asparagine at position corresponding to K446 in SP722. Further, the B. stearothermophilus ⁇ -amylase has a Histidine at position corresponding to K446 in SP722. Therefore, for said a-amylases these substitutions are not relevant.
  • the B. licheniformis ⁇ -amylase, the B. amyloliquefaciens ⁇ -amylase and the B. stearothermophilus ⁇ -amylase have a Serine at position corresponding to P459 in SP722. Further, the Bacillus strain 12512 ⁇ -amylase has a Threonine at position corresponding to P459 in SP722. Therefore, for said ⁇ -amylases these substitutions are not relevant.
  • the invention relates to improving the stability of low temperature ⁇ -amylases (e.g., Alteromonas haloplanctis (Feller et al., (1994), Eur. J. Biochem 222:441-447), and medium temperature ⁇ -amylases (e.g., SP722 and SP690) possessing medium temperature activity, i.e., commonly known as psychrophilic enzymes and mesophilic enzymes.
  • the stability can for this particular enzyme class be understood either as thermostability or the stability at Calcium depletion conditions.
  • enzymes displaying the high activity at medium temperatures also display severe problems under conditions that stress the enzyme, such as temperature or Calcium depletion.
  • the objective is to provide enzymes that at the same time display the desired high activity at medium temperatures without loosing their activity under slightly stressed conditions.
  • the activity of the stabilized variant measured at medium temperatures should preferably be between 100% or more and 50%, and more preferably between 100% or more and 70%, and most preferably between 100% or more and 85% of the original activity at that specific temperature before stabilization of the enzyme and the resulting enzyme should withstand longer incubation at stressed condition than the wild type enzyme.
  • Contemplated enzymes include ⁇ -amylases of, e.g., bacterial or fungal origin.
  • the A. haloplanctis alpha-amylase (5 in alignment shown in FIG. 4 ) has a homology of approximately 66% to porcine pancreatic alpha-amylase (PPA) (3 in the alignment shown in FIG. 4 ).
  • PPA pancreatic alpha-amylase
  • the PPA 3D structure is known, and can be obtained from Brookhaven database under the name 1OSE or 1DHK. Based on the homology to other mare stable alpha amylases, stabilization of “the low temperature highly active enzyme” from Alteromonas haloplanctis alpha-amylase, can be obtained and at the same time retaining the desired high activity at medium temperatures.
  • FIG. 4 shown a multiple sequence alignments of five ⁇ -amylases, including the AHA and the PPA ⁇ -amylase. Specific mutations giving increased stability in Alteromoxias haloplantis alpha-amylase:
  • the DNA sequence encoding a parent ⁇ -amylase may be isolated from any cell or microorganism producing the ⁇ -amylase in question, using various methods well known in the art.
  • a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the ⁇ -amylase to be studied.
  • homologous, labeled oligonucleotide probes may be synthesized and used to identify ⁇ -amylase-encoding clones from a genomic library prepared from the organism in question.
  • oligonucleotide probe containing sequences homologous to a known ⁇ -amylase gene could be used as a probe to identify ⁇ -amylase-encoding clones, using hybridisation and washing conditions of lower stringency.
  • Yet another method for identifying ⁇ -amylase-encoding clones would involve inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming ⁇ -amylase-negative bacteria with the resulting genomic DMA library, and then plating the transformed bacteria onto agar containing a substrate for ⁇ -amylase, thereby allowing clones expressing the ⁇ -amylase to be identified.
  • an expression vector such as a plasmid
  • transforming ⁇ -amylase-negative bacteria with the resulting genomic DMA library
  • the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g., the phosphoroamidite method described by S. L. Beaucage and M. H. Caruthers (1981) or the method described by Matthes et al. (1984).
  • the phosphoroamidite method oligonucleotides are synthesized, e.g., in an automatic DMA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.
  • the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, the fragments corresponding to various parts of the entire DNA sequence), in accordance with standard techniques.
  • the DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in U.S. Pat. No. 4,683,202 or R. K. Saiki et al. (1988).
  • 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 ⁇ -amylase variant of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or an artificial chromosome.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the DNA sequence should be operably connected to a suitable promoter sequence.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for directing the transcription of the DNA sequence encoding an ⁇ -amylase variant of the invention, especially in a bacterial host are the promoter of the lac operon of E.
  • the Streptomyces coelicolor agarase gene dagA promoters the promoters of the Bacillus licheniformis ⁇ -amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens ⁇ -amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc.
  • useful promoters are those derived from the gene encoding A, oryzae TAKA amylase, Rhizomucor miehei aspartie proteinase, A.
  • niger neutral ⁇ -amylase A. niger acid stable ⁇ -amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.
  • the expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, poly-adenylation sequences operably connected to the DNA sequence encoding the ⁇ -amylase variant, of the invention. Termination and polyadenyiation sequences may suitably be derived from the same sources as the promoter.
  • the vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pJJ702.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B. licheniformis , or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance.
  • a selectable marker e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B. licheniformis , or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance.
  • the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by cotransformation, e.g., as described in WO 91/17243.
  • Bacillus ⁇ -amylases mentioned herein comprise a pre-region permitting secretion of the expressed protease into the culture medium. If desirable, this preregion may be replaced by a different preregion or signal sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective preregions.
  • the cell of the invention is advantageously used as a host cell in the recombinant production of an ⁇ -amylase variant of the invention.
  • the cell may be transformed with the DNA construct of the invention encoding the variant, conveniently by integrating the DMA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.
  • the cell of the invention may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g. a bacterial or a fungal (including yeast) cell.
  • a microbial cell e.g. a bacterial or a fungal (including yeast) cell.
  • suitable bacteria are Gram positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalo - philus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis , or Streptomyces lividans or Streptomyces murinus , or gramnegative bacteria such as E. coli .
  • the transformation of the bacteria may, for instance, be effected by protoplast transformation or by using competent cells in a manner known per se.
  • the yeast organism may favorably be selected from a species of Saccharomyces or Schizosaccharomyces , e.g. Saccharomyces cerevisiae .
  • the filamentous fungus may advantageously belong to a species of Aspergillus , e.g., Aspergillus oryzae or Aspergillus niger .
  • Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023.
  • the present invention relates to a method of producing an ⁇ -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 ⁇ -amylase variant of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. as described in catalogues of the American Type Culture Collection).
  • the ⁇ -amylase variant secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
  • the ⁇ -amylase variants of this invention possesses valuable properties allowing for a variety of industrial applications.
  • enzyme variants of the invention are applicable as a component in washing, dishwashing and hard-surface cleaning detergent compositions.
  • variants of the invention may suitably be incorporated in detergent, compositions.
  • Detergent compositions comprising a variant of the invention may additionally comprise one or more other enzymes, such as a lipase, cutinase, protease, cellulase, peroxidase or laccase, and/or another ⁇ -amylase.
  • enzymes such as a lipase, cutinase, protease, cellulase, peroxidase or laccase, and/or another ⁇ -amylase.
  • ⁇ -amylase variants of the invention may be incorporated in detergents at conventionally employed concentrations. It is at present contemplated that a variant of the invention may be incorporated in an amount corresponding to 0.00001-1 mg (calculated as pure, active enzyme protein) of ⁇ -amylase per liter of wash/dishwash liquor using conventional dosing levels of detergent.
  • the invention also relates to a method of providing ⁇ -amylases with 1) altered pH optimum, and/or 2) altered temperature optimum, and/or 3) improved stability, comprising the following steps:
  • identifying (a) target position(s) and/or region(s) for mutation of the ⁇ -amylase by comparing the molecular dynamics of two or more ⁇ -amylase 3D structures having substantially different pH, temperature and/or stability profiles, ii) substituting, adding and/or deleting one or more amino acids in the identified position(s) and/or region(s).
  • a medium temperature ⁇ -amylase is compared with a high temperature ⁇ -amylase.
  • a low temperature ⁇ -amylase is compared with either a medium or a high temperature ⁇ -amylase.
  • the ⁇ -amylases compared should preferably be at least 70%, preferably 80%, up to 90%, such as up to 95%, especially 95% homologous with each other.
  • the ⁇ -amylases compared may be Termamyl-like ⁇ -amylases as defined above.
  • the ⁇ -amylases compared are the ⁇ -amylases shown in SEQ ID NO; 1 to SEQ ID NO: 8.
  • the stability profile of the ⁇ -amylases in question compared are the Ca 2+ dependency profile.
  • SP722 (SEQ ID NO: 2, available from Novo Nordisk) TermamylTM (SEQ ID NO: 4, available from Novo Nordisk)
  • SP690 (SEQ ID NO: 1, available from Novo Nordisk)
  • Bacillus subtilis SHA273 see WO 95/10603
  • pJE1 contains the gene encoding a variant of SP722 ⁇ -amylase (SEQ ID NO: 2): vis. deletion of 6 nucleotides corresponding to amino acids D183-G184 in the mature protein. Transcription of the JE1 gene is directed from the amyL promoter.
  • the plasmid further more contains the origin of replication and cat-gene conferring resistance towards kanamycin obtained from plasmid pUB110 (Gryczan, T J et al. (1978), J. Bact. 134:318-329).
  • the E. coli/Bacillus shuttle vector pDorK101 (described below) can be used to introduce mutations without expression of ⁇ -amylase in E. coli and then be modified in such way that the ⁇ -amylase is active in Bacillus .
  • the vector was constructed as follows: The JE1 encoding gene (SP722 with the deletion of D183-G184) was inactivated in pJE1 by gene interruption in the PstI site in the 5′coding region of the SEQ ID NO: 2: SP722 by a 1.2 kb fragment containing an E. coli origin of replication.
  • This fragment was PCR amplified from the pUC19 (GenBank Accession #:X02514) using the forward primer: 5′-gacctgcagtcaggcaacta-3′ and the reverse primer: 5′-tagagtcgacctgcaggcat-3′.
  • the PCR amplicon and the pJE1 vector were digested with PstI at 37° C. for 2 hours.
  • the pJE1 vector fragment and the PCR fragment were ligated at room temperature, for 1 hour and transformed in E. coli by electrotransformation.
  • the resulting vector is designated pDorK101.
  • the assay can be used to screening of Termamyl-like ⁇ -amylase variants having an improved stability at high pH compared to the parent enzyme and Termamyl-like ⁇ -amylase variants having an improved stability at high pH and medium temperatures compared to the parent enzyme depending of the screening temperature setting
  • Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67, Schleicher & Sehuell, Dassel, Germany)—and nitrocellulose filters (Protran-Ba 85, Schleicher & Sehuell, Dassel, Germany) on TY agar plates with 10 ⁇ g/ml kanamycin at 37° C. for at least 21 hours.
  • the cellulose acetate layer is located on the TY agar plate.
  • Each filter sandwich is specifically marked with a needle after plating, but before incubation in order to be able to localize positive variants on the filter and the nitrocellulose filter with bound variants is transferred to a container with glycin-NaOH buffer, pH 8.6-10.6 and incubated at room temperature (can be altered from 10°-60° C.) for 15 min.
  • the cellulose acetate filters with colonies are stored on the TY-plates at room temperature until use. After incubation, residual activity is detected on plates containing 1% agarose, 0.2% starch in glycin-NaOH buffer, pH 8.6-10.6.
  • the assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for 2 hours, at room temperature. After removal of the filters the assay plates are stained with 10% Lugol solution. Starch degrading variants are detected as white spots on dark blue background and then identified on the storage plates. Positive variants are rescreened twice under the same conditions as the first screen.
  • the Bacillus library are plated on a sandwich of cellulose acetate (OE 67, Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plates with a relevant antibiotic, e.g., kanatnycin or chloramphenicol, at 37° C. for at least 21 hours.
  • a relevant antibiotic e.g., kanatnycin or chloramphenicol
  • Each filter sandwich is specifically marked with a needle after plating, but before incubation in order to be able to localize positive variants on the filter and the nitrocellulose filter with bound variants is transferred to a container with carbonate/bicarbonate buffer pH 8.5-10 and with different EDTA concentrations (0.001 mM-100 mM).
  • the filters are incubated at room temperature for 1 hour.
  • the cellulose acetate filters with colonies are stored on the TY-plates at room temperature until use.
  • residual activity is detected on plates containing 1% agarose, 0.2% starch in carbonate/bicarbonate buffer pH 8.5-10.
  • the assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for 2 hours, at room temperature. After removal of the filters the assay plates are stained with 10% Lugol solution. Starch degrading variants are detected as white spots on dark blue background and then identified on the storage plates. Positive variants are rescreened twice under the same conditions as the first screen.
  • Fermentation and purification may be performed by methods well known in the art.
  • the enzyme is incubated under the relevant conditions (1-4). Samples are taken at various time points, e.g., after 0, 5, 10, 15 and 30 minutes and diluted 2 5 times (same dilution for all taken samples) in assay buffer (0.1M 50 mM Britton buffer pH 7.3) and the activity is measured using the Phadebas assay (Pharmacia) under standard conditions pH 7.3, 37° C.
  • assay buffer 0.1M 50 mM Britton buffer pH 7.3
  • the activity measured before incubation (0 minutes) is used as reference (100%).
  • the decline in percent is calculated as a function of the incubation time.
  • the table shows the residual activity after, e.g., 30 minutes of incubation.
  • the specific activity is determined using the Phadebas assay (Pharmacia) as activity/mg enzyme.
  • the manufactures instructions are followed (see also below under “Assay for ⁇ -amylase activity).
  • Phadebas® tablets Phadebas tablets (Phadebas® Amylase Test, supplied by Pharmacia Diagnostic) contain a cross-linked insoluble blue-colored starch polymer which has been mixed with bovine serum albumin and a buffer substance and tabletted.
  • the measured 620 nm absorbance after 10or 15 minutes of incubation is in the range of 0.2 to 2.0 absorbance units at 620 nm. In this absorbance range there is linearity between activity and absorbance (Lambert-Beer law). The dilution of the enzyme must therefore be adjusted to fit this criterion. Under a specified set of conditions (temp., pH, reaction time, buffer conditions) 1 trig of a given ⁇ -amylase will hydrolyze a certain amount of substrate and a blue colour will be produced. The colour intensity is measured at 620 nm. The measured absorbance is directly proportional to the specific activity (activity/mg of pure ⁇ -amylase protein) of the ⁇ -amylase in question under the given set of conditions.
  • ⁇ -amylase activity is determined by a method employing the PNP-G7 substrate.
  • PNP-G7 which is a abbreviation for p nitrophenyl- ⁇ ,D-maltoheptaoside is a blocked oligosaccharide which can be cleaved by an endo-amylase.
  • Kits containing PNP-G7 substrate and ⁇ -Glucosidase is manufactured by Boehringer-Mannheim, (cat.No. 1054635).
  • BM 1442309 To prepare the substrate one bottle of substrate (BM 1442309) is added to 5 ml buffer (BM1442309).
  • BM 1462309 To prepare the ⁇ -Glucosidase one bottle of ⁇ -Glueosidase (BM 1462309) is added to 45 ml buffer (BM1442309).
  • the working solution is made by mixing 5 ml ⁇ -Glucosidase solution with 0.5 ml substrate.
  • the assay is performed by transforming 20 ⁇ l enzyme solution to a 96 well microtitre plate and incubating at 25° C. 200 ⁇ l working solution, 25° C. is added. The solution is mixed and pre-incubated 1 minute and absorption is measured every 15 sec. over 3 minutes at OD 405 nm.
  • the slope of the time dependent absorption-curve is directly proportional to the specific activity (activity per rag enzyme) of the ⁇ -amylase in question under the given set of conditions.
  • the random mutagenesis may be carried out by the following steps:
  • Suitable dope algorithms for use in step 6 are well known in the art.
  • One algorithm is described by Tomandl, D, et al., Journal of Computer-Aided Molecular Design, 11 (1997), pp. 29-38).
  • Another algorithm, DOPE is described in the following:
  • the “DOPE” program is a computer algorithm useful to optimize the nucleotide composition of a codon triplet in such a way that it encodes an amino acid distribution which resembles most the wanted amino acid distribution. In order to assess which of the possible distributions is the most similar to the wanted amino acid distribution, a scoring function is needed. In the “Dope” program the following function was found to be suited:
  • x i 's are the obtained amounts of amino acids and groups of amino acids as calculated by the program
  • y i 's are the wanted amounts of amino acids and groups of amino acids as defined by the user of the program (e.g. specify which of the 20 amino acids or stop codons are wanted to be introduced, e.g. with a certain percentage (e.g. 90% Ala, 3% Ile, 7% Val), and w i 's are assigned weight factors as defined by the user of the program (e.g., depending on the importance of having a specific amino acid residue inserted into the position in question).
  • N is 21 plus the number of amino acid groups as defined by the user of the program. For purposes of this function 0° is defined as being 1.
  • a cycle normally consists of 1000 Iterations as described above in which d is decreasing linearly from 1 to 0. One hundred or more cycles are performed in an optimization process. The nucleotide composition resulting in the highest score is finally presented.
  • TermamylTM The overall homology of the B. licheniformis ⁇ -amylase (in the following referred to as TermamylTM) to other Termamyl-like ⁇ -amylases is high and the percent similarity is extremely high.
  • TERM has a deletion of 2 residues between residue G180 and K181 compared to BANTM and BSG.
  • BSG has a deletion of 3 residues between G371 and 1372 in comparison with BANTM and TermamylTM. Further BSG has a C-terminal extension of more than 20 residues compared to BANTM and TermamylTM. BANTM has 2 residues less and Termamyl has one residue less in the N-terminal compared to BSG.
  • TermamylTM differs in that it lacks two residues around 178-182.
  • the HOMOLOGY program from BIOSYM was used to substitute the residues in equivalent positions in the structure (not only structurally conserved regions) except for the deletion point.
  • a peptide bond was established between G179(G177) and K180(K180) in TermamylTM (BANTM), The close structural relationship between the solved structure and the model structure (and thus the validity of the latter) is indicated by the presence of only very few atoms found to be too close together in the model.
  • the X-ray structure and/or the model build structure of the enzyme of interest, here SP722 and TermamylTM are subjected to molecular dynamics simulations.
  • the molecular dynamics simulation are made using the CHARMM (from Molecular simulations (MSI)) program or other suited program like, e.g., DISCOVER (from MSI).
  • the molecular dynamic analysis is made in vacuum, or more preferred including crystal waters, or with the enzyme embedded in water, e.g., a water sphere or a water box.
  • the simulation are run for 300 pico seconds (ps) or more, e.g., 300-1200 ps.
  • the isotropic fluctuations are extracted for the CA carbons of the structures and compared between the structures. Where the sequence has deletions and/or insertions the isotropic fluctuations from the other structure are inserted thus giving 0 as difference in isotropic fluctuation.
  • isotropic fluctuations see the CHARMM manual (obtainable from MSI).
  • the molecular dynamics simulation can be done using standard charges on the chargeable amino acids. This is Asp and Glu are negatively charged and Lys and Arg are positively charged. This condition resembles the medium pH of approximately 7.
  • titration of the molecule can be done to obtain the altered pKa's of the standard titrateable residues normally within pH 2-10; Lys, Arg, Asp, Glu, Tyr and His. Also Ser, Thr and Cys are titrateable but are not taking into account here.
  • the altered charges due to the pH has been described as both Asp and Glu are negative at high pH, and both Arg and Lys are uncharged. This imitates a pH around 10 to 11 where the titration of Lys and Arg starts, as the normal pKa of these residues are around 9-11.
  • the important regions for constructing variants with improved pH stability are the regions which at the extreme pH display the highest mobility, i.e., regions having the highest isotropic fluctuations.
  • Such regions are identified by carrying out two molecular dynamics simulations; i) a high pH run at which the basic amino acids, Lys and Arg, are seen as neutral (i.e. not protonated) and the acidic amino acids, Asp and Glu, have the charge ( ⁇ 1) and ii) a neutral pH run with the basic amino acids, Lys and Arg, having the net charge of (+1) and the acidic amino acids having a charge of ( ⁇ 1).
  • the two run are compared and regions displaying the relatively higher mobility at high pH compared to neutral pH analysis were identified.
  • residues improving general stability e.g., hydrogen bonding, making the region more rigid (by mutations such as Proline substitutions or replacement of Glycine residues), or improving the charges or their interaction, improves the high pH stability of the enzyme.
  • the important regions for constructing variants with increased activity at medium temperature was found as the difference between the isotropic fluctuations in SP722 and Termamyl, i.e., SP722 minus Termamyl CA isotrophic fluctuations, The regions with the highest mobility in the isotrophic fluctuations were selected. These regions and there residues were expected to increase the activity at medium temperatures.
  • the activity of an alpha-amylase is only expressed if the correct mobility of certain residues are present. If the mobility of the residues is too low the activity is decreased or abandoned.
  • the DOPE software (see Materials and Methods) was used to determine spiked codons for each suggested change in the SA1 region minimizing the amount of stop codons (see table 1). The exact, distribution of nucleotides was calculated in the three positions of the codon to give the suggested population of amino acid changes. The doped regions were doped specifically in the indicated positions to have a high chance of getting the desired residues, but still allow other possibilities.
  • the resulting doped oligonucleotide strand is shown in table 2 as sense strand: with the wild type nucleotide and amino acid sequences and the distribution of nucleotides for each doped position.
  • the spiked oligonucleotides apparent from Table 2 (which by a common term is designated FSA) and reverse primers RSA for the SA1 region and specific SEQ ID NO: 2: SP722 primers covering the SacII and the Drain sites are used to generate PGR-library-fragments by the overlap extension method (Horton et al., Gene, 77 (1989), pp. 61-68) with an overlap of 21 base pairs.
  • Plasmid pJE1 is template for the Polymerase Chain Reaction.
  • the PGR fragments are cloned in the E. coli/Bacillus shuttle vector pDork101 (see Materials and Methods) enabling mutagenesis in E.
  • the library may be screened in the low calcium filter assays described in the “Material and Methods” section above.
  • the gene encoding the amylase from SEQ ID NO; 1 is located in a plasmid pTVB106 described in WO96/23873.
  • the amylase is expressed from the amyL promoter in this construct in Bacillus subtilis.
  • a variant of the protein is delta(T183-G184)+Y243F+Q3 91E+K444Q. Construction of this variant is described in WO96/23873.
  • Gene specific primer B1 (SEQ ID NO: 17) and mutagenic primer 101458 (SEQ ID NO: 19) were used to amplify by PCR an approximately 645 bp DNA fragment from a pTVB106-like plasmid (with the delta(T183-G184) mutations in the gene encoding the amylase from SEQ ID NO: 1).
  • the 645 bp fragment was purified from an agarose gel and used as a mega-primer together with primer Y2 (SEQ ID NO: 18) in a second PCR carried out on the same template.
  • the resulting approximately 1080 bp fragment was digested with restriction enzymes BstEII and AflIII and the resulting approximately 510 bp DNA fragment was purified and ligated with the pTVB106-like plasmid (with the delta (T183-G184) mutations in the gene encoding the amylase from SEQ ID NO: 1) digested with the same enzymes.
  • Competent Bacillus subtilis SHA273 (amylase and protease low) cells were transformed with the ligation and Chlorampenicol resistant transformants and was checked by DNA sequencing to verify the presence of the correct mutations on the plasmid.
  • primer B1 (SEQ ID NO: 17) 5′ CGA TTG CTG ACG CTG TTA TTT GCG 3′ primer Y2: (SEQ ID NO: 18) 5′ CTT GTT CCC TTG TCA GAA CCA ATG 3′ primer 101458 (SEQ ID NO: 19): 5′ GT CAT AGT TGC CGA AAT CTG TAT CGA CTT C 3′
  • delta(T183-G184)+K185R+A186T was carried out in a similar way except that mutagenic primer 101638 was used.
  • primer 101638 (SEQ ID NO: 20) 5′ CC CAG TCC CAC GTA CGT CCC CTG AAT TTA TAT ATT TTG 3′
  • delta(T183-G184)+A186T, delta(T183-G184)+A186I, delta(T183-G184)+A186S, delta(T183-G184)+A186N are constructed by a similar method except that pTVB106-like plasmid (carrying variant delta(T183-G184)+K185R+A186T) is used as template and as the vector for the cloning purpose.
  • the mutagenic oligonucleotide (Oligo 1) is:
  • N represents a mixture of the four bases: A, C, G, and T used in the synthesis of the mutagenicoli-gonucleotide.
  • delta(T183-G184)+K185R+A186T+N195F is constructed as follows:
  • PGR is carried out with primer x2 (SEQ ID NO: 22) and primer 101458 (SEQ ID NO: 19) on pTVB106-like plasmid (with mutations delta(T183-G184)+K185R+A186T).
  • the resulting DNA fragment is used as a mega-primer together with primer Y2 (SEQ ID NO: 18) in a PGR on pTVB106-like plasmid (with mutations delta(T183-G184)+N195).
  • the product of the second PGR is digested with restriction endonucleases Acc651 and AflIII and cloned into pTVB106 like plasmid (delta(T183-G184)+N195F) digested with the same enzymes.
  • primer x2 (SEQ ID NO: 22) 5′ GCG TGG ACA AAG TTT GAT TTT CCT G 3′
  • delta(T183-G184)+K185R+A186T+N195F+Y243F+Q391E+K444Q is constructed as follows:
  • PGR is carried out with primer x2 and primer 101458 on pTVB106-like plasmid (with mutations delta(T183-G184)+K185R+A186T).
  • the resulting DNA fragment is used as a mega-primer together with primer Y2 in a PGR on pTVB106 like plasmid (with mutations delta(T183-G184)+Y243F+Q39TE+K444Q).
  • the product of the second PGR is digested with restriction endonucleases Acc65I and AflIII and cloned into pTVB106 like plasmid (delta(T183-G184)+Y243F+Q391E+K444Q) digested with the same enzymes.
  • the gene encoding the amylase from SEQ ID NO: 2 is located in a plasmid pTVB112 described in WO 96/23873.
  • the amylase is expressed from the amyL promoter in this construct in Bacillus subtilis.
  • Gene specific primer DAQ3 and mutagenic primer DA07 are used to amplify by PGR an approximately 820 bp DNA fragment from a pTVB112-like plasmid (with the delta(D183-G184) mutations in the gene encoding the ⁇ -amylase shown in SEQ ID NO: 2.
  • the 820 bp fragment is purified from an agarose gel and used as a mega-primer together with primer DA01 in a second PGR carried out on the same template.
  • the resulting approximately 920 bp fragment is digested with restriction enzymes NgoM I and Aat II and the resulting approximately 170 bp DNA fragment is purified and ligated with the pTVB112-like plasmid (with the delta(D183-G184) mutations in the gene encoding the amylase shown in SEQ ID NO: 2) digested with the same enzymes.
  • Competent Bacillus subtilis SHA273 (amylase and protease low) cells are transformed with the ligation and Chlorampenicol resistant transformants are checked by DNA sequencing to verify the presence of the correct mutations on the plasmid.
  • primer DA01 (SEQ ID NO: 22) 5′ CCTAATGATGGGAATCACTGG 3′ primer DA03: (SEQ ID NO: 24) 5′ GCATTGGATGCTTTTGAACAACCG 3′ primer DA07 (SEQ ID NO: 25): 5′ CGCAAAATGATATCGGGTATGGAGCC 3′ Variants: delta(D183-G184)+K108L, delta(D183-G184)+K108Q, delta(D183-G184)+K108E, delta(D183-G184)+K108V, were constructed by the mega-primer method as described by Sarkar and Sommer, 1990 (BioTechniques 8; 404-407):
  • PCR is carried out with primer DA03 and mutagenesis primer DA20 on pTVB112-like plasmid (with mutations delta(D183-G184)).
  • the resulting DNA fragment is used as a mega-primer together with primer DA01 in a PCR on pTVB112-like plasmid (with mutations delta(D183-G184)).
  • the approximately 920 bp product of the second PCR is digested with restriction endonucleases Aat II and Mlu I and cloned into pTVB112-like plasmid (delta(D183-G1845) digested with the same enzymes,
  • primer DA20 (SQ ID NO: 26): 5′ GTGATGAACCACSWAGGTGGAGCTGATGC 3′
  • S represents a mixture of the two bases: C and G used in the synthesis of the mutagenic oligonucleotide and W represents a mixture of the two bases: A and T used in the synthesis of the mutagenic oligonucleotide.
  • Sequencing of transformants identifies the correct codon for amino acid position 108 in the mature amylase.
  • delta(D183-G184)+D168A, delta(D183-G184)+D168I, delta(D183-G184)+D168V, delta(D183-G184)+D168T is carried out in a similar way except that mutagenic primer DA14 is used.
  • primer DA14 (SEQ ID NO: 27): 5′ GATGGTGTATGGRYCAATCACGACAATTCC 3′
  • R represents a mixture of the two bases
  • a and G used in the synthesis of the mutagenic oligonucleotide and Y represents a mixture of the two bases;
  • C and T used in the synthesis of the mutagenic oligonucleotide.
  • Sequencing of transformants identifies the correct codon for amino acid position 168 in the mature amylase.
  • delta(D183-G184)+Q169N is carried out in a similar way except that mutagenic primer DA15 is used.
  • primer DA15 (SEQ ID NO: 28): 5′ GGTGTATGGGATAACTCACGACAATTCC 3′
  • primer DA16 (SEQ ID NO: 29): 5′ GGTGTATGGGATCTCTCACGACAATTCC 3′
  • primer DA17 (SEQ ID NO: 30): 5′ GGGATCAATCACGAAATTTCCAAAATCGTATC 3′
  • delta(D183-G184)+Q172L is carried out in a similar way except that mutagenic primer DAIS is used.
  • primer DA18 (SEQ ID NO: 31): 5′ GGGATCAATCACGACTCTTCCAAAATCGTATC 3′
  • delta(D183-G184)+L201I is carried out in a similar way except that mutagenic primer DA06 is used.
  • primer DA06 (SEQ ID NO: 32): 5′ GGAAATTATGATTATATCATGTATGCAGATGTAG 3′
  • delta(D183-G184)+K269S is carried out in a similar way except that mutagenic primer DA09 is used.
  • primer DA09 (SEQ ID NO: 33): 5′ GCTGAATTTTGGTCGAATGATTTAGGTGCC 3′
  • primer DA11 (SEQ ID NO: 34): 5′ GCTGAATTTTGGTCGAATGATTTAGGTGCC 3′
  • primer DA21 (SEQ ID NO: 35): 5′ GAATTTTGGAAGTACGATTTAGGTCGG 3′
  • delta(D183-G184)+L272A, delta(D183-G184)+L272I, delta(D183-G184)+L272V, delta(D183-G184)+L272T is carried out in a similar way except that mutagenic primer DA12 is used,
  • primer DA12 (SEQ ID NO: 36): 5′ GGAAAAACGATRYCGGTGCCTTGGAGAAC 3′
  • R represents a mixture of the two bases: A and G used in the synthesis of the mutagenic oligonucleotide and Y represents a mixture of the two bases: C and T used in the synthesis of the mutagenic oligonucleotide.
  • Sequencing of transformants identifies the correct codon for amino acid position 272 in the mature amylase.
  • delta(D183-G184)+L275A, delta(D183-G184)+L275I, delta(D183-G184)+L275V, delta(D183-G184)+L275T is carried out in a similar way except that mutagenic primer DA13 is used.
  • primer DA13 (SEQ ID NO: 37): 5′ GATTTAGGTGCCTRYCAGAACTATTTA 3′ R represents a mixture of the two bases; A and G used in the synthesis of the mutagenic oligonucleotide and Y represents a mixture of the two bases; C and T used in the synthesis of the mutagenic oligonucleotide. Sequencing of transformants identifies the correct codon for amino acid position 275 in the mature amylase.
  • primer DA08 (SEQ ID NO: 38): 5′ CCCCCTTCATGAGAATCTTTATAACG 3′
  • Gene specific primer DA04, annealing 214-231 bp downstream relative to the STOP-codon and mutagenic primer DA10 were used to amplify by PGR an approximately 350 bp DNA fragment from a pTVB112-like plasmid (with the delta(D183-G184) mutations in the gene encoding the amylase depicted in SEQ ID NO: 2).
  • the resulting DNA fragment is used as a mega-primer together with primer DA05 in a PGR on pTVB112 like plasmid (with mutations delta(D183-G184)).
  • the app. 460 bp product of the second PGR is digested with restriction endonucleases SnaB I and Not I and cloned into pTVB112 like plasmid (delta(D183-G184)) digested with the same enzymes,
  • primer DA04 (SEQ ID NO: 39): 5′ GAATCCGAACCTCATTACACATTCG 3′ primer DA05 (SEQ ID NO: 40) 5′ CGGATGGACTCGAGAAGGAAATACCACG 3′ primer DA10 (SEQ ID NO: 41): 5′ CGTAGGGCAAAATCAGGCCGGTCAAGTTTGG 3′
  • delta(D183-G184)+K458R is carried out in a similar way except that mutagenic primer DA22 is used.
  • primer DA22 (SEQ ID NO: 42): 5′ CATAACTGGAAATCGCCCGGGAACAGTTACG 3′
  • delta(D183-G184)+P459S and delta(D183-G184)+P459T is carried out in a similar way except that mutagenic primer DA19 is used.
  • primer DA19 (SEQ ID NO: 43): 5′ CTGGAAATAAAWCCGGAACAGTTACG 3′ W represents a mixture of the two bases: A and T used in the synthesis of the mutagenic oligonucleotide. Sequencing of transformants identifies the correct codon for amino acid position 459 in the mature amylase.
  • delta(D183-G184)+T461P is carried out in a similar way except that mutagenic primer DA23 is used.
  • primer DA23 (SEQ ID NO: 44): 5′ GGAAATAAACCAGGACCCGTTACGATCAATGC 3′
  • delta(D183-G184)+K142R is carried out in a similar way except that mutagenic primer DA32 is used.
  • Primer DA32 (SEQ ID NO: 45): 5′ GAGGCTTGGACTAGGTTTGATTTTCCAG 3′
  • Primer DA31 (SEQ ID NO: 46): 5′ GCTGAATTTTGGCGCAATGATTTAGGTGCC 3′
  • amyL gene encoding the Termamyl ⁇ -amylase is located in plasmid pDN1528 described in WO 95/10603 (Novo Nordisk).
  • Variants with substitutions N265R and N265D, respectively, of said parent, ⁇ -amylase are constructed by methods described in WO 97/41213 or by the “megaprimer” approach described above.
  • the percentage of the initial activity as a function is shown in the table below for the parent enzyme (SEQ ID NO; 2) and for the variants in question.
  • Residual activity Residual activity Variant after 20 min after 30 min ⁇ (D183-G184) + M323L 56% 44% ⁇ (D183-G184) + M323L + R181S 67% 55% ⁇ (D183-G184) + M323L + A186T 62% 50%
  • the solutions were incubated at 50° C.
  • Residual activity Residual activity Variant after 20 min after 30 min ⁇ (T183-G184) 32% 19% ⁇ (T183-G184) + A186T 36% 23% ⁇ (T183-G184) + K185R + A186T 45% 29% ⁇ (T183-G184) + A186I 35% 20% ⁇ (T183-G184) + N195F 44% n.d. n.d. Not determinated
  • the percentage of the initial activity as a function is shown in the table below for the parent enzyme (SEQ ID NO: 2) and for the variants in question.
  • Residual activity Residual activity Variant after 20 min after 30 min ⁇ (D183-G184) + M323L 21% 13% ⁇ (D183-G184) + M323L + R181S 32% 19% ⁇ (D183-G184) + M323L + A186T 28% 17% ⁇ (D183-G184) + M323L + A186R 30% 18% ⁇ (D183-G184) 30% 20% ⁇ (D183-G184) + N195F 55% 44%
  • the percentage of the initial activity as a function is shown in the table below for the parent enzyme (SEQ ID NO: 2) and for the variants in question.
  • the measurement were made using solutions of the respective variants in 100 mM CAPS buffer adjusted to pH 10.5 to which polyphosphate was added (at time t0) to give a final concentration of 2400 ppm.
  • the solutions were incubated at 60° C. for 20 minutes.
  • the measurement were made using solutions of the respective variants in 50 mM Britton Robinson buffer adjusted to pH 7.3 and using the Phadebas assay described above.
  • the activity in the samples was measured at 37° C. using 50 mM Britton Robinson buffer pH 7.3 and at 25° C. using 50 mM CAPS buffer pH 10.5.
  • the temperature dependent activity and the percentage of the activity at 25° C. relative to the activity at 37° C. is shown in the table below for the parent enzyme (SEQ ID NO: 1) and for the variants in question.
  • Another measurement was made using solutions of the respective variants in 50 mM Britton Robinson buffer adjusted to pH 7.3 and using the Phadebas assay described above. The activity in the samples was measured at 37° C. and 50° C. using 50 mM Britton Robinson buffer pH 7.3.
  • the temperature dependent activity and the percentage of the activity at 37° C. relative to the activity at 50° C. is shown in the table below for the parent enzyme (SEQ ID NO: 1) and for the variants in question.
  • NU(37° C.)/ Variant NU/mg 37° C. NU/mg 50° C. NU(50° C.) SP690 (seq ID NO: 1) 13090 21669 60% K269Q 7804 10063 78%
  • the measurement were made using solutions of the respective variants in 50 mM Britton Robinson buffer adjusted to pH 7.3 and using the Phadebas assay described above.
  • the activity in the samples was measured at both 25° C. and 37° C. using 50 mM Britton Robinson buffer pH 7.3.
  • the temperature dependent activity and the percentage of the activity at 25° C. relative to the activity at 37° C. Is shown in the table below for the parent enzyme (SEQ ID NO: 2) and for the variants in question.
  • the measurement were made using solutions of the respective variants in 50 mM Britton Robinson buffer adjusted to pH 7.3 and using the Phadebas assay described above.
  • the activity in the samples was measured at both 37° C. using 50 mM Britton Robinson buffer pH 7.3 and at 60° C. using 50 mM CAPS buffer pH 10.5.
  • the temperature dependent activity and the percentage of the activity at 37° C. relative to the activity at 60° C. is shown in the table below for the parent enzyme (SEQ ID NO: 4) and for the variants in question.
  • Plasmid pTVB191 contains the gene encoding hybrid ⁇ -amylase BAN:1-300/Termamyl:301-483 as well as an origin of replication functional in Bacillus subtilis and the cat gene conferring chloramphenicol resistance.
  • Variant BM4 (F290E) was constructed using the megaprimer approach (Sarkar and Sommer, 1990) with plasmid pTVB191 as template.
  • Primer p1 (SEQ ID NO; 52) and mutagenic oligonucleotide bm4 (SEQ ID NO: 47) were used to amplify a 444 bp fragment with polymerase chain reaction (PCR) under standard conditions.
  • This fragment was purified from an agarose gel and used as ‘Megaprimer’ in a second PCR with primer p2 (SEQ ID NO: 5.3) resulting in a 531 bp fragment.
  • This fragment was digested with restriction endonucleases HinDIII and Tth111I.
  • the 389 bp fragment produced by this was ligated into plasmid pTVB191 that had been cleaved with the same two enzymes.
  • the resulting plasmid was transformed into S. subtilis SHA273. Chloramphenicol resistant clones were selected by growing the transformants on plates containing chloramphenicol as well as insoluble starch. Clones expressing an active ⁇ -amylase were isolated by selecting clones that formed halos after staining the plates with iodine vapour. The identity of the introduced mutations was confirmed by DNA sequencing.
  • Variants BM5(F290K), BM6(F290A), BM8(Q360E) and BM11(N102D) were constructed in a similar way. Details of their construction are given below.
  • mutagenic oligonucleotide bm5 (SEQ ID NO: 48)

Abstract

The invention relates to a variant of a parent Termamyl-like α-amylase, which exhibits an alteration in at least one of the following properties relative to said parent α-amylase: i) improved pH stability at a pH from 8 to 10.5; and/or ii) improved Ca2+ stability at pH 8 to 10.5, and/or iii) increased specific activity at temperatures from 10 to 60° C.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a divisional of U.S. application Ser. No. 10/980,923, filed Nov. 4, 2004 which is a continuation of U.S. application no, 10/665,667, filed Sep. 19, 2003, which is a divisional of U.S. application Ser. No. 09/769,864, filed on Jan. 25, 2001, which is a divisional of U.S. application Ser. No. 09/183,412, filed on Oct. 30, 1998, and claims priority under 35 U.S.C. 119 of Danish application no. 1240/97, filed on Oct. 30, 1997, Danish application no. PA 1998 00936, filed on Jul. 14, 1998, U.S. provisional application No. 60/064,662, filed on Nov. 6, 1997 and U.S. provisional application No. 60/093,234, filed on Jul. 17, 1998, the contents of which are fully incorporated herein by reference.
    • FIELD OP THE INVENTION
  • The present invention relates to variants (mutants) of parent Termamyl-like α-amylases with higher activity at medium temperatures and/or high pH.
    • BACKGROUND OP THE INVENTION
  • α-Amylases (α-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute a group of enzymes which catalyze hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides.
  • There is a very extensive body of patent and scientific literature relating to this industrially very important class of enzymes. A number of α-amylases such as Termamyl-like α-amylases variants are known from e.g. WO 90/11352, WO 95/10603, WO 95/26397, WO 96/23873 and WO 96/23874.
  • Among more recent disclosures relating to α-amylases, WO 96/23874 provides three-dimensional, X-ray crystal structural data for a Termamyl-like α-amylase which consists of the 300 N-terminal amino acid residues of the B. amyloliquefaciens α-amylase (BAN™) and amino acids 301-483 of the C-terminal end of the B. licheniformis α-amylase comprising the amino acid sequence (the latter being available commercially under the tradename Termamyl™), and which is thus closely related to the industrially important Bacillus α-amylases (which in the present context are embraced within the meaning of the term “Termamyl-like α-amylases”, and which include, inter alia, the B. licheniformis, B. amyloliquefaciens (BAN™) and B. stearothermophilus (BSG™) α-amylases). WO 96/23874 further describes methodology for designing, on the basis of an analysis of the structure of a parent Termamyl-like α-amylase, variants of the parent Termamyl-like α-amylase which exhibit altered properties relative to the parent.
    • BRIEF DISCLOSURE OF THE INVENTION
  • The present invention relates to novel α-amylolytic variants (mutants) of a Termamyl-like α-amylase which exhibit improved wash performance (relative to the parent α-amaylase) at high pH and at a medium temperature.
  • The term “medium temperature” means in the context of the invention a temperature from 10° C. to 60° C., preferably 20° C. to 50° C., especially 30-40° C.
  • The term “high pH” means the alkaline pH which today are used for washing, more specifically from about pH 8 to 10.5.
  • In the context of the invention a “low temperature α-amylase” means an α-amylase which has an relative optimum activity in the temperature range from 0-30° C.
  • In the context of the invention a “medium temperature α-amylase” means an α-amylase which has an optimum activity in the temperature range from 30-60° C. For instance, SP690 and SP722 α-amylases, respectively, are “medium temperature α-amylases.
  • In the context of the invention a “high temperature α-amylase” is an α-amylase having the optimum activity in the temperature range from 60-110° C. For instance, Termamyl is a “high temperature α-amylase.
  • Alterations in properties which may be achieved in variants (mutants) of the invention are alterations in:
  • the stability of the Termamyl-like α-amylase at a pH from 8 to 10.5, and/or the Ca2+ stability at pH 8 to 10.5, and/or
    the specific activity at temperatures from 10 to 60° C., preferably 20-50° C., especially 30-40° C.
  • It should be noted that the relative temperature optimum often is dependent on the specific pH used. In other words the relative temperature optimum determined at, e.g., pH 8 may be substantially different from the relative temperature optimum determined at, e.g., pH 10.
    • The Temperature's Influence on the Enzymatic Activity
  • The dynamics in the active site and surroundings are dependent on the temperature and the amino acid composition and of strong importance for the relative temperature optimum of an enzyme. By comparing the dynamics of medium and high temperature α-amylases, regions of importance for the function of high temperature α-amylases at medium temperatures can be determined. The temperature activity profile of the SP722 α-amaylase (SEQ ID NO: 2) and the B. licheniformis α-amylase (available from Novo Nordisk as Termamyl®) (SEQ ID NO: 4) are shown in FIG. 2.
  • The relative temperature optimum of SP722 in absolute activities are shown to be higher at medium range temperatures (30-60° C.) than the homologous B. licheniformis α-amylase, which have an optimum activity around. 60-100° C. The profiles are mainly dependent on the temperature stability and the dynamics of the active site residues and their surroundings. Further, the activity profiles are dependent on the pH used and the pKa of the active site residues.
  • In the first aspect the invention relates to a variant of a parent Termamyl-like α-amylase, which variant has α-amylase activity, said variant comprises one or more mutations corresponding to the following mutations in the amino acid sequence shown in SEQ ID NO: 2:
    • T141, K142, F143, D144, F145, P146, G147, R148, G149, Q174, R181, G182, D183, G184, K185, A186, W189, S193, N195, H107, K108, G109, D166, W167, D168, Q169, S170, R171, Q172, F173, F267, W268, K269, N270, D271, L272, G273, A274, L275, K311, E346, F385, G456, N457, K458, P459, G460, T461, V462, T463.
  • A variant of the invention have one or more of the following substitutions or deletions:
  • T141A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, W, Y, V
    K142A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S,
    T, W, Y, V;
    F143A, D, R, N, C, E, Q, G, H, I, L, K, M, P, S,
    T, W, Y, V;
    D144A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    F145A, D, R, N, C, E, Q, G, H, I, L, K, M, P, S,
    T, W, Y, V;
    P146A, D, R, N, C, E, Q, G, H, I, L, K, M, F, S,
    T, W, Y, V;
    G147A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    R148A, D, N, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    G149A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    R181*, A, D, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, T, W, Y, V;
    G182*, A, D, R, N, C, E, Q, H, I, L, K, M, F, P,
    S, T, W, Y, V;
    D183*, A, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, T, W, Y, V;
    G184*, A, R, D, N, C, E, Q, H, I, L, K, M, F, P,
    S, T, W, Y, V;
    K185A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S,
    T, W, Y, V;
    A186D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    W189A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, T, Y, V;
    S193A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    T, W, Y, V;
    N195A, D, R, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    H107A, D, R, N, C, E, Q, G, I, L, K, M, F, P, S,
    T, W, Y, V;
    K108A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    G109A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    D166A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    W167A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, T, Y, V;
    D168A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    Q169A, D, R, N, C, E, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    S170A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    T, W, Y, V;
    R171A, D, N, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    Q172A, D, R, N, C, E, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    F173A, D, R, N, C, E, Q, G, H, I, L, K, M, P, S,
    T, W, Y, V;
    Q174*, A, D, R, N, C, E, G, H, I, L, K, M, F, P,
    S, T, W, Y, V;
    F267A, D, R, N, C, E, Q, G, H, I, L, K, M, P, S,
    T, W, Y, V;
    W268A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, T, Y, V;
    K269A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S,
    T, W, Y, V;
    N270A, D, R, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    D271A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    L272A, D, R, N, C, E, Q, G, H, I, K, M, F, P, S,
    T, W, Y, V;
    G273A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    A274D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    L275A, D, R, N, C, E, Q, G, H, I, K, M, F, P, S,
    T, W, Y, V;
    K311A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S,
    T, W, Y, V;
    E346A, D, R, N, C, Q, G, H, I, K, L, M, F, P, S,
    T, W, Y, V;
    K385A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S,
    T, W, Y, V;
    G456A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    N457A, D, R, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    K458A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S,
    T, W, Y, V;
    P459A, D, R, N, C, E, Q, G, H, I, L, K, M, F, S,
    T, W, Y, V;
    G460A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    T461A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, W, Y, V;
    V462A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, T, W, Y;
    T463A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, W, Y, V.
  • Preferred are variants having one or more of the following substitutions or deletions:
    • K142R; S193P; R195F; K269R,Q; N270Y,R,D; K311R; E346Q; K385R; K458R; P459T; T461P; Q174*; R181Q,N,S; G182T,S,N; D183*; G184*; K185A,R,D,C,E,Q,G,H,I,L,M,N,F,P,S,T,W,Y,V; A186T,S,N,I,V,R; W189T,S,N,Q.
  • Especially preferred are variants having a deletion in positions D183 and G184 and further one or more of the following substitutions or deletions;
    • K142R; S193P; N195F; K269R,Q; N270Y,R,D; K311R; E346Q; K385R; K458R; P459T; T461P; Q174*; R181Q,N,S; G182T,S,N; K185A,R,D,C,E,Q,G,H,I,L,M,N,F,P,S,T,W,Y,V; A186T,S,N,I,V,R; W189T,S,N,Q.
  • The variants of the invention mentioned above exhibits an alteration in at least one of the following properties relative to the parent α-amylase:
  • i) improved pH stability at a pH from 8 to 10.5; and/or
    ii) improved Ca2+ stability at pH 8 to 10.5, and/or
    iii) increased specific activity at temperatures from 10 to 60° C., preferably 20-50° C., especially 30-40° C. Further, details will be described below.
  • The invention further relates to DNA constructs encoding variants of the invention; to methods for preparing variants of the invention; and to the use of variants of the invention, alone or in combination with other enzymes, in various industrial products or processes, e.g., in detergents or for starch liquefaction.
  • In a final aspect the invention relates to a method of providing α-amylases with altered pH optimum, and/or altered temperature optimum, and/or improved stability,
    • Nomenclature
  • In the present, description and claims, the conventional one-letter and three-letter codes for amino acid residues are used. For ease of reference, α-amylase variants of the invention are described by use of the following nomenclature:
  • Original amino acid(s):position(s):substituted amino acid is)
  • According to this nomenclature, for instance the substitution of alanine for asparagine in position 30 is shown as:
  • Ala30Asn or A30N
  • a deletion of alanine in the same position is shown as:
  • Ala30*or A30*
  • and insertion of an additional amino acid residue, such as lysine, is shown as:
  • Ala30AlaLys or A3 OAK
  • A deletion of a consecutive stretch of amino acid residues, such as amino acid residues 30-33, is indicated as (30-33)* or Δ(A30-N33).
  • Where a specific α-amylase contains a “deletion” in comparison with other α-amylases and an insertion is made in such a position this is indicated as:
  • *36Asp or *36D
  • 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.
  • When one or more alternative amino acid residues may be inserted in a given position it is indicated as
  • A30N,E or
  • A30N or A30E
  • Furthermore, when a position suitable for modification is identified herein without any specific modification being suggested, it is to be understood that any amino acid residue may be substituted for the amino acid residue present in the position.
  • Thus, for instance, when a modification of an alanine in position 30 is mentioned, but not specified, it is to be understood that the alanine may be deleted or substituted for any other amino acid, i.e., any one of:
    • R,N,D,A,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.
    • BRIEF DESCRIPTION OF THE DRAWING
  • FIG. 1 is an alignment of the amino acid sequences of six parent Termamyl-like α-amylases. The numbers on the extreme left designate the respective amino acid sequences as follows;
    • 1: SEQ ID NO: 2 2: Kaoamyl 3: SEQ ID NO: 1 4: SEQ ID NO; 5 5: SEQ ID NO: 4 6: SEQ ID NO: 3.
  • FIG. 2 shows the temperature activity profile of SP722 (SEQ ID NO: 2) (at pH 9) and B. licheniformis α-amylase (SEQ ID NO: 4) (at pH 7.3).
  • FIG. 3 shows the temperature profile for SP690 (SEQ ID NO: 1), SP722 (SEQ ID NO: 2), B. licheniformis α-amylase (SEQ ID NO: 4) at pH 10.
  • FIG. 4 is an alignment of the amino acid sequences of five α-amylases. The numbers on the extreme left designate the respective amino acid sequences as follows:
  • 1; amyp_mouse
    2: amyp_rat
    3: amyp_pig porcine pancreatic alpha-amylase (PPA)
    4: amyp_human
    5: amy_altha A. haloplanctis alpha-amylase (AHA)
    • DETAILED DISCLOSURE OF THE INVENTION The Termamyl-Like α-Amylase
  • It is well known that a number of α-amylases produced by Bacillus spp. are highly homologous on the amino acid level. For instance, the B. licheniformis α-amylase comprising the amino acid sequence shown in SEQ ID NO: 4 (commercially available as Termamyl™) has been found to be about 89% homologous with the B. amyloliquefaciens α-amylase comprising the amino acid sequence shown in SEQ ID NO: 5 and about 79% homologous with the B. stearothermophilus α-amylase comprising the amino acid sequence shown in SEQ ID NO: 3. Further homologous α-amylases include an α-amylase derived from a strain of the Bacillus Bp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of which are described in detail in WO 95/26397, and the α-amylase described by Tsukamoto et al., Biochemical and Biophysical Research Communications, 151 (1988), pp. 25-31, (see SEQ ID NO: 6).
  • Still further homologous α-amylases include the α-amylase produced by the B. licheniformis strain described in BP 0252666 (ATCC 27811), and the α-amylases identified in WO 91/00353 and WO 94/18314. Other commercial Termamyl-like B. licheniformis α-amylases are comprised in the products Optitherm™ and Takatherm™ (available from Solvay), Maxamyl™ (available from Gist-brocades/Genencor), Spezyro AA™ and Spezyme Delta AA™ (available from Genencor), and Keistase™ (available from Daiwa).
  • Because of the substantial homology found between these α-amylases, they are considered to belong to the same class of α-amylases, namely the class of “Termamyl-like α-amylases”.
  • Accordingly, in the present context, the term “Termamyl-like a amylase” is intended to indicate an α-amylase which, at the amino acid level, exhibits a substantial homology to Termamyl™, i.e., the B. licheniformis α-amylase having the amino acid sequence shown in SEQ ID NO:4 herein. In other words, all the following α-amylases which has the amino acid sequences shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 or 8 herein, or the amino acid sequence shown in SEQ ID NO: 1 of WO 95/26397 (the same as the amino acid sequence shown as SEQ ID NO: 7 herein) or in SEQ ID NO: 2 of WO 95/26397 (the same as the amino acid sequence shown as SEQ ID NO: 8 herein) or in Tsukamoto et al., 1988, (which amino acid sequence is shown in SEQ ID NO: 6 herein) are considered to be “Termamyl-like α-amylase”. Other Termamyl-like α-amylases are α-amylases i) which displays at least 60%, such as at least 70%, e.g., at least 75%, or at least 80%, e.g., at least 85%, at least 90% or at least 95% homology with at least one of said amino acid sequences shown in SEQ ID NOS: 1-8 and/or ii) displays immunological cross-reactivity with an antibody raised against at least one of said α-amylases, and/or iii) is encoded by a DMA sequence which hybridizes to the DNA sequences encoding the above-specified a amylases which are apparent from SEQ ID NOS: 9, 10, 11, or 12 of the present application (which encoding sequences encode the amino acid sequences shown in SEQ ID NOS: 1, 2, 3, 4 and 5 herein, respectively), from SEQ ID NO: 4 of WO 95/26397 (which DNA sequence, together with the stop codon TAA, is shown in SEQ ID NO: 13 herein and encodes the amino acid sequence shown in SEQ ID NO; 8 herein) and from SEQ ID NO: 5 of WO 95/26397 (shown in SEQ ID NO: 14 herein), respectively.
  • In connection with property i), 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, Wis., USA 53711).
  • A structural alignment between Termamyl (SEQ ID NO: 4) and a Termamyl-like α-amylase may be used to identify equivalent/corresponding positions in other Termamyl-like α-amylases. One method of obtaining said structural alignment is to use the Pile Up programme from the GCG package using default values of gap penalties, i.e., a gap creation penalty of 3.0 and gap extension penalty of 0.1. Other structural alignment methods include the hydrophobic cluster analysis (Gaboriaud et al., (1987), FSBS LETTERS 224, pp. 149-155) and reverse threading (Huber, T; Torda, A E, PROTEIN SCIENCE Vol. 7, No. 1 pp. 142-149 (1998).
  • Property ii) of the α-amylase, i.e., the immunological cross reactivity, may be assayed using an antibody raised against, or reactive with, at least one epitope of the relevant Termamyl-like α-amylase. The antibody, which may either be monoclonal or polyclonal, may be produced by methods known in the art, e.g., as described by Hudson et al., Practical Immunology, Third edition (1989), Blackwell Scientific Publications. The immunological cross-reactivity may be determined using assays known in the art, examples of which are Western Blotting or radial immunodiffusion assay, e.g., as described by Hudson et al., 1989. In this respect, immunological cross-reactivity between the α-amylases having the amino acid sequences SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, or 8, respectively, has been found.
  • The oligonucleotide probe used in the characterisation of the Termamyl-like α-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 α-amylase in question.
  • Suitable conditions for testing hybridisation involve pre-soaking in 5×SSC and prehybridising for 1 hour at −40° C. in a solution of 20% formamide, 5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 mg of denatured sonicated calf thymus DNA, followed by hybridisation in the same solution supplemented with 100 mM ATP for 18 hours at −40° C., followed by three times washing of the filter in 2×SSC, 0.2% SDS at 40° C. for 30 minutes (low stringency), preferred at 50° C. (medium stringency), more preferably at 65° C. (high stringency), even more preferably at ˜75° C. (very high stringency). More details about the hybridisation method can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.
  • In the present context, “derived from” is intended not only to indicate an α-amylase produced or producible by a strain of the organism in question, but also an α-amylase encoded by a DNA sequence isolated from such strain and produced in a host organism transformed with said DNA sequence. Finally, the term is intended to indicate an α-amylase which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the α-amylase in question. The term is also intended to indicate that the parent α-amylase may be a variant of a naturally occurring α-amylase, i.e. a variant which is the result of a modification (insertion, substitution, deletion) of one or more amino acid residues of the naturally occurring α-amylase.
    • Parent Hybrid α-Amylases
  • The parent α-amylase (i.e., backbone α-amylase) may be a hybrid α-amylase, i.e., an α-amylase which comprises a combination of partial amino acid sequences derived from at least two α-amylases.
  • The parent hybrid α-amylase may be one which on the basis of amino acid homology and/or immunological cross-reactivity and/or DNA hybridisation (as defined above) can be determined to belong to the Termamyl-like α-amylase family. In this case, the hybrid α-amylase is typically composed of at least one part of a Termamyl-like α-amylase and part(s) of one or more other α-amylases selected from Termamyl-like α-amylases or non-Termamyl-like α-amylases of microbial (bacterial or fungal) and/or mammalian origin.
  • Thus, the parent hybrid α-amylase may comprise a combination of partial amino acid sequences deriving from at least two Termamyl-like α-amylases, or from at least one Termamyl-like and at least one non-Termamyl-like bacterial α-amylase, or from at least one Termamyl-like and at least one fungal α-amylase. The Termamyl-like α-amylase from which a partial amino acid sequence derives may, e.g., be any of those specific Termamyl-like α-amylase referred to herein.
  • For instance, the parent α-amylase may comprise a C-terminal part: of an α-amylase derived from a strain of B. licheniformis, and a N-terminal part of an α-amylase derived from a strain of B. amyloliquefaciens or from a strain of B. stearothermophilus. For instance, the parent α-amylase may comprise at least 430 amino acid residues of the C-terminal part of the B. licheniformis α-amylase, and may, e.g., comprise a) an amino acid segment corresponding to the 37 N-terminal amino acid residues of the B. amyloliquefaciens α-amylase having the amino acid sequence shown in SEQ ID NO: 5 and an amino acid segment corresponding to the 445 C-terminal amino acid residues of the B. licheniformis α-amylase having the amino acid sequence shown in SEQ ID NO: 4, or a hybrid Termamyl-like α-amylase being identical to the Termamyl sequence, i.e., the Bacillus licheniformis α-amylase shown in SEQ ID NO: 4, except that the N-terminal 35 amino acid residues (of the mature protein) has been replaced by the N-terminal 33 residues of BAN (mature protein), i.e., the Bacillus amyloliquefaciens α-amylase shown in SEQ ID NO: 5; or b) an amino acid, segment corresponding to the 68 N-terminal amino acid residues of the B. stearothermophilus α-amylase having the amino acid sequence shown in SEQ ID NO: 3 and an amino acid segment corresponding to the 415 C-terminal amino acid residues of the B. licheniformis α-amylase having the amino acid sequence shown in SEQ ID NO: 4.
  • Another suitable parent hybrid α-amylase is the one previously described in WO 96/23374 (from Novo Nordisk) constituting the N-terminus of BAN, Bacillus amyloliquefaciens α-amylase (amino acids 1-300 of the mature protein) and the C-terminus from Termamyl (amino acids 301-483 of the mature protein). Increased activity was achieved by substituting one or more of the following positions of the above hybrid α-amylase (BAN:1-300/Termamyl:301-483); Q360, F290, and N102. Particularly interesting substitutions are one or more of the following substitutions: Q360E,D; F290A, C, D, E, G, H, I, K, L, M, N, P,Q, R,S,T; N102D, E;
  • The corresponding positions in the SP722 α-amylase shown in SEQ ID NO: 2 are one or more of: S365, Y2 95, N106. Corresponding substitutions of particular interest in said α-amylase shown in SEQ ID NO: 2 are one or more of: S365D,E; Y295A,C,D,E,G,H,I,K,L,M,N,P,Q,R,S,T; and N106D,E.
  • The corresponding positions in the SP690 α-amylase shown in SEQ ID NO: 1 are one or more of: S365, Y295, N106. The corresponding substitutions of particular interest are one or more of: S365D,E; Y295 A,C,D,E,G,H,I,K,L,M,N,P,Q,R,S,T; N106D.E.
  • The above mentioned non-Termamyl-like α-amylase may, e.g., be a fungal α-amylase, a mammalian or a plant α-amylase or a bacterial α-amylase (different from a Termamyl-like α-amylase). Specific examples of such α-amylases include the Aspergillus oryzae TAKA α-amylase, the A. niger acid α-amylase, the Bacillus subtilis α-amylase, the porcine pancreatic α-amylase and a barley α-amylase. All of these α-amylases have elucidated structures which are markedly different from the structure of a typical Termamyl-like α-amylase as referred to herein.
  • The fungal α-amylases mentioned above, i.e., derived from A. niger and A. oryzae, are highly homologous on the amino acid level and generally considered to belong to the same family of α-amylases. The fungal α-amylase derived from Aspergillus oryzae is commercially available under the tradename Fungamyl™.
  • Furthermore, when a particular variant of a Termamyl-like α-amylase (variant of the invention) is referred to—in a conventional manner—by reference to modification (e.g., deletion or substitution) of specific amino acid residues in the amino acid sequence of a specific Termamyl-like α-amylase, it is to be understood that variants of another Termamyl-like α-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.
  • In a preferred embodiment of the invention the α-amylase backbone is derived from B. licheniformis (as the parent Termamyl-like α-amylase), e.g., one of those referred to above, such as the B. licheniformis α-amylase having the amino acid sequence shown in SEQ ID NO: 4.
    • Altered Properties of Variants of the Invention
  • The following discusses the relationship between mutations which are present in variants of the invention, and desirable alterations in properties (relative to those a parent Termamyl-like α-amylase) which may result therefrom.
    • Improved Stability at pH 8-10.5
  • In the context of the present invention, mutations (including amino acid substitutions) of importance with respect to achieving improved stability at high pH (i.e., pH 8-10.5) include mutations corresponding to mutations in one or more of the following positions in SP722 α-amylase (having the amino acid sequence shown in SEQ ID NO: 2): T141, K142, F143, D144, F145, P146, G147, R148, G149, R181, A186, S193, N195, K269, N270, K311, K458, P459, T461.
  • The variant of the invention have one or more of the following substitutions (using the SEQ ID NO: 2 numbering):
  • T141A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, W, Y, V;
    K142A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S,
    T, W, Y, V;
    F143A, D, R, N, C, E, Q, G, H, I, L, K, M, P, S,
    T, W, Y, V;
    D144A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    F145A, D, R, N, C, E, Q, G, H, I, L, K, M, P, S,
    T, W, Y, V;
    P146A, D, R, N, C, E, Q, G, H, I, L, K, M, F, S,
    T, W, Y, V;
    G147A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    R148A, D, N, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    G149A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    K181A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S,
    T, W, Y, V;
    A186D, R, N, C, E, Q, G, H, I, L, P, K, M, F, S,
    T, W, Y, V;
    S193A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    T, W, Y, V;
    N195A, D, R, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    K269A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S,
    T, W, Y, V;
    N270A, D, R, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    K311A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S,
    T, W, Y, V;
    K458A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S,
    T, W, Y, V;
    P459A, D, R, N, C, E, Q, G, H, I, L, K, M, F, S,
    T, W, Y, V;
    T461A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, W, Y, V.
  • Preferred, high pH stability variants include one or more of the following substitutions in the SP722 α-amylase (having the amino acid sequence shown in SEQ ID NO: 2):
    • K142R, R1813, A186T, S193P, N195F, K269R, N270Y, K311R, K458R, P459T and T461P.
  • In specific embodiments the Bacillus strain NCIB 12512 α-amylase having the sequence shown in SEQ ID NO: 1, or the B. stearothermophilus α-amylase having the sequence shown in SEQ ID NO: 3, or the B. licheniformis α-amylase having the sequence shown in SEQ ID NO: 4, or the S, amyloliquefaciens α-amylase having the sequence shown in SEQ ID NO: 5 is used as the backbone, i.e., parent Termamyl-like α-amylase, for these mutations.
  • As can been seen from the alignment in FIG. 1 the B. stearothermophilus α-amylase already has a Tyrosine at position corresponding to N270 in SP722. Further, the Bacillus strain NCIB 12512 α-amylase, the B. stearothermophilus α-amylase, the B. licheniformis α-amylase and the B. amyloliquefaciens α-amylase already have Arginine at position corresponding to K458 in SP722. Furthermore, the B. licheniformis α-amylase already has a Proline at position corresponding to T461 in SP722. Therefore, for said α-amylases these substitutions are not relevant.
  • α-amylase variants with improved stability at high pH can be constructed by making substitutions in the regions found using the molecular dynamics simulation mentioned in Example 2. The simulation depicts the region(s) that has a higher flexibility or mobility at high pH (i.e., pH 8-10.5) when compared to medium pH.
  • By using the structure of any bacterial alpha-amylase with homology (as defined below) to the Termamyl-like α-amylase (BA2), of which the 3D structure is disclosed in Appendix 1 of WO 96/23874 (from Novo Nordisk), it is possible to model build the structure of such alpha-amylase and to subject it to molecular dynamics simulations. The homology of said bacterial α-amylase may be at least 60%, preferably be more than 70%, more preferably more than 80%, most preferably more than 90% homologous to the above mentioned Termamyl-like α-amylase (BA2), measured using the UWGCG GAP program from the GCG package version 7.3 (June 1993) using default values for GAP penalties [Genetic Computer Group (1991) Programme Manual for the GCG Package, version 7, 575 Science Drive, Madison, Wis., USA 53711], Substitution of the unfavorable residue for another would be applicable.
    • Improved Ca2+ Stability at pH 8-10.5.
  • Improved Ca stability means the stability of the enzyme under Ca+ depletion has been improved. In the context of the present invention, mutations (including amino acid substitutions) of importance with respect to achieving improved Ca2+ stability at high pH include mutation or deletion in one or more positions corresponding to the following positions in the SP722 α-amylase having the amino acid sequence shown in SEQ ID NO; 2: R181, G182, D183, G184, K185, A18S, W189, M195, N270, E346, K385, K458, P459.
  • A variant of the invention have one or more of the following substitutions or deletions:
  • R181*, A, D, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, T, W, Y, V;
    G182*, A, D, R, N, C, E, Q, H, I, L, K, M, F, P,
    S, T, W, Y, V;
    D183*, A, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, T, W, Y, V;
    G184*, A, R, D, N, C, E, Q, H, I, L, K, M, F, P,
    S, T, W, Y, V;
    K185A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S,
    T, W, Y, V;
    A186D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    W189A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, T, Y, V;
    N195A, D, R, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    N270A, R, D, N, C, E, Q, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    E346A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    K385A, R, D, N, C, E, Q, G, H, I, L, M, F, P, S,
    T, W, Y, V;
    K458A, R, D, N, C, E, Q, G, H, I, L, M, F, P, S,
    T, W, Y, V;
    P459A, R, D, N, C, E, Q, G, H, I, L, K, M, F, S,
    T, W, Y, V.
  • Preferred are variants having one or more of the following substitutions or deletions:
    • R181Q,N; G182T,S,N; D183*; G184*; K185A,R,D,C,E,Q,G,H,I,L,F,P,S,T,W,Y,V; A186T,S,N,I,V; W189T,S,N,Q; N195F, N27GR,D; E346Q; K385R; K458R; P459T.
  • In specific embodiments the Bacillus strain NCIB 12512 α-amylase having the sequence shown in SEQ ID NO: 1, or the B. amyloliquefaciens α-amylase having the sequence shown in SEQ ID NO: 5, or the B. licheniformis α-amylase having the sequence shown in SEQ ID NO: 4 are used as the backbone for these mutations.
  • As can been seen from the alignment in FIG. 1 the B. licheniformis α-amylase does not have the positions corresponding to D183 and G184 in SP722. Therefore for said α-amylases these deletions are not relevant.
  • In a preferred embodiment the variant is the Bacillus strain NCTB 12512 α-amylase with deletions in D183 and G184 and further one of the following substitutions: R181Q,N and/or G182T,S,N and/or D183*; G184* and/or
  • K185A,R, D, C, E, Q, G, H,I,L,M,N,F,P,S,T,W,Y,V and/or A186T,S,N,I,V and/or W189T,S,N,Q and/or N195F and/or N270R,D and/or E346Q and/or F385R and/or K458R and/or P459T,
    • Increased Specific Activity at Medium Temperature
  • In a further aspect of the present invention, important mutations with respect to obtaining variants exhibiting increased specific activity at temperatures from 10-60° C., preferably 20-50° C., especially 30-40° C., include mutations corresponding to one or more of the following positions in the SP722 α-amylase having the amino acid sequence shown in SEQ ID NO: 2:
  • H107, K108, G109, D166, W167, D168, Q169, S170, R171, Q172, F173, Q174, D183, G184, N195, F267, W268, K269, N270, D271, L272, G273, A274, L275, G456, N457, K458, P459, G460, T461, V462, T463.
  • The variant, of the invention have one or more of the following substitutions:
  • H107A, D, R, N, C, E, Q, G, I, L, K, M, F, P, S,
    T, W, Y, V;
    K108A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S,
    T, W, Y, V;
    G109A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    D166A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    W167A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, T, Y, V;
    D168A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    Q169A, D, R, N, C, E, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    S170A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    T, W, Y, V;
    R171A, D, N, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    Q172A, D, R, N, C, E, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    F173A, D, R, N, C, E, Q, G, H, I, L, K, M, P, S,
    T, W, Y, V;
    Q174*, A, D, R, N, C, E, G, H, I, L, K, M, F, P,
    S, T, W, Y, V;
    D183*, A, D, R, N, C, E, Q, G, H, I, L, K, M, F,
    P, S, W, Y, V;
    G184*, A, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, T, W, Y, V;
    N195A, D, R, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    F267A, D, R, N, C, E, Q, G, H, I, L, K, M, P, S,
    T, W, Y, V;
    W268A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, T, Y, V;
    K269A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S,
    T, W, Y, V;
    N270A, D, R, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    D271A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    L272A, D, R, N, C, E, Q, G, H, I, K, M, F, P, S,
    T, W, Y, V;
    G273A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    A274D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    L275A, D, R, N, C, E, Q, G, H, I, K, M, F, P, S,
    T, W, Y, V;
    G456A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    N457A, D, R, C, E, Q, G, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    K458A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S,
    T, W, Y, V;
    P459A, D, R, N, C, E, Q, G, H, I, L, K, M, F, S,
    T, W, Y, V;
    G460A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S,
    T, W, Y, V;
    T461A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, W, Y, V;
    V462A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, T, W, Y;
    T463A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P,
    S, W, Y, V.
  • Preferred variants has one or more of the following substitutions or deletions: □174*, D183*, G184*, K269S.
  • In a specific embodiment the B. licheniformis α-amylase having the sequence shown in SEQ ID NO: 4 is used as the backbone for these mutations.
    • General Mutations in Variants Invention: Increased Specific Activity at Medium Temperatures
  • The particularly interesting amino acid substitution are those that increase the mobility around the active site of the enzyme. This is accomplished by changes that disrupt stabilizing interaction in the vicinity of the active site, i.e., within preferably 10 Å or 8 Å or 6 Å or 4 Å from any of the residues constituting the active site.
  • Examples are mutations that reduce the size of side chains, such as
    • Ala to Gly, Val to Ala or Gly, Ile or Leu to Val, Ala, or Gly Thr to Ser
  • Such mutations are expected to cause increased flexibility in the active site region either by the introduction of cavities or by the structural rearrangements that fill the space left by the mutation.
  • It may be preferred that a variant of the invention comprises one or more modifications in addition to those outlined above. Thus, it may be advantageous that one or more Proline residues present in the part of the α-amylase variant which is modified is/are replaced with a non-Proline residue which may be any of the possible, naturally occurring non-Proline residues, and which preferably is an Alanine, Glycine, Serine, Threonine, Valine or Leucine.
  • Analogously, it may be preferred that one or more Cysteine residues present among the amino acid residues with which the parent α-amylase is modified is/are replaced with a non-Cysteine 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 α-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 point-mutations in any of the variants described herein.
    • α-Amylase Variants Having Increased Mobility Around the Active Site:
  • The mobility of α-amylase variants of the invention may be increased by replacing one or more amino acid residue at one or more positions close to the substrate site. These positions are (using the SP722 α-amylase (SEQ ID NO: 2) numbering): V56, K108, D168, Q169, Q172, L201, K269, L272, L275, K446, P45S.
  • Therefore, in an aspect the invention relates to variants being mutated in one or more of the above mentioned positions.
  • Preferred substitutions are one or more of the following:
  • V56A, G, S, T;
    K108A, D, E, Q, G, H, I, L, M, N, S, T, V;
    D168A, G, I, V, N, S, T;
    Q169A, D, G, H, I, L, M, N, S, T, V;
    Q172A, D, G, H, I, L, M, N, S, T, V;
    L201A, G, I, V, S, T;
    K269A, D, E, Q, G, H, I, L, M, N, S, T, V;
    L272A, G, I, V, S, T;
    L275A, G, I, V, S, T;
    Y295A, D, E, Q, G, H, I, L, M, N, F, S, T, V;
    K446A, D, E, Q, G, H, I, L, M, N, S, T, V;
    P459A, G, I, L, S, T, V.
  • In specific embodiments of the invention the Bacillus strain NCIB 12512 α-amylase having the sequence shown in SEQ ID NO: 1, or the B. stearothermophilus ex-amylase having the sequence shown in SEQ ID NO: 3, or the B. licheniformis α-amylase having the sequence shown in SEQ ID NO; 4, or the B. amyloliquefaciens α-amylase having the sequence shown in SEQ ID NO: 5 are used as the backbone for these mutations.
  • As can been seen from the alignment in FIG. 1 the B. licheniformis α-amylase and the B. amyloliquefaciens α-amylase have a Glut amine at position corresponding to K269 in SP722. Further, the B. stearothermophilus α-amylase has a Serine at position corresponding to K269 in SP722Therefore, for said α-amylases these substitutions are not relevant.
  • Furthermore, as can been seen from the alignment in FIG. 1 the B. amyloliquefaciens α-amylase has an Alanine at position corresponding to L272 in SP722, and the B. stearothermophilus α-amylase has a Isoleucine at the position corresponding to L272 in SP722. Therefore, for said α-amylases these substitutions are not relevant.
  • As can been seen from the alignment in FIG. 1, the Bacillus strain 12512 α-amylase has a Isoleucine at position corresponding to L275 in SP722. Therefore for said α-amylase this substitution is not relevant.
  • As can been seen from the alignment in FIG. 1 the B. amyloliquefaciens α-amylase has a Phenylalanine at position corresponding to 295 in SP722. Further, the B. stearothermophilus α-amylase has an Asparagine at position corresponding to Y295 in SP722. Therefore, for said α-amylases these substitutions are not relevant.
  • As can been seen from the alignment in FIG. 1 the B. licheniformis α-amylase and the B. amyloliquefaciens α-amylase have a Asparagine at position corresponding to K446 in SP722. Further, the B. stearothermophilus α-amylase has a Histidine at position corresponding to K446 in SP722. Therefore, for said a-amylases these substitutions are not relevant.
  • As can been seen from the alignment in FIG. 1 the B. licheniformis α-amylase, the B. amyloliquefaciens α-amylase and the B. stearothermophilus α-amylase have a Serine at position corresponding to P459 in SP722. Further, the Bacillus strain 12512α-amylase has a Threonine at position corresponding to P459 in SP722. Therefore, for said α-amylases these substitutions are not relevant.
    • Stabilization of Enzymes Having High Activity at Medium Temperatures.
  • In a further embodiment, the invention relates to improving the stability of low temperature α-amylases (e.g., Alteromonas haloplanctis (Feller et al., (1994), Eur. J. Biochem 222:441-447), and medium temperature α-amylases (e.g., SP722 and SP690) possessing medium temperature activity, i.e., commonly known as psychrophilic enzymes and mesophilic enzymes. The stability can for this particular enzyme class be understood either as thermostability or the stability at Calcium depletion conditions.
  • Typically, enzymes displaying the high activity at medium temperatures also display severe problems under conditions that stress the enzyme, such as temperature or Calcium depletion.
  • Consequently, the objective is to provide enzymes that at the same time display the desired high activity at medium temperatures without loosing their activity under slightly stressed conditions.
  • The activity of the stabilized variant measured at medium temperatures should preferably be between 100% or more and 50%, and more preferably between 100% or more and 70%, and most preferably between 100% or more and 85% of the original activity at that specific temperature before stabilization of the enzyme and the resulting enzyme should withstand longer incubation at stressed condition than the wild type enzyme.
  • Contemplated enzymes include α-amylases of, e.g., bacterial or fungal origin.
  • An example of such a low temperature α-amylase is the one isolated from Alteromonas haloplanctis (Feller et. al., (1994), Eur. J. Biochem 222:441-447). The crystal structure of this alpha-amylase has been solved (Aghajari et al., (1998), Protein Science 7:564-572).
  • The A. haloplanctis alpha-amylase (5 in alignment shown in FIG. 4) has a homology of approximately 66% to porcine pancreatic alpha-amylase (PPA) (3 in the alignment shown in FIG. 4). The PPA 3D structure is known, and can be obtained from Brookhaven database under the name 1OSE or 1DHK. Based on the homology to other mare stable alpha amylases, stabilization of “the low temperature highly active enzyme” from Alteromonas haloplanctis alpha-amylase, can be obtained and at the same time retaining the desired high activity at medium temperatures.
  • FIG. 4 shown a multiple sequence alignments of five α-amylases, including the AHA and the PPA α-amylase. Specific mutations giving increased stability in Alteromoxias haloplantis alpha-amylase:
    • T66P, Q69P, R155P, Q177R, A205P, A232P, L243R, V295P, S315R. Methods for Preparing α-Amylase Variants
  • Several methods for introducing mutations into genes are known in the art. After a brief discussion of the cloning of α-amylase-encoding DNA sequences, methods for generating mutations at specific sites within the α-amylase-encoding sequence will be discussed.
  • Cloning a DNA Sequence Encoding an α-AmylaseCloning a DMA, Sequence Encoding an a-AmylaseCloning a DNA Sequence Encoding an a-AmylaseCloning a DNA Sequence Encoding an a-AmylaseCloning a DNA Sequence Encoding an a-AmylaseCloning a DNA Sequence Encoding an a-AmylaseCloning a DNA Sequence Encoding an a-AmylaseCloning a DNA Sequence Encoding an a-AmylaseCloning a DNA Sequence Encoding an a-Amylase
  • The DNA sequence encoding a parent α-amylase may be isolated from any cell or microorganism producing the α-amylase 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 α-amylase to be studied. Then, if the amino acid sequence of the α-amylase is known, homologous, labeled oligonucleotide probes may be synthesized and used to identify α-amylase-encoding clones from a genomic library prepared from the organism in question. Alternatively, a labeled oligonucleotide probe containing sequences homologous to a known α-amylase gene could be used as a probe to identify α-amylase-encoding clones, using hybridisation and washing conditions of lower stringency.
  • Yet another method for identifying α-amylase-encoding clones would involve inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming α-amylase-negative bacteria with the resulting genomic DMA library, and then plating the transformed bacteria onto agar containing a substrate for α-amylase, thereby allowing clones expressing the α-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, e.g., in an automatic DMA 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 U.S. Pat. No. 4,683,202 or R. K. Saiki et al. (1988).
    • Expression of α-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 α-amylase variant of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or an artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • In the vector, the DNA sequence should be operably 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 suitable promoters for directing the transcription of the DNA sequence encoding an α-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 α-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens α-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 aspartie proteinase, A. niger neutral α-amylase, A. niger acid stable α-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.
  • The expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, poly-adenylation sequences operably connected to the DNA sequence encoding the α-amylase variant, of the invention. Termination and polyadenyiation sequences may suitably be derived from the same sources as the promoter.
  • The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pJJ702.
  • The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B. licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. Furthermore, 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 cotransformation, e.g., as described in WO 91/17243.
  • While intracellular expression may be advantageous in some respects, e.g., when using certain bacteria as host cells, it is generally preferred that the expression is extracellular. In general, the Bacillus α-amylases mentioned herein comprise a pre-region permitting secretion of the expressed protease into the culture medium. If desirable, this preregion may be replaced by a different preregion or signal sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective preregions.
  • The procedures used to ligate the DNA construct of the invention encoding an α-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 α-amylase variant of the invention. The cell may be transformed with the DNA construct of the invention encoding the variant, conveniently by integrating the DMA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.
  • The cell of the invention may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g. a bacterial or a fungal (including yeast) cell.
  • Examples of suitable bacteria are Gram positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalo-philus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyces lividans or Streptomyces murinus, or gramnegative bacteria such as E. coli. The transformation of the bacteria may, for instance, be effected by protoplast transformation or by using competent cells in a manner known per se.
  • The yeast organism may favorably be selected from a species of Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae. The filamentous fungus may advantageously belong to a species of Aspergillus, e.g., Aspergillus oryzae or Aspergillus niger. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023.
  • In a yet further aspect, the present invention relates to a method of producing an α-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 α-amylase variant of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. as described in catalogues of the American Type Culture Collection).
  • The α-amylase variant secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
    • INDUSTRIAL APPLICATIONS
  • The α-amylase variants of this invention possesses 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 from starch, and/or for textile desizing. Conditions for conventional starch-conversion processes, including starch liquefaction and/or saccharification processes, are described in, e.g., U.S. Pat. No. 3,912,590 and in BP patent publications Nos. 252,730 and 63,909,
    • Detergent Compositions
  • As mentioned above, variants of the invention may suitably be incorporated in detergent, compositions. Reference is made, for example, to WO 96/23874 and WO 97/07202 for further details concerning relevant ingredients of detergent compositions (such as laundry or dishwashing detergents), appropriate methods of formulating the variants in such detergent compositions, and for examples of relevant types of detergent compositions.
  • Detergent compositions comprising a variant of the invention may additionally comprise one or more other enzymes, such as a lipase, cutinase, protease, cellulase, peroxidase or laccase, and/or another α-amylase.
  • α-amylase variants of the invention may be incorporated in detergents at conventionally employed concentrations. It is at present contemplated that a variant of the invention may be incorporated in an amount corresponding to 0.00001-1 mg (calculated as pure, active enzyme protein) of α-amylase per liter of wash/dishwash liquor using conventional dosing levels of detergent.
  • The invention also relates to a method of providing α-amylases with 1) altered pH optimum, and/or 2) altered temperature optimum, and/or 3) improved stability, comprising the following steps:
  • i) identifying (a) target position(s) and/or region(s) for mutation of the α-amylase by comparing the molecular dynamics of two or more α-amylase 3D structures having substantially different pH, temperature and/or stability profiles,
    ii) substituting, adding and/or deleting one or more amino acids in the identified position(s) and/or region(s).
  • In embodiment of the invention a medium temperature α-amylase is compared with a high temperature α-amylase. In another embodiment a low temperature α-amylase is compared with either a medium or a high temperature α-amylase.
  • The α-amylases compared should preferably be at least 70%, preferably 80%, up to 90%, such as up to 95%, especially 95% homologous with each other.
  • The α-amylases compared may be Termamyl-like α-amylases as defined above. In specific embodiment the α-amylases compared are the α-amylases shown in SEQ ID NO; 1 to SEQ ID NO: 8.
  • In another embodiment the stability profile of the α-amylases in question compared are the Ca2+ dependency profile.
    • Materials and Methods Enzymes:
  • SP722: (SEQ ID NO: 2, available from Novo Nordisk)
    Termamyl™ (SEQ ID NO: 4, available from Novo Nordisk)
    SP690: (SEQ ID NO: 1, available from Novo Nordisk)
    Bacillus subtilis SHA273: see WO 95/10603
    • Plasmids
  • pJE1 contains the gene encoding a variant of SP722 α-amylase (SEQ ID NO: 2): vis. deletion of 6 nucleotides corresponding to amino acids D183-G184 in the mature protein. Transcription of the JE1 gene is directed from the amyL promoter. The plasmid further more contains the origin of replication and cat-gene conferring resistance towards kanamycin obtained from plasmid pUB110 (Gryczan, T J et al. (1978), J. Bact. 134:318-329).
    • Methods:
  • Construction of Library Vector pDorK101
  • The E. coli/Bacillus shuttle vector pDorK101 (described below) can be used to introduce mutations without expression of α-amylase in E. coli and then be modified in such way that the α-amylase is active in Bacillus. The vector was constructed as follows: The JE1 encoding gene (SP722 with the deletion of D183-G184) was inactivated in pJE1 by gene interruption in the PstI site in the 5′coding region of the SEQ ID NO: 2: SP722 by a 1.2 kb fragment containing an E. coli origin of replication. This fragment was PCR amplified from the pUC19 (GenBank Accession #:X02514) using the forward primer: 5′-gacctgcagtcaggcaacta-3′ and the reverse primer: 5′-tagagtcgacctgcaggcat-3′. The PCR amplicon and the pJE1 vector were digested with PstI at 37° C. for 2 hours. The pJE1 vector fragment and the PCR fragment were ligated at room temperature, for 1 hour and transformed in E. coli by electrotransformation. The resulting vector is designated pDorK101.
    • Filter Screening Assays
  • The assay can be used to screening of Termamyl-like α-amylase variants having an improved stability at high pH compared to the parent enzyme and Termamyl-like α-amylase variants having an improved stability at high pH and medium temperatures compared to the parent enzyme depending of the screening temperature setting
    • High pH Filter Assay
  • Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67, Schleicher & Sehuell, Dassel, Germany)—and nitrocellulose filters (Protran-Ba 85, Schleicher & Sehuell, Dassel, Germany) on TY agar plates with 10 μg/ml kanamycin at 37° C. for at least 21 hours. The cellulose acetate layer is located on the TY agar plate.
  • Each filter sandwich is specifically marked with a needle after plating, but before incubation in order to be able to localize positive variants on the filter and the nitrocellulose filter with bound variants is transferred to a container with glycin-NaOH buffer, pH 8.6-10.6 and incubated at room temperature (can be altered from 10°-60° C.) for 15 min. The cellulose acetate filters with colonies are stored on the TY-plates at room temperature until use. After incubation, residual activity is detected on plates containing 1% agarose, 0.2% starch in glycin-NaOH buffer, pH 8.6-10.6. The assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for 2 hours, at room temperature. After removal of the filters the assay plates are stained with 10% Lugol solution. Starch degrading variants are detected as white spots on dark blue background and then identified on the storage plates. Positive variants are rescreened twice under the same conditions as the first screen.
    • Low Calcium Filter Assay
  • The Bacillus library are plated on a sandwich of cellulose acetate (OE 67, Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plates with a relevant antibiotic, e.g., kanatnycin or chloramphenicol, at 37° C. for at least 21 hours. The cellulose acetate layer is located on the TY agar plate.
  • Each filter sandwich is specifically marked with a needle after plating, but before incubation in order to be able to localize positive variants on the filter and the nitrocellulose filter with bound variants is transferred to a container with carbonate/bicarbonate buffer pH 8.5-10 and with different EDTA concentrations (0.001 mM-100 mM). The filters are incubated at room temperature for 1 hour. The cellulose acetate filters with colonies are stored on the TY-plates at room temperature until use. After incubation, residual activity is detected on plates containing 1% agarose, 0.2% starch in carbonate/bicarbonate buffer pH 8.5-10. The assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for 2 hours, at room temperature. After removal of the filters the assay plates are stained with 10% Lugol solution. Starch degrading variants are detected as white spots on dark blue background and then identified on the storage plates. Positive variants are rescreened twice under the same conditions as the first screen.
    • Method to Obtaining the Regions of Interest:
  • There are three known 3D structures of bacterial α-amylases. Two of B. licheniformis α-amylase, Brookhaven database 1BPL (Machius et al. (1995), J. Mol. Biol. 246, p. 545-559) and 1VJS (Song et al. (1996), Enzymes for Carbohydrate 163 Engineering (Prog. Biotechnol. V 12). These two structures are lacking an important piece of the structure from the so-called B-domain, in the area around the two Calcium ions and one Sodium ion binding sites. We have therefore used a 3D structure of an α-amylase BA2 (WO 96/23874 which are a hybrid between BAN™ (SEQ ID NO, 5) and B. licheniformis α-amylase (SEQ ID NO. 4). On basis of the structure a model of B. licheniformis alpha amylase and the SP722α-amylase has been build.
  • Fermentation and Purification of α-amylase Variants
  • Fermentation and purification may be performed by methods well known in the art.
    • Stability Determination
  • All stability trials are made using the same set up. The method are:
  • The enzyme is incubated under the relevant conditions (1-4). Samples are taken at various time points, e.g., after 0, 5, 10, 15 and 30 minutes and diluted 2 5 times (same dilution for all taken samples) in assay buffer (0.1M 50 mM Britton buffer pH 7.3) and the activity is measured using the Phadebas assay (Pharmacia) under standard conditions pH 7.3, 37° C.
  • The activity measured before incubation (0 minutes) is used as reference (100%). The decline in percent is calculated as a function of the incubation time. The table shows the residual activity after, e.g., 30 minutes of incubation.
    • Specific Activity Determination
  • The specific activity is determined using the Phadebas assay (Pharmacia) as activity/mg enzyme. The manufactures instructions are followed (see also below under “Assay for α-amylase activity).
    • Assays for α-Amylase Activity 1. Phadebas Assay
  • α-amylase activity is determined by a method employing Phadebas® tablets as substrate. Phadebas tablets (Phadebas® Amylase Test, supplied by Pharmacia Diagnostic) contain a cross-linked insoluble blue-colored starch polymer which has been mixed with bovine serum albumin and a buffer substance and tabletted.
  • 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 CaCl2, pH adjusted to the value of interest with NaOH). The test is performed in a water bath at the temperature of interest. The α-amylase to be tested is diluted in x ml of 50 mM Britton-Robinson buffer. 1 ml of this α-amylase solution is added to the 5 ml 50 mM Britton-Robinson buffer. The starch is hydrolyzed by the α-amylase giving soluble blue fragments. The absorbance of the resulting blue solution, measured spectrophotometrically at 620 nm, is a function of the α-amylase activity.
  • It is important that the measured 620 nm absorbance after 10or 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 trig of a given α-amylase will hydrolyze a certain amount of substrate and a blue colour will be produced. The colour intensity is measured at 620 nm. The measured absorbance is directly proportional to the specific activity (activity/mg of pure α-amylase protein) of the α-amylase in question under the given set of conditions.
    • 2. Alternative Method
  • α-amylase activity is determined by a method employing the PNP-G7 substrate. PNP-G7 which is a abbreviation for p nitrophenyl-α,D-maltoheptaoside is a blocked oligosaccharide which can be cleaved by an endo-amylase. Following the cleavage, the α-Glucosidase included in the kit digest the substrate to liberate a free PNP molecule which has a yellow colour and thus can be measured by visible spectophometry at λ=405 nm. (400-420 nm.). Kits containing PNP-G7 substrate and α-Glucosidase is manufactured by Boehringer-Mannheim, (cat.No. 1054635).
  • To prepare the substrate one bottle of substrate (BM 1442309) is added to 5 ml buffer (BM1442309). To prepare the α-Glucosidase one bottle of α-Glueosidase (BM 1462309) is added to 45 ml buffer (BM1442309). The working solution is made by mixing 5 ml α-Glucosidase solution with 0.5 ml substrate.
  • The assay is performed by transforming 20 μl enzyme solution to a 96 well microtitre plate and incubating at 25° C. 200 μl working solution, 25° C. is added. The solution is mixed and pre-incubated 1 minute and absorption is measured every 15 sec. over 3 minutes at OD 405 nm.
  • The slope of the time dependent absorption-curve is directly proportional to the specific activity (activity per rag enzyme) of the α-amylase in question under the given set of conditions.
    • General Method for Random Mutagenesis by Use of the DOPE Program
  • The random mutagenesis may be carried out by the following steps:
  • 1. Select regions of interest for modification in the parent enzyme
    2. Decide on mutation sites and non-mutated sites in the selected region
    3. Decide on which kind of mutations should be carried out, e.g. with respect to the desired stability and/or performance of the variant to be constructed
    4. Select structurally reasonable mutations.
    5. Adjust the residues selected by step 3 with regard to step 4.
    6. Analyze by use of a suitable dope algorithm the nucleotide distribution.
    7. If necessary, adjust the wanted residues to genetic code realism (e.g., taking into account constraints resulting from the genetic code (e.g. in order to avoid introduction of stop codons))(the skilled person will be aware that some codon combinations cannot be used in practice and will need to be adapted)
    8. Make primers
    9. Perform random mutagenesis by use of the primers
    10. Select resulting α-amylase variants by screening for the desired improved properties.
  • Suitable dope algorithms for use in step 6 are well known in the art. One algorithm is described by Tomandl, D, et al., Journal of Computer-Aided Molecular Design, 11 (1997), pp. 29-38). Another algorithm, DOPE, is described in the following:
    • The Dope Program
  • The “DOPE” program is a computer algorithm useful to optimize the nucleotide composition of a codon triplet in such a way that it encodes an amino acid distribution which resembles most the wanted amino acid distribution. In order to assess which of the possible distributions is the most similar to the wanted amino acid distribution, a scoring function is needed. In the “Dope” program the following function was found to be suited:
  • s ≡ ∏ i = 1 N ( x i yi ( 1 - x i ) 1 - y i y i yi ( 1 - y i ) 1 - y i ) w i ,
  • where the xi's are the obtained amounts of amino acids and groups of amino acids as calculated by the program, yi's are the wanted amounts of amino acids and groups of amino acids as defined by the user of the program (e.g. specify which of the 20 amino acids or stop codons are wanted to be introduced, e.g. with a certain percentage (e.g. 90% Ala, 3% Ile, 7% Val), and wi's are assigned weight factors as defined by the user of the program (e.g., depending on the importance of having a specific amino acid residue inserted into the position in question). N is 21 plus the number of amino acid groups as defined by the user of the program. For purposes of this function 0° is defined as being 1.
  • A Monte-Carlo algorithm (one example being the one described by Valleau, J. P. & Whittington, S. G. (1977) A guide to Mont Carlo for statistical mechanics; 1 Highways. In “Stastistieal Mechanics, Part A” Equilibrium Techniqeues ed. B. J. Berne, New York: Plenum) is used for finding the maximum value of this function. In each Iteration the following steps are performed:
    • 1. A new random nucleotide composition is chosen for each base, where the absolute difference between the current and the new composition is smaller than or equal to d for each of the four nucleotides G,A,T,C in all three positions of the codon (see below for definition of d).
    • 2. The scores of the new composition and the current composition are compared by the use of the function s as described above. If the new score is higher or equal to the score of the current composition, the new composition is kept and the current composition is changed to the new one. If the new score is smaller, the probability of keeping the new composition is exp(1000)(new_score−current_score)).
  • A cycle normally consists of 1000 Iterations as described above in which d is decreasing linearly from 1 to 0. One hundred or more cycles are performed in an optimization process. The nucleotide composition resulting in the highest score is finally presented.
    • EXAMPLES Example 1 Example on Homology Building of Termamyl™
  • The overall homology of the B. licheniformis α-amylase (in the following referred to as Termamyl™) to other Termamyl-like α-amylases is high and the percent similarity is extremely high. The similarity calculated for Termamyl™ to BSG (the B. stearothermophilus α-amylase having SEQ ID NO: 3), and BAN (the B. amyloliquefaciens α-amylase having SEQ ID NO: 5) using the University of Wisconsin Genetics Computer Group's program GCG gave 89% and 78%, respectively. TERM has a deletion of 2 residues between residue G180 and K181 compared to BAN™ and BSG. BSG has a deletion of 3 residues between G371 and 1372 in comparison with BAN™ and Termamyl™. Further BSG has a C-terminal extension of more than 20 residues compared to BAN™ and Termamyl™. BAN™ has 2 residues less and Termamyl has one residue less in the N-terminal compared to BSG.
  • The structure of the B. licheniformis (Termamyl™) and of the B. amyloliquefaciens α-amylase (BAN™), respectively, was model built on the structure disclosed in Appendix 1 of WO 96/23974. The structure of other Termamyl-like α-amylases (e.g. those disclosed herein) may be built analogously.
  • In comparison with the α-amylase used for elucidating the present structure, Termamyl™ differs in that it lacks two residues around 178-182. In order to compensate for this in the model structure, the HOMOLOGY program from BIOSYM was used to substitute the residues in equivalent positions in the structure (not only structurally conserved regions) except for the deletion point. A peptide bond was established between G179(G177) and K180(K180) in Termamyl™ (BAN™), The close structural relationship between the solved structure and the model structure (and thus the validity of the latter) is indicated by the presence of only very few atoms found to be too close together in the model.
  • To this very rough structure of Termamyl™was then added all waters (605) and ions (4 Calcium and 1 Sodium) from the solved structure (See Appendix 1 of WO 96/23874) at the same coordinates as for said solved structure using the INSIGHT program. This could be done with only few overlaps—in other words with a very nice fit. This model structure were then minimized using 200 steps of Steepest descent and 600 steps of Conjugated gradient (see Brooks et al 1983, J. Computational Chemistry 4, p. 187-217). The minimized structure was then subjected to molecular dynamics, Bps heating followed by up to 200 ps equilibration but more than 3 Bps. The dynamics as run with the Verlet algorithm and the e-quilibration temperature 300K were kept using the Behrendsen coupling to a water bath (Berendsen et. al., 1984, J. Chemical Physics 81, p. 3684-3690). Rotations and translations were removed every pico second.
    • Example 2 Method of Extracting Important Regions for Identifying α-Amylase Variants with Improved pH Stability and Altered Temperature Activity
  • The X-ray structure and/or the model build structure of the enzyme of interest, here SP722 and Termamyl™ are subjected to molecular dynamics simulations. The molecular dynamics simulation are made using the CHARMM (from Molecular simulations (MSI)) program or other suited program like, e.g., DISCOVER (from MSI). The molecular dynamic analysis is made in vacuum, or more preferred including crystal waters, or with the enzyme embedded in water, e.g., a water sphere or a water box. The simulation are run for 300 pico seconds (ps) or more, e.g., 300-1200 ps. The isotropic fluctuations are extracted for the CA carbons of the structures and compared between the structures. Where the sequence has deletions and/or insertions the isotropic fluctuations from the other structure are inserted thus giving 0 as difference in isotropic fluctuation. For explanation of isotropic fluctuations see the CHARMM manual (obtainable from MSI).
  • The molecular dynamics simulation can be done using standard charges on the chargeable amino acids. This is Asp and Glu are negatively charged and Lys and Arg are positively charged. This condition resembles the medium pH of approximately 7. To analyze a higher or lower pH, titration of the molecule can be done to obtain the altered pKa's of the standard titrateable residues normally within pH 2-10; Lys, Arg, Asp, Glu, Tyr and His. Also Ser, Thr and Cys are titrateable but are not taking into account here. Here the altered charges due to the pH has been described as both Asp and Glu are negative at high pH, and both Arg and Lys are uncharged. This imitates a pH around 10 to 11 where the titration of Lys and Arg starts, as the normal pKa of these residues are around 9-11.
  • 1. The approach used for extracting important regions for identifying α-amylase variants with high pH stability:
  • The important regions for constructing variants with improved pH stability are the regions which at the extreme pH display the highest mobility, i.e., regions having the highest isotropic fluctuations.
  • Such regions are identified by carrying out two molecular dynamics simulations; i) a high pH run at which the basic amino acids, Lys and Arg, are seen as neutral (i.e. not protonated) and the acidic amino acids, Asp and Glu, have the charge (−1) and ii) a neutral pH run with the basic amino acids, Lys and Arg, having the net charge of (+1) and the acidic amino acids having a charge of (−1).
  • The two run are compared and regions displaying the relatively higher mobility at high pH compared to neutral pH analysis were identified.
  • Introduction of residues improving general stability, e.g., hydrogen bonding, making the region more rigid (by mutations such as Proline substitutions or replacement of Glycine residues), or improving the charges or their interaction, improves the high pH stability of the enzyme.
  • 2. The approach used for extracting regions for identifying α-amylase variants with increased activity at medium temperatures:
  • The important regions for constructing variants with increased activity at medium temperature was found as the difference between the isotropic fluctuations in SP722 and Termamyl, i.e., SP722 minus Termamyl CA isotrophic fluctuations, The regions with the highest mobility in the isotrophic fluctuations were selected. These regions and there residues were expected to increase the activity at medium temperatures. The activity of an alpha-amylase is only expressed if the correct mobility of certain residues are present. If the mobility of the residues is too low the activity is decreased or abandoned.
    • Example 3 Construction, by Localized Random, Doped Mutagenesis, of Termamyl-Like α-Amylase Variants having an Improved Ca2+ Stability at Medium Temperatures Compared to the Parent Enzymes
  • To improve the stability at low calcium concentration of α-amylases random mutagenesis in pre-selected region was performed.
    • Region: Residue: SAT: R181-W189
  • The DOPE software (see Materials and Methods) was used to determine spiked codons for each suggested change in the SA1 region minimizing the amount of stop codons (see table 1). The exact, distribution of nucleotides was calculated in the three positions of the codon to give the suggested population of amino acid changes. The doped regions were doped specifically in the indicated positions to have a high chance of getting the desired residues, but still allow other possibilities.
  • TABLE 1
    Distribution of amino acid residues for each position
    R181: 72% R, 2% N, 7% Q, 4% H, 4% K, 11% S
    G182: 73% G, 13% A, 12% S, 2% T
    K185: 95% K, 5% R
    A186: 50% A, 4% N, 6% D, 1% E, 1% G, 1% K, 5% S, 31% T
    W187: 100% W
    D188: 100% D
    W189: 92% W, 8% S
  • The resulting doped oligonucleotide strand is shown in table 2 as sense strand: with the wild type nucleotide and amino acid sequences and the distribution of nucleotides for each doped position.
  • TABLE 2
    Position 181 182 185 186 187 188 189
    Amino acid seq. Arg Gly Lys Ala Thr Asp Thr
    Wt nuc. seq. cga ggt aaa gct tgg gat tgg
    Forward primer:
    (SEQ ID NO: 15)
    FSA:
    5′-caa aat cgt atc tac aaa ttc 123 456 a7g 8910
    tgg gat t11g gaa gta gat tcg gaa aat-3′
    Distribution of nucleotides for each doped
    Position
     1: 35% A, 65% C
     2: 83% G, 17% A
     3: 63% G, 37% T
     4: 86% G, 14% A
     5: 85% G, 15% C
     6: 50% T, 50% C
     7: 95% A, 5%G
     8: 58% G, 37% A, 5% T
     9: 86% C, 13% A, 1% G
    10: 33% T, 17% G
    11: 92% G, 8% C
    Reverse primer:
    (SEQ ID NO: 16)
    RSA:
    5′-gaa ttt gta gat acg att ttg-3′
    • Random Mutagenesis
  • The spiked oligonucleotides apparent from Table 2 (which by a common term is designated FSA) and reverse primers RSA for the SA1 region and specific SEQ ID NO: 2: SP722 primers covering the SacII and the Drain sites are used to generate PGR-library-fragments by the overlap extension method (Horton et al., Gene, 77 (1989), pp. 61-68) with an overlap of 21 base pairs. Plasmid pJE1 is template for the Polymerase Chain Reaction. The PGR fragments are cloned in the E. coli/Bacillus shuttle vector pDork101 (see Materials and Methods) enabling mutagenesis in E. coli and immediate expression in Bacillus subtilis preventing lethal accumulation of amylases in E. coli. After establishing the cloned PCR fragments in E. coli, a modified pUC19 fragment is digested out of the plasmid and the promoter and the mutated Termamyl gene is physically connected and expression can take place in Bacillus.
    • Screening
  • The library may be screened in the low calcium filter assays described in the “Material and Methods” section above.
    • Example 4 Construction of Variants of Amylase SEQ ID NO: 1 (SP690)
  • The gene encoding the amylase from SEQ ID NO; 1 is located in a plasmid pTVB106 described in WO96/23873. The amylase is expressed from the amyL promoter in this construct in Bacillus subtilis.
  • A variant of the protein is delta(T183-G184)+Y243F+Q3 91E+K444Q. Construction of this variant is described in WO96/23873.
  • Construction of delta(T183-G184)+N195F by the mega-primer method as described by Sarkar and Sommer, (1990), BioTechniques 8: 404-407.
  • Gene specific primer B1 (SEQ ID NO: 17) and mutagenic primer 101458 (SEQ ID NO: 19) were used to amplify by PCR an approximately 645 bp DNA fragment from a pTVB106-like plasmid (with the delta(T183-G184) mutations in the gene encoding the amylase from SEQ ID NO: 1).
  • The 645 bp fragment was purified from an agarose gel and used as a mega-primer together with primer Y2 (SEQ ID NO: 18) in a second PCR carried out on the same template.
  • The resulting approximately 1080 bp fragment was digested with restriction enzymes BstEII and AflIII and the resulting approximately 510 bp DNA fragment was purified and ligated with the pTVB106-like plasmid (with the delta (T183-G184) mutations in the gene encoding the amylase from SEQ ID NO: 1) digested with the same enzymes. Competent Bacillus subtilis SHA273 (amylase and protease low) cells were transformed with the ligation and Chlorampenicol resistant transformants and was checked by DNA sequencing to verify the presence of the correct mutations on the plasmid.
  • primer B1: (SEQ ID NO: 17)
    5′ CGA TTG CTG ACG CTG TTA TTT GCG 3′
    primer Y2: (SEQ ID NO: 18)
    5′ CTT GTT CCC TTG TCA GAA CCA ATG 3′
    primer 101458 (SEQ ID NO: 19):
    5′ GT CAT AGT TGC CGA AAT CTG TAT CGA CTT C 3′
  • The construction of variant: delta(T183-G184)+K185R+A186T was carried out in a similar way except that mutagenic primer 101638 was used.
  • primer 101638: (SEQ ID NO: 20)
    5′ CC CAG TCC CAC GTA CGT CCC CTG AAT TTA TAT ATT
    TTG
    3′
  • Variants: delta(T183-G184)+A186T, delta(T183-G184)+A186I, delta(T183-G184)+A186S, delta(T183-G184)+A186N are constructed by a similar method except that pTVB106-like plasmid (carrying variant delta(T183-G184)+K185R+A186T) is used as template and as the vector for the cloning purpose. The mutagenic oligonucleotide (Oligo 1) is:
  • (SEQ ID NO: 21)
    5′ CC CAG TCC CAG NTCTTT CCC CTG AAT TTA TAT ATT
    TTG
    3′
  • N represents a mixture of the four bases: A, C, G, and T used in the synthesis of the mutagenicoli-gonucleotide.
    • Sequencing of Transformants Identifies the Correct Codon for Amino Acid Position 186 in the Mature Amylase.
  • variant: delta(T183-G184)+K185R+A186T+N195F is constructed as follows:
  • PGR is carried out with primer x2 (SEQ ID NO: 22) and primer 101458 (SEQ ID NO: 19) on pTVB106-like plasmid (with mutations delta(T183-G184)+K185R+A186T). The resulting DNA fragment is used as a mega-primer together with primer Y2 (SEQ ID NO: 18) in a PGR on pTVB106-like plasmid (with mutations delta(T183-G184)+N195). The product of the second PGR is digested with restriction endonucleases Acc651 and AflIII and cloned into pTVB106 like plasmid (delta(T183-G184)+N195F) digested with the same enzymes.
  • primer x2: (SEQ ID NO: 22)
    5′ GCG TGG ACA AAG TTT GAT TTT CCT G 3′
  • Variant: delta(T183-G184)+K185R+A186T+N195F+Y243F+Q391E+K444Q is constructed as follows:
  • PGR is carried out with primer x2 and primer 101458 on pTVB106-like plasmid (with mutations delta(T183-G184)+K185R+A186T). The resulting DNA fragment is used as a mega-primer together with primer Y2 in a PGR on pTVB106 like plasmid (with mutations delta(T183-G184)+Y243F+Q39TE+K444Q). The product of the second PGR is digested with restriction endonucleases Acc65I and AflIII and cloned into pTVB106 like plasmid (delta(T183-G184)+Y243F+Q391E+K444Q) digested with the same enzymes.
    • Example 5 Construction of Site-Directed α-Amylase Variants in the Parent SP722 α-Amylase (SEQ ID NO: 2)
  • Construction of variants of amylase SEQ ID NO: 2 (SP722) is carried out as described below.
  • The gene encoding the amylase from SEQ ID NO: 2 is located in a plasmid pTVB112 described in WO 96/23873. The amylase is expressed from the amyL promoter in this construct in Bacillus subtilis.
  • Construction of delta(D183-G184)+V56I by the mega-primer method as described by Sarkar and Sommer, 1990 (BioTechniques 8: 404-407).
  • Gene specific primer DAQ3 and mutagenic primer DA07 are used to amplify by PGR an approximately 820 bp DNA fragment from a pTVB112-like plasmid (with the delta(D183-G184) mutations in the gene encoding the α-amylase shown in SEQ ID NO: 2.
  • The 820 bp fragment is purified from an agarose gel and used as a mega-primer together with primer DA01 in a second PGR carried out on the same template.
  • The resulting approximately 920 bp fragment is digested with restriction enzymes NgoM I and Aat II and the resulting approximately 170 bp DNA fragment is purified and ligated with the pTVB112-like plasmid (with the delta(D183-G184) mutations in the gene encoding the amylase shown in SEQ ID NO: 2) digested with the same enzymes. Competent Bacillus subtilis SHA273 (amylase and protease low) cells are transformed with the ligation and Chlorampenicol resistant transformants are checked by DNA sequencing to verify the presence of the correct mutations on the plasmid.
  • primer DA01: (SEQ ID NO: 22)
    5′ CCTAATGATGGGAATCACTGG 3′
    primer DA03: (SEQ ID NO: 24)
    5′ GCATTGGATGCTTTTGAACAACCG 3′
    primer DA07 (SEQ ID NO: 25):
    5′ CGCAAAATGATATCGGGTATGGAGCC 3′

    Variants: delta(D183-G184)+K108L, delta(D183-G184)+K108Q, delta(D183-G184)+K108E, delta(D183-G184)+K108V, were constructed by the mega-primer method as described by Sarkar and Sommer, 1990 (BioTechniques 8; 404-407):
  • PCR is carried out with primer DA03 and mutagenesis primer DA20 on pTVB112-like plasmid (with mutations delta(D183-G184)). The resulting DNA fragment is used as a mega-primer together with primer DA01 in a PCR on pTVB112-like plasmid (with mutations delta(D183-G184)). The approximately 920 bp product of the second PCR is digested with restriction endonucleases Aat II and Mlu I and cloned into pTVB112-like plasmid (delta(D183-G1845) digested with the same enzymes,
  • primer DA20 (SQ ID NO: 26):
    5′ GTGATGAACCACSWAGGTGGAGCTGATGC 3′
  • S represents a mixture of the two bases: C and G used in the synthesis of the mutagenic oligonucleotide and W represents a mixture of the two bases: A and T used in the synthesis of the mutagenic oligonucleotide.
  • Sequencing of transformants identifies the correct codon for amino acid position 108 in the mature amylase.
  • Construction of the variants; delta(D183-G184)+D168A, delta(D183-G184)+D168I, delta(D183-G184)+D168V, delta(D183-G184)+D168T is carried out in a similar way except that mutagenic primer DA14 is used.
  • primer DA14 (SEQ ID NO: 27):
    5′ GATGGTGTATGGRYCAATCACGACAATTCC 3′
  • R represents a mixture of the two bases; A and G used in the synthesis of the mutagenic oligonucleotide and Y represents a mixture of the two bases; C and T used in the synthesis of the mutagenic oligonucleotide.
  • Sequencing of transformants identifies the correct codon for amino acid position 168 in the mature amylase.
  • Construction of the variant: delta(D183-G184)+Q169N is carried out in a similar way except that mutagenic primer DA15 is used.
  • primer DA15 (SEQ ID NO: 28):
    5′ GGTGTATGGGATAACTCACGACAATTCC 3′
  • Construction of the variant; delta(D183-G184)+Q169L is carried out in a similar way except that mutagenic primer DA16 is used.
  • primer DA16 (SEQ ID NO: 29):
    5′ GGTGTATGGGATCTCTCACGACAATTCC 3′
  • Construction of the variant; delta(D183-G184)+Q172N is carried out in a similar way except that mutagenic primer DA17 is used.
  • primer DA17 (SEQ ID NO: 30):
    5′ GGGATCAATCACGAAATTTCCAAAATCGTATC 3′
  • Construction of the variant: delta(D183-G184)+Q172L is carried out in a similar way except that mutagenic primer DAIS is used.
  • primer DA18 (SEQ ID NO: 31):
    5′ GGGATCAATCACGACTCTTCCAAAATCGTATC 3′
  • Construction of the variant: delta(D183-G184)+L201I is carried out in a similar way except that mutagenic primer DA06 is used.
  • primer DA06 (SEQ ID NO: 32):
    5′ GGAAATTATGATTATATCATGTATGCAGATGTAG 3′
  • Construction of the variant: delta(D183-G184)+K269S is carried out in a similar way except that mutagenic primer DA09 is used.
  • primer DA09 (SEQ ID NO: 33):
    5′ GCTGAATTTTGGTCGAATGATTTAGGTGCC 3′
  • Construction of the variant; delta(D183-G184)+K269Q is carried out in a similar way except that mutagenic primer DA11 is used.
  • primer DA11 (SEQ ID NO: 34):
    5′ GCTGAATTTTGGTCGAATGATTTAGGTGCC 3′
  • Construction of the variant; delta(D183-G184)+N270Y is carried out in a similar way except that mutagenic primer DA21 is used.
  • primer DA21 (SEQ ID NO: 35):
    5′ GAATTTTGGAAGTACGATTTAGGTCGG 3′
  • Construction of the variants: delta(D183-G184)+L272A, delta(D183-G184)+L272I, delta(D183-G184)+L272V, delta(D183-G184)+L272T is carried out in a similar way except that mutagenic primer DA12 is used,
  • primer DA12 (SEQ ID NO: 36):
    5′ GGAAAAACGATRYCGGTGCCTTGGAGAAC 3′

    R represents a mixture of the two bases: A and G used in the synthesis of the mutagenic oligonucleotide and Y represents a mixture of the two bases: C and T used in the synthesis of the mutagenic oligonucleotide.
    Sequencing of transformants identifies the correct codon for amino acid position 272 in the mature amylase.
  • Construction of the variants: delta(D183-G184)+L275A, delta(D183-G184)+L275I, delta(D183-G184)+L275V, delta(D183-G184)+L275T is carried out in a similar way except that mutagenic primer DA13 is used.
  • primer DA13 (SEQ ID NO: 37):
    5′ GATTTAGGTGCCTRYCAGAACTATTTA 3′

    R represents a mixture of the two bases; A and G used in the synthesis of the mutagenic oligonucleotide and Y represents a mixture of the two bases; C and T used in the synthesis of the mutagenic oligonucleotide.
    Sequencing of transformants identifies the correct codon for amino acid position 275 in the mature amylase.
  • Construction of the variant: delta(D183-G184)+Y295E is carried out In a similar way except that mutagenic primer DA08 is used.
  • primer DA08 (SEQ ID NO: 38):
    5′ CCCCCTTCATGAGAATCTTTATAACG 3′
  • Construction of delta(D183-G184)+K446Q by the mega-primer method as described by Sarkar and Sommer, 1990 (BioTechniques 8: 404-407):
  • Gene specific primer DA04, annealing 214-231 bp downstream relative to the STOP-codon and mutagenic primer DA10 were used to amplify by PGR an approximately 350 bp DNA fragment from a pTVB112-like plasmid (with the delta(D183-G184) mutations in the gene encoding the amylase depicted in SEQ ID NO: 2).
  • The resulting DNA fragment is used as a mega-primer together with primer DA05 in a PGR on pTVB112 like plasmid (with mutations delta(D183-G184)). The app. 460 bp product of the second PGR is digested with restriction endonucleases SnaB I and Not I and cloned into pTVB112 like plasmid (delta(D183-G184)) digested with the same enzymes,
  • primer DA04 (SEQ ID NO: 39):
    5′ GAATCCGAACCTCATTACACATTCG 3′
    primer DA05 (SEQ ID NO: 40)
    5′ CGGATGGACTCGAGAAGGAAATACCACG 3′
    primer DA10 (SEQ ID NO: 41):
    5′ CGTAGGGCAAAATCAGGCCGGTCAAGTTTGG 3′
  • Construction of the variants: delta(D183-G184)+K458R is carried out in a similar way except that mutagenic primer DA22 is used.
  • primer DA22 (SEQ ID NO: 42):
    5′ CATAACTGGAAATCGCCCGGGAACAGTTACG 3′
  • Construction of the variants: delta(D183-G184)+P459S and delta(D183-G184)+P459T is carried out in a similar way except that mutagenic primer DA19 is used.
  • primer DA19 (SEQ ID NO: 43):
    5′ CTGGAAATAAAWCCGGAACAGTTACG 3′

    W represents a mixture of the two bases: A and T used in the synthesis of the mutagenic oligonucleotide.
    Sequencing of transformants identifies the correct codon for amino acid position 459 in the mature amylase.
  • Construction of the variants: delta(D183-G184)+T461P is carried out in a similar way except that mutagenic primer DA23 is used.
  • primer DA23 (SEQ ID NO: 44):
    5′ GGAAATAAACCAGGACCCGTTACGATCAATGC 3′
  • Construction of the variant: delta(D183-G184)+K142R is carried out in a similar way except that mutagenic primer DA32 is used.
  • Primer DA32 (SEQ ID NO: 45):
    5′ GAGGCTTGGACTAGGTTTGATTTTCCAG 3′
  • Construction of the variant; delta(D183-G184)+K269R is carried out in a similar way except that mutagenic primer DA31 is used.
  • Primer DA31 (SEQ ID NO: 46):
    5′ GCTGAATTTTGGCGCAATGATTTAGGTGCC 3′
    • Example 6 Construction of Site-Directed α-Amylase Variants in the Parent Termamyl α-Amylase (SEQ ID NO: 4)
  • The amyL gene, encoding the Termamyl α-amylase is located in plasmid pDN1528 described in WO 95/10603 (Novo Nordisk). Variants with substitutions N265R and N265D, respectively, of said parent, α-amylase are constructed by methods described in WO 97/41213 or by the “megaprimer” approach described above.
    • Mutagenic Oligonucleotides are:
  • Primer b11 for the N265R substitution:
  • (SEQ ID NO: 56)
    5′ PCC AGC GCG CCT AGG TCA CGC TGC CAA TAT TCA G

    Primer b12 for the N265D substitution;
  • (SEQ ID NO: 57)
    5′ PCC AGC GCG CCT AGG TCA TCC TGC CAA TAT TCA G

    P represents a phosphate group.
    • Example 7 Determination of pH Stability at Alkaline pH of Variants of the Parent α-Amylase having the Amino Acid Sequence Shown in SEQ ID NO: 2
  • In this series of analysis purified enzyme samples were used. The measurements were made using solutions of the respective variants in 100 mM CAPS buffer adjusted to pH 10.5. The solutions were incubated at 75° C.
  • After incubation for 20 and 30 min the residual activity was measured using the PNP-G7 assay (described in the “Materials and Methods” section above). The residual activity in the samples was measured using Britton Robinson buffer pH 7.3. The decline in residual activity was measured relative to a corresponding reference solution of the same enzyme at 0 minutes, which has not been incubated at high pH and 75° C.
  • The percentage of the initial activity as a function is shown in the table below for the parent enzyme (SEQ ID NO; 2) and for the variants in question.
  • Residual activity Residual activity
    Variant after 20 min after 30 min
    Δ(D183-G184) + M323L 56% 44%
    Δ(D183-G184) + M323L + R181S 67% 55%
    Δ(D183-G184) + M323L + A186T 62% 50%
  • In an other series of analysis culture supernatants were used. The measurements were made using solutions of the respective variants in 100 mM CAPS buffer adjusted to pH 10.5. The solutions were incubated at 80° C.
  • After incubation for 30 minutes the residual activity was measured using the Phadebas assay (described in the “Materials and Method” section above. The residual activity in the samples was measured using Britton Robinson buffer pH 7.3. The decline in residual activity was measured relative to a corresponding reference solution of the same enzyme at 0 minutes, which has not been incubated at high pH and 80° C.
  • The percentage of the initial activity as a function is shown in the table below for the parent enzyme (SEQ ID NO: 2) and for the variants in question,
  • Variant Residual activity after 30 min
    Δ(D183-G184) 4%
    Δ(D183-G184) + P459T 25%
    Δ(D183-G184) + K458R 31%
    Δ(D183-G184) + K311R 10%
    • Example 8 Determination of Calcium Stability at Alkaline pH of Variants of the Parent α-Amylase having the Amino Acid Sequence Shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:4 A: Calcium Stability of Variants of the Sequence in SEQ ID NO: 1
  • The measurement were made using solutions of the respective variants in 100 mM CAPS buffer adjusted to pH 10.5 to which polyphosphate was added (at time t=0) to give a final concentration of 2400 ppm. The solutions were incubated at 50° C.
  • After incubation for 20 and 30 minutes the residual activity was measured using the PNP-G7 assay (described above). The residual activity in the samples was measured using Britton Robinson buffer pH 7.3, The decline in residual activity was measured relative to a corresponding reference solution of the same enzyme at 0 minutes, which has not been incubated at high pH and 50° C.
  • The percentage of the initial activity as a function is shown in the table below for the parent enzyme (SEQ ID NO: 1) and for the variants in question,
  • Residual activity Residual activity
    Variant after 20 min after 30 min
    Δ(T183-G184) 32% 19%
    Δ(T183-G184) + A186T 36% 23%
    Δ(T183-G184) + K185R + A186T 45% 29%
    Δ(T183-G184) + A186I 35% 20%
    Δ(T183-G184) + N195F 44% n.d.
    n.d. = Not determinated
    • B: Calcium Stability of Variants of the Sequence in SEQ ID NO: 2
  • In this series of analysis purified samples of enzymes were used. The measurement were made using solutions of the respective variants in 100 mM CAPS buffer adjusted to pH 10.5 to which polyphosphate was added (at time t=0) to give a final concentration of 2400 ppm. The solutions were incubated at 50° C.
  • After incubation for 20 and 30 minutes the residual activity was measured using the PNP-G7 assay (described above). The residual activity in the samples was measured using Britton Robinson buffer pH 7.3. The decline in residual activity was measured relative to a corresponding reference solution of the same enzyme at 0 minutes, which has not been incubated at high pH and 50° C.
  • The percentage of the initial activity as a function is shown in the table below for the parent enzyme (SEQ ID NO: 2) and for the variants in question.
  • Residual activity Residual activity
    Variant after 20 min after 30 min
    Δ(D183-G184) + M323L 21% 13%
    Δ(D183-G184) + M323L + R181S 32% 19%
    Δ(D183-G184) + M323L + A186T 28% 17%
    Δ(D183-G184) + M323L + A186R 30% 18%
    Δ(D183-G184) 30% 20%
    Δ(D183-G184) + N195F 55% 44%
  • In this series of analysis culture supernatants were used. The measurement were made using solutions of the respective variants in 100 mM CAPS buffer adjusted to pH 10.5 to which polyphosphate was added (at time t=0) to give a final concentration of 2400 ppm. The solutions were Incubated at 50° C.
  • After incubation for 30 minutes the residual activity was measured using the Phadebas assay as described above. The residual activity in the samples was measured using Britton Robinson buffer pH 7.3. The decline in residual activity was measured relative to a corresponding reference solution of the same enzyme at 0 minutes, which has not been incubated at high pH and 50° C.
  • The percentage of the initial activity as a function is shown in the table below for the parent enzyme (SEQ ID NO: 2) and for the variants in question.
  • Variant Residual activity after 30 min
    Δ(D183-G184) 0%
    Δ(D183-G184) + P459T 19%
    Δ(D183-G184) + K458R 18%
    Δ(D183-G184) + T461P 13%
    Δ(D183-G184) + E346Q + K385R 4%
    • C: Calcium Stability of Variants of the Sequence in SEQ ID NO: 4
  • The measurement were made using solutions of the respective variants in 100 mM CAPS buffer adjusted to pH 10.5 to which polyphosphate was added (at time t0) to give a final concentration of 2400 ppm. The solutions were incubated at 60° C. for 20 minutes.
  • After incubation for 20 minutes the residual activity was measured using the PNP-G7 assay (described above). The residual activity in the samples was measured using Britton Robinson buffer pH 7.3. The decline in residual activity was measured relative to a corresponding reference solution of the same enzyme at 0 minutes, which has not been incubated at high pH and 60° C.
  • The percentage of the initial activity as a function is shown in the table below for the parent enzyme (SEQ ID NO: 4) and for the variants in question,
  • Residual
    activity after
    Variant 20 min
    Termamyl (SEQ ID NO: 4) 17%
    N265R 28%
    N265D 25%
    • Example 9 Activity Measurement at Medium Temperature of α-Amylases having the Amino Acid Sequence Shown in SEQ ID NO: 1.
  • A: α-Amylase activity of Variants of the Sequence in SEQ ID NO: 1
  • The measurement were made using solutions of the respective variants in 50 mM Britton Robinson buffer adjusted to pH 7.3 and using the Phadebas assay described above. The activity in the samples was measured at 37° C. using 50 mM Britton Robinson buffer pH 7.3 and at 25° C. using 50 mM CAPS buffer pH 10.5.
  • The temperature dependent activity and the percentage of the activity at 25° C. relative to the activity at 37° C. is shown in the table below for the parent enzyme (SEQ ID NO: 1) and for the variants in question.
  • NU(25° C.)/
    Variant NU/mg 25° C. NU/mg 37° C. NU(37° C.)
    SP690 1440 35000 4.1%
    Δ(T183-G184) 2900 40000 7.3%
    Δ(T183-G184) + K269S 1860 12000 15.5%
    Δ(Q174) 3830 38000 7.9%

    Another measurement was made using solutions of the respective variants in 50 mM Britton Robinson buffer adjusted to pH 7.3 and using the Phadebas assay described above. The activity in the samples was measured at 37° C. and 50° C. using 50 mM Britton Robinson buffer pH 7.3.
  • The temperature dependent activity and the percentage of the activity at 37° C. relative to the activity at 50° C. is shown in the table below for the parent enzyme (SEQ ID NO: 1) and for the variants in question.
  • NU(37° C.)/
    Variant NU/mg 37° C. NU/mg 50° C. NU(50° C.)
    SP690 (seq ID NO: 1) 13090 21669 60%
    K269Q 7804 10063 78%
    • B: αAmylase Activity of Variants of the Sequence in SEQ ID NO:2
  • The measurement were made using solutions of the respective variants in 50 mM Britton Robinson buffer adjusted to pH 7.3 and using the Phadebas assay described above. The activity in the samples was measured at both 25° C. and 37° C. using 50 mM Britton Robinson buffer pH 7.3.
  • The temperature dependent activity and the percentage of the activity at 25° C. relative to the activity at 37° C. Is shown in the table below for the parent enzyme (SEQ ID NO: 2) and for the variants in question.
  • NU/mg NU/mg NU(25° C.)/
    Variant 25° C. 37° C. NU(37° C.)
    Δ(D183-G184) + M323L 3049 10202 30%
    Δ(D183-G184) + M323L + R181S 18695 36436 51%
    • C: α-Amylase Activity of Variants of the Sequence in SEQ ID NO:4
  • The measurement were made using solutions of the respective variants in 50 mM Britton Robinson buffer adjusted to pH 7.3 and using the Phadebas assay described above. The activity in the samples was measured at both 37° C. using 50 mM Britton Robinson buffer pH 7.3 and at 60° C. using 50 mM CAPS buffer pH 10.5.
  • The temperature dependent activity and the percentage of the activity at 37° C. relative to the activity at 60° C. is shown in the table below for the parent enzyme (SEQ ID NO: 4) and for the variants in question.
  • Variant NU/mg 37° C. NU/mg 60° C. NU(37° C.)/NU(60° C.)
    Termamyl 7400 4350 170%
    Q264S 10000 4650 215%
    • Example 10 Construction of Variants of Parent Hybrid BAN:1-300/Termamyl:301-483 α-Amylase
  • Plasmid pTVB191 contains the gene encoding hybrid α-amylase BAN:1-300/Termamyl:301-483 as well as an origin of replication functional in Bacillus subtilis and the cat gene conferring chloramphenicol resistance.
  • Variant BM4 (F290E) was constructed using the megaprimer approach (Sarkar and Sommer, 1990) with plasmid pTVB191 as template.
  • Primer p1 (SEQ ID NO; 52) and mutagenic oligonucleotide bm4 (SEQ ID NO: 47) were used to amplify a 444 bp fragment with polymerase chain reaction (PCR) under standard conditions. This fragment was purified from an agarose gel and used as ‘Megaprimer’ in a second PCR with primer p2 (SEQ ID NO: 5.3) resulting in a 531 bp fragment. This fragment was digested with restriction endonucleases HinDIII and Tth111I. The 389 bp fragment produced by this was ligated into plasmid pTVB191 that had been cleaved with the same two enzymes. The resulting plasmid was transformed into S. subtilis SHA273. Chloramphenicol resistant clones were selected by growing the transformants on plates containing chloramphenicol as well as insoluble starch. Clones expressing an active α-amylase were isolated by selecting clones that formed halos after staining the plates with iodine vapour. The identity of the introduced mutations was confirmed by DNA sequencing.
  • Variants BM5(F290K), BM6(F290A), BM8(Q360E) and BM11(N102D) were constructed in a similar way. Details of their construction are given below.
    • Variant: BM5(F290K)
  • mutagenic oligonucleotide: bm5 (SEQ ID NO: 48)
    • Primer (1st PGR); p1 (SEQ ID NO; 52)
  • Size of resulting fragment: 444 bp
    • Primer (2nd PCR): p2 (SEQ ID NO: 53)
  • Restriction endonucleases: HinDIII, Tth111I
    Size of cleaved fragment: 389 bp
    • Variant: BM6(F290A)
  • mutagenic oligonucleotide; bm6 (SEQ ID NO: 49)
    • Primer (1st PGR): p1 (SEQ ID NO: 52)
  • Size of resulting fragment: 444 bp
    • Primer (2nd PGR): p2 (SEQ ID NO: 53)
  • Restriction endonucleases: HinDIII, Tth111I
    Size of cleaved fragment: 389 bp
    • Variant: BM8(Q360E)
  • mutagenic oligonucleotide; bm8 (SEQ ID NO: 50)
    • Primer (1st PGR): p1 (SEQ ID NO: 52)
  • Size of resulting fragment; 230 bp
    • Primer (2nd PGR); p2 (SEQ ID NO; 53)
  • Restriction endonucleases: HinDIII, Tth111I
    Size of cleaved fragment: 389 bp
    • Variant: BM11(N102D)
  • mutagenic oligonucleotide; bm11 (SEQ ID NO; 51)
    • Primer (1st PGR): p3 (SEQ ID NO: 54)
  • Size of resulting fragment; 577
    • Primer (2nd PGR): p4 (SEQ ID NO: 55)
  • Restriction endonucleases: HinDIII, PvuI
    Size of cleaved fragment: 576
    • Mutagenic Oligonucleotides:
  • bm4 (SEQ ID NO: 47): F290E
    primer
    5′ GTG TTT GAC GTC CCG CTT CAT GAG AAT TTA
    CAG G
    bm5 (SEQ ID NO: 48): F290K
    primer
    5′ GTG TTT GAC GTC CCG CTT CAT AAG AAT TTA
    CAG G
    bm6 (SEQ ID NO: 49): F290A
    primer
    5′ GTG TTT GAC GTC CCG CTT CAT GCC AAT TTA
    CAG G
    bm8 (SEQ ID NO: 50): Q360E
    primer
    5′ AGG GAA TCC GGA TAC CCT GAG GTT TTC TAC
    GG
    bm11 (SEQ ID NO: 51): N102D
    primer
    5′ GAT GTG GTT TTG GAT CAT AAG GCC GGC GCT
    GAT G
    • Other Primers:
  • (SEQ ID N0: 52)
    p1: 5′ CTG TTA TTA ATG CCG CCA AAC C
    (SEQ ID NO: 53)
    p2: 5′ G GAA AAG AAA TGT TTA CGG TTG CG
    (SEQ ID NO: 54)
    p3: 5′ G AAA TGA AGC GGA ACA TCA AAC ACG
    (SEQ ID NO: 55)
    p4: 5′ GTA TGA TTT AGG AGA ATT CC
    • Example 11 α-Amylase Activity at Alkaline pH of Variants of BAN:1-300/Termamyl:301-483 Hybrid α-Amylase
  • The measurements were made using solutions for the respective enzymes and utilizing the Phadebas assay (described above). The activity was measured after incubating for 15 minutes at 30° C. in 50 mM Britton-Robinson buffer adjusted to the indicated pH by NaOH,
  • NU/mg enzyme
    pH wt Q360E F290A F290K F290E N102D
    8.0 5300 7800 8300 4200 6600 6200
    9.0 1600 2700 3400 2100 1900 1900
    • REFERENCES CITED
    • Klein, C, et al., Biochemistry 1992, 31, 8740-8746,
    • Mizuno, H., et al., J. Mol. Biol. (1993) 234, 1282-1283,
    • Chang, C, et al, J. Mol. Biol. (1993) 229, 235-238,
    • Larson, S. B., J. Mol. Biol. (1994) 235, 1560-1584,
    • Lawson, C. L., J. Mol. Biol. (1994) 236, 590-600,
    • Qian, M., et al., J. Mol. Biol. (1993) 231, 785-799,
    • Brady, R. L., et al. Acta Crystallogr. sect. B, 47, 527-535,
    • Swift, H. J., 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
    • MacGregor, E. A., Food Hydrocolloids, 1987, Vol. 1, No. 5-6, p.
    • B. Diderichsen and L. Christiansen, Cloning of a maltogenic α-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
    • 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.

Claims (11)

1-39. (canceled)
40. A variant alpha-amylase, which variant has alpha-amylase activity, has at least 90% homology with the amino acid sequence shown in SEQ ID. NO. 3, and which comprises one or more of the following substitutions (using SEQ SD NO:3 for numbering):
(a) R179A, D, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V; (b) G180A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, or V; (c) I181A, R, N, C, E, Q, G, H, L, K, M, F, P, S, T, W, Y, or V; (d) G182A, R, D, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, or V.
41. A variant alpha-amylase, which variant has alpha-amylase activity, has at least 95% homology with the amino acid sequence shown in SEQ ID. NO. 3, and which comprises one or more of the following substitutions (using SEQ ID NO:3 for numbering):
(a) R179A, D, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or v; (b) G180A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, or V; (c) I181A, R, N, C, E, Q, G, H, L, K, M, F, P, S, T, W, Y, or V; (d) G182A, R, D, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, or V.
42. The variant of claim 40, wherein the variant comprises a substitution of R179A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, or V.
43. The variant of claim 40, wherein the variant comprises a substitution of G180A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y, or V.
44. The variant of claim 40, wherein the variant comprises a substitution of I181A,R,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y, or V.
45. The variant of claim 40, wherein the variant comprises a substitution of G182A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y, or V.
46. The variant of claim 41, wherein the variant comprises a substitution of R179A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,TlW,Y, or V.
47. The variant of claim 41, wherein the variant comprises a substitution of G180A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y, or V.
48. The variant of claim 41, wherein the variant comprises a substitution of I181A,R,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,or V.
49. The variant of claim 41, wherein the variant comprises a substitution of G182A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y, or V.
US11/953,532 1997-10-30 2007-12-10 Alpha-Amylase Mutants Abandoned US20090263881A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US11/953,532 US20090263881A1 (en) 1997-10-30 2007-12-10 Alpha-Amylase Mutants
US13/785,190 US20140017715A1 (en) 1997-10-30 2013-03-05 Alpha-amylase Mutants
US14/163,585 US20140248685A1 (en) 1997-10-30 2014-01-24 Alpha-amylase Mutants
US15/012,229 US20160222365A1 (en) 1997-10-30 2016-02-01 Alpha-amylase mutants

Applications Claiming Priority (11)

Application Number Priority Date Filing Date Title
DK1240/97 1997-10-30
DK124097 1997-10-30
US6466297P 1997-11-06 1997-11-06
DK199800936 1998-07-14
DKPA199800936 1998-07-14
US9323498P 1998-07-17 1998-07-17
US09/183,412 US6204232B1 (en) 1997-10-30 1998-10-30 α-amlase mutants
US09/769,864 US6673589B2 (en) 1997-10-30 2001-01-25 α-amylase mutants
US10/665,667 US20040038368A1 (en) 1997-10-30 2003-09-19 Alpha-amylase mutants
US10/980,923 US20050084937A1 (en) 1997-10-30 2004-11-04 Alpha-amylase mutants
US11/953,532 US20090263881A1 (en) 1997-10-30 2007-12-10 Alpha-Amylase Mutants

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US10/980,923 Division US20050084937A1 (en) 1997-10-30 2004-11-04 Alpha-amylase mutants

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US13/785,190 Division US20140017715A1 (en) 1997-10-30 2013-03-05 Alpha-amylase Mutants
US14/163,585 Continuation US20140248685A1 (en) 1997-10-30 2014-01-24 Alpha-amylase Mutants

Publications (1)

Publication Number Publication Date
US20090263881A1 true US20090263881A1 (en) 2009-10-22

Family

ID=55701615

Family Applications (8)

Application Number Title Priority Date Filing Date
US09/183,412 Expired - Lifetime US6204232B1 (en) 1997-10-30 1998-10-30 α-amlase mutants
US09/769,864 Expired - Lifetime US6673589B2 (en) 1997-10-30 2001-01-25 α-amylase mutants
US10/665,667 Abandoned US20040038368A1 (en) 1997-10-30 2003-09-19 Alpha-amylase mutants
US10/980,923 Abandoned US20050084937A1 (en) 1997-10-30 2004-11-04 Alpha-amylase mutants
US11/953,532 Abandoned US20090263881A1 (en) 1997-10-30 2007-12-10 Alpha-Amylase Mutants
US13/785,190 Abandoned US20140017715A1 (en) 1997-10-30 2013-03-05 Alpha-amylase Mutants
US14/163,585 Abandoned US20140248685A1 (en) 1997-10-30 2014-01-24 Alpha-amylase Mutants
US15/012,229 Abandoned US20160222365A1 (en) 1997-10-30 2016-02-01 Alpha-amylase mutants

Family Applications Before (4)

Application Number Title Priority Date Filing Date
US09/183,412 Expired - Lifetime US6204232B1 (en) 1997-10-30 1998-10-30 α-amlase mutants
US09/769,864 Expired - Lifetime US6673589B2 (en) 1997-10-30 2001-01-25 α-amylase mutants
US10/665,667 Abandoned US20040038368A1 (en) 1997-10-30 2003-09-19 Alpha-amylase mutants
US10/980,923 Abandoned US20050084937A1 (en) 1997-10-30 2004-11-04 Alpha-amylase mutants

Family Applications After (3)

Application Number Title Priority Date Filing Date
US13/785,190 Abandoned US20140017715A1 (en) 1997-10-30 2013-03-05 Alpha-amylase Mutants
US14/163,585 Abandoned US20140248685A1 (en) 1997-10-30 2014-01-24 Alpha-amylase Mutants
US15/012,229 Abandoned US20160222365A1 (en) 1997-10-30 2016-02-01 Alpha-amylase mutants

Country Status (11)

Country Link
US (8) US6204232B1 (en)
EP (4) EP2388267A1 (en)
JP (1) JP4426094B2 (en)
CN (2) CN1163597C (en)
AT (1) ATE490311T1 (en)
AU (1) AU9737398A (en)
BR (2) BRPI9816290B1 (en)
CA (2) CA2308119C (en)
DE (1) DE69842027D1 (en)
ES (1) ES2515218T3 (en)
WO (1) WO1999023211A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011084695A1 (en) 2009-12-21 2011-07-14 Novozymes, Inc. Methods for producing heterologous polypeptides in thiol-disulfide oxidoreductase-deficient bacterial mutant cells
WO2022074037A3 (en) * 2020-10-07 2022-07-07 Novozymes A/S Alpha-amylase variants

Families Citing this family (346)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6440716B1 (en) 1995-02-03 2002-08-27 Novozymes A/S α-amylase mutants
US6187576B1 (en) * 1997-10-13 2001-02-13 Novo Nordisk A/S α-amylase mutants
US6361989B1 (en) * 1997-10-13 2002-03-26 Novozymes A/S α-amylase and α-amylase variants
US6410295B1 (en) * 1999-03-30 2002-06-25 Novozymes A/S Alpha-amylase variants
JP4745503B2 (en) * 1999-03-31 2011-08-10 ノボザイムス アクティーゼルスカブ Polypeptides having alkaline α-amylase activity and nucleic acids encoding them
US6623948B1 (en) * 1999-03-31 2003-09-23 Novozymes A/S Nucleic acid sequences encoding alkaline alpha-amylases
CN1212395C (en) * 1999-06-10 2005-07-27 花王株式会社 Mutation alph-amylase
CN1654641A (en) * 1999-11-10 2005-08-17 诺维信公司 Fungamyl-like alpha-amylase variants
US7005288B1 (en) 1999-11-10 2006-02-28 Novozymes A/S Fungamyl-like alpha-amylase variants
US6350599B1 (en) 2000-01-12 2002-02-26 Novozymes A/S Pullulanase variants and methods for preparing such variants with predetermined properties
WO2001064852A1 (en) * 2000-03-03 2001-09-07 Novozymes A/S Polypeptides having alkaline alpha-amylase activity and nucleic acids encoding same
CN101532001A (en) * 2000-03-08 2009-09-16 诺维信公司 Variants with altered properties
DE10036752C2 (en) * 2000-07-28 2003-02-20 Henkel Kgaa Detergent and cleaning agent with a new amylolytic enzyme from Bacillus sp. A 7-7 (DSM 12368)
US7153818B2 (en) * 2000-07-28 2006-12-26 Henkel Kgaa Amylolytic enzyme extracted from bacillus sp. A 7-7 (DSM 12368) and washing and cleaning agents containing this novel amylolytic enzyme
US20020155574A1 (en) * 2000-08-01 2002-10-24 Novozymes A/S Alpha-amylase mutants with altered properties
JP4426716B2 (en) * 2000-10-11 2010-03-03 花王株式会社 High productivity α-amylase
PL361363A1 (en) 2000-11-28 2004-10-04 Henkel Kommanditgesellschaft Auf Aktien Novel cyclodextrin glucanotransferase (cgtase), obtained from bacillus agaradherens (dsm 9948) and detergents and cleaning agents containing said novel cyclodextrin glucanotransferase
CA2438205C (en) * 2001-02-21 2015-11-03 Diversa Corporation Enzymes having alpha amylase activity and methods of use thereof
EP3000881A3 (en) 2001-05-15 2016-07-20 Novozymes A/S Alpha-amylase variant with altered properties
DE10138753B4 (en) * 2001-08-07 2017-07-20 Henkel Ag & Co. Kgaa Detergents and cleaners with hybrid alpha-amylases
JP4897186B2 (en) * 2002-03-27 2012-03-14 花王株式会社 Mutant alkaline cellulase
US7226771B2 (en) 2002-04-19 2007-06-05 Diversa Corporation Phospholipases, nucleic acids encoding them and methods for making and using them
BR0309391A (en) 2002-04-19 2005-10-25 Diversa Corp Phospholipases, nucleic acids encoding them and methods for preparing and using them
BR0312121A (en) 2002-06-14 2007-04-03 Diversa Corp xinalases, nucleic acids encoding them and methods for preparing and employing them
MX282672B (en) * 2002-10-10 2011-01-10 Diversa Corp Proteases, nucleic acids encoding them and methods for making and using them.
DE10362172B4 (en) * 2003-03-05 2009-04-02 Henkel Ag & Co. Kgaa Detergents and cleaners with alpha-amylase variants with improved alkalinity
ES2713024T3 (en) 2003-03-06 2019-05-17 Basf Enzymes Llc Amylases, nucleic acids that encode them and methods for their manufacture and use
US8298799B2 (en) 2003-03-07 2012-10-30 Dsm Ip Assets B. V. Hydrolases, nucleic acids encoding them and methods for making and using them
US20070056053A1 (en) * 2003-03-20 2007-03-08 Diversa Corporation Glucosidases, nucleic acids encoding them and methods for making and using them
CA2521402C (en) 2003-04-04 2015-01-13 Diversa Corporation Pectate lyases, nucleic acids encoding them and methods for making and using them
PL1654355T3 (en) 2003-06-13 2010-09-30 Dupont Nutrition Biosci Aps Variant pseudomonas polypeptides having a non-maltogenic exoamylase activity and their use in preparing food products
AU2004254640B2 (en) 2003-07-02 2010-08-26 Bp Corporation North America Inc. Glucanases, nucleic acids encoding them and methods for making and using them
DE602004016658D1 (en) 2003-07-07 2008-10-30 Danisco As Danisco Intellectua VARIATIONS OF NON-MALTIC EXOAMYLASE FROM PSEUDOMONAS COMPLEMENTARY FOOD ADDITIVE
US8143048B2 (en) 2003-07-07 2012-03-27 Danisco A/S Exo-specific amylase polypeptides, nucleic acids encoding those polypeptides and uses thereof
US7741089B2 (en) 2003-08-11 2010-06-22 Verenium Corporation Laccases, nucleic acids encoding them and methods for making and using them
BRPI0512776A (en) * 2004-07-05 2008-04-08 Novozymes As originating termamyl alpha-amylase variant, DNA construct, recombinant expression vector, cell, composition, detergent additive, detergent composition, manual or automatic laundry composition, use of an alpha-amylase variant or composition, and method of producing a variant
BRPI0513297A (en) * 2004-07-07 2008-05-06 Danisco polypeptide
DE102004047777B4 (en) 2004-10-01 2018-05-09 Basf Se Alpha-amylase variants with increased solvent stability, process for their preparation and their use
EP1861506B1 (en) 2005-03-15 2015-04-15 BP Corporation North America Inc. Cellulases, nucleic acids encoding them and methods for making and using them
US8030050B2 (en) 2005-07-07 2011-10-04 Danisco A/S Modified amylases from Pseudomonas species
DE102006038448A1 (en) 2005-12-28 2008-02-21 Henkel Kgaa Enzyme-containing cleaning agent
EP1987142A4 (en) 2006-02-02 2009-07-15 Verenium Corp Esterases and related nucleic acids and methods
NZ595499A (en) 2006-02-10 2013-05-31 Verenium Corp Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them
US8043839B2 (en) 2006-02-14 2011-10-25 Verenium Corporation Xylanases, nucleic acids encoding them and methods for making and using them
CA2656313C (en) 2006-06-19 2018-07-03 Danisco A/S Ps4 exoamylase h307k/r variant
BRPI0714876B1 (en) 2006-08-04 2022-04-19 Verenium Corporation Isolated, synthetic or recombinant nucleic acid, expression cassette, cloning vector or vehicle, transformed bacterial, fungal or yeast cell, isolated, synthetic or recombinant polypeptide, composition, as well as production methods and uses thereof
WO2008036863A2 (en) 2006-09-21 2008-03-27 Verenium Corporation Phospholipases, nucleic acids encoding them and methods for making and using them
EP2479266B1 (en) 2006-12-21 2016-03-09 BASF Enzymes LLC Amylases and glucoamylases, nucleic acids encoding them and methods for making and using them
NZ598285A (en) 2007-01-30 2013-10-25 Syngenta Participations Ag Enzymes for the treatment of lignocellulosics, nucleic acids encoding them and methods for making and using them
BRPI0808513A2 (en) 2007-03-09 2014-08-19 Danisco Us Inc Genencor Div ALPHA-AMILASE VARIANTS OF ALKALIFYL BACILLUS SPECIES, COMPOSITIONS UNDERSTANDING ALPHA-AMYLASE VARIANTS AND METHODS OF USE
WO2008153935A2 (en) * 2007-06-06 2008-12-18 Danisco Us, Inc., Genencor Division Methods for improving protein properties
EP2205744B1 (en) 2007-10-03 2015-01-14 BP Corporation North America Inc. Xylanases, nucleic acids encoding them and methods for making and using them
US8066818B2 (en) 2008-02-08 2011-11-29 The Procter & Gamble Company Water-soluble pouch
US20090209447A1 (en) * 2008-02-15 2009-08-20 Michelle Meek Cleaning compositions
US20090233830A1 (en) 2008-03-14 2009-09-17 Penny Sue Dirr Automatic detergent dishwashing composition
EP2100947A1 (en) 2008-03-14 2009-09-16 The Procter and Gamble Company Automatic dishwashing detergent composition
US8153391B2 (en) 2008-08-29 2012-04-10 Bunge Oils, Inc. Hydrolases, nucleic acids encoding them and methods for making and using them
US8198062B2 (en) 2008-08-29 2012-06-12 Dsm Ip Assets B.V. Hydrolases, nucleic acids encoding them and methods for making and using them
US8357503B2 (en) 2008-08-29 2013-01-22 Bunge Oils, Inc. Hydrolases, nucleic acids encoding them and methods for making and using them
EP2166092A1 (en) 2008-09-18 2010-03-24 The Procter and Gamble Company Detergent composition
EP2166073A1 (en) * 2008-09-23 2010-03-24 The Procter & Gamble Company Cleaning composition
EP2166075A1 (en) 2008-09-23 2010-03-24 The Procter and Gamble Company Cleaning composition
EP2166076A1 (en) 2008-09-23 2010-03-24 The Procter & Gamble Company Cleaning composition
US20100125046A1 (en) 2008-11-20 2010-05-20 Denome Frank William Cleaning products
EP2216393B1 (en) 2009-02-09 2024-04-24 The Procter & Gamble Company Detergent composition
EP2401371A1 (en) 2009-02-27 2012-01-04 Novozymes A/S Mutant cells having reduced expression of metallopeptidase, suitable for recombinant polypeptide production
MX2012000480A (en) 2009-07-09 2012-01-27 Procter & Gamble A catalytic laundry detergent composition comprising relatively low levels of water-soluble electrolyte.
MX2012000486A (en) 2009-07-09 2012-01-27 Procter & Gamble A catalytic laundry detergent composition comprising relatively low levels of water-soluble electrolyte.
HUE029942T2 (en) 2009-08-13 2017-04-28 Procter & Gamble Method of laundering fabrics at low temperature
UA111708C2 (en) 2009-10-16 2016-06-10 Бандж Ойлз, Інк. METHOD OF OIL REFINING
UA109884C2 (en) 2009-10-16 2015-10-26 A POLYPEPTIDE THAT HAS THE ACTIVITY OF THE PHOSPHATIDYLINOSYTOL-SPECIFIC PHOSPHOLIPASE C, NUCLEIC ACID, AND METHOD OF METHOD
WO2011072117A1 (en) 2009-12-09 2011-06-16 The Procter & Gamble Company Fabric and home care products
EP2333039B2 (en) 2009-12-10 2020-11-11 The Procter & Gamble Company Method and use of a dishwasher composition
ES2399311T5 (en) 2009-12-10 2020-06-19 Procter & Gamble Detergent composition
PL2333042T3 (en) 2009-12-10 2015-12-31 Procter & Gamble Automatic dishwashing product and use thereof
EP2333041B1 (en) 2009-12-10 2013-05-15 The Procter & Gamble Company Method and use of a dishwasher composition
EP2521772A1 (en) 2010-01-04 2012-11-14 Novozymes A/S Alpha-amylases
EP3404087A1 (en) 2010-02-10 2018-11-21 Novozymes A/S Alpha-amylase variants with high stability in presence of a chelating agent
EP2357220A1 (en) * 2010-02-10 2011-08-17 The Procter & Gamble Company Cleaning composition comprising amylase variants with high stability in the presence of a chelating agent
PL2361964T3 (en) 2010-02-25 2013-05-31 Procter & Gamble Detergent composition
EP2380962B1 (en) 2010-04-23 2016-03-30 The Procter and Gamble Company Particle
PL2380963T3 (en) 2010-04-23 2016-07-29 Procter & Gamble Method of perfuming
EP2383329A1 (en) 2010-04-23 2011-11-02 The Procter & Gamble Company Particle
TR201810936T4 (en) 2010-04-23 2018-08-27 Industrial Chemicals Group Ltd Detergent composition.
ES2533368T3 (en) 2010-04-23 2015-04-09 The Procter & Gamble Company Dishwasher product
EP2380478A1 (en) 2010-04-23 2011-10-26 The Procter & Gamble Company Automatic dishwashing product
HUE045202T2 (en) 2010-05-06 2019-12-30 Procter & Gamble Consumer products with protease variants
PL2420558T3 (en) 2010-08-17 2017-12-29 The Procter And Gamble Company Stable sustainable hand dish-washing detergents
CA2806265C (en) 2010-08-17 2016-10-18 The Procter & Gamble Company Method for hand washing dishes having long lasting suds
AR083354A1 (en) 2010-10-06 2013-02-21 Bp Corp North America Inc VARIABLE POLYPEPTIDES CBH I (CELOBIOHIDROLASAS I) WITH REDUCED PRODUCT INHIBITION
BR112013027963A2 (en) 2011-05-05 2016-11-29 Danisco Us Inc "Subtilisin variant with proteolytic activity, nucleic acid, expression vector, host cell, composition and cleaning method".
MX357386B (en) 2011-05-05 2018-07-06 Procter & Gamble Compositions and methods comprising serine protease variants.
US20140371435A9 (en) 2011-06-03 2014-12-18 Eduardo Torres Laundry Care Compositions Containing Thiophene Azo Dyes
EP2537918A1 (en) 2011-06-20 2012-12-26 The Procter & Gamble Company Consumer products with lipase comprising coated particles
KR20140056237A (en) 2011-06-30 2014-05-09 노보자임스 에이/에스 Alpha-amylase variants
EP2540824A1 (en) * 2011-06-30 2013-01-02 The Procter & Gamble Company Cleaning compositions comprising amylase variants reference to a sequence listing
CN112662734A (en) 2011-06-30 2021-04-16 诺维信公司 Method for screening alpha-amylase
EP2551335A1 (en) 2011-07-25 2013-01-30 The Procter & Gamble Company Enzyme stabilized liquid detergent composition
ES2633292T3 (en) 2011-10-19 2017-09-20 The Procter & Gamble Company Particle
EP3246404B1 (en) 2011-10-28 2019-02-27 Danisco US Inc. Variant maltohexaose-forming alpha-amylase variants
KR101882182B1 (en) * 2011-11-22 2018-07-27 삼성전자주식회사 Dish washer and method for controlling the same
MX353896B (en) 2012-02-03 2018-02-01 Procter & Gamble Compositions and methods for surface treatment with lipases.
IN2014DN07763A (en) 2012-03-19 2015-05-15 Milliken & Co
CN104204198B (en) 2012-04-02 2018-09-25 诺维信公司 Lipase Variant and the polynucleotides for encoding it
PL2662436T3 (en) 2012-05-11 2018-02-28 The Procter And Gamble Company Detergent composition
EP2674475A1 (en) 2012-06-11 2013-12-18 The Procter & Gamble Company Detergent composition
CN104471048B (en) 2012-07-12 2018-11-16 诺维信公司 Polypeptide with lipase active and the polynucleotides for encoding it
EP2700703B1 (en) 2012-08-24 2018-05-02 The Procter and Gamble Company Dishwashing method
EP2700704B1 (en) 2012-08-24 2018-05-09 The Procter and Gamble Company Dishwashing method
JP2016506442A (en) 2012-12-20 2016-03-03 ザ プロクター アンド ギャンブルカンパニー Detergent composition comprising a silicate-coated bleach
EP2746381A1 (en) 2012-12-21 2014-06-25 The Procter & Gamble Company Cleaning pack
WO2014106593A1 (en) * 2013-01-03 2014-07-10 Novozymes A/S Alpha-amylase variants and polynucleotides encoding same
ES2652301T3 (en) 2013-03-05 2018-02-01 The Procter & Gamble Company Mixed sugar-based amide surfactant compositions
WO2014164834A1 (en) * 2013-03-11 2014-10-09 Danisco Us Inc. Alpha-amylase combinatorial variants
CN105051174B (en) 2013-03-21 2018-04-03 诺维信公司 Polypeptide and their polynucleotides of coding with lipase active
AU2014241193B2 (en) 2013-03-28 2016-10-20 The Procter And Gamble Company Cleaning compositions containing a polyetheramine
CN105209614A (en) * 2013-05-17 2015-12-30 诺维信公司 Polypeptides having alpha amylase activity
JP6077177B2 (en) 2013-05-28 2017-02-08 ザ プロクター アンド ギャンブル カンパニー Surface treatment composition containing photochromic dye
EP3004314B1 (en) 2013-05-29 2018-06-20 Danisco US Inc. Novel metalloproteases
US20160108387A1 (en) 2013-05-29 2016-04-21 Danisco Us Inc. Novel metalloproteases
EP3882346A1 (en) 2013-05-29 2021-09-22 Danisco US Inc. Novel metalloproteases
EP3110833B1 (en) 2013-05-29 2020-01-08 Danisco US Inc. Novel metalloproteases
WO2014202782A1 (en) * 2013-06-21 2014-12-24 Novozymes A/S Polypeptides having amylase activity and polynucleotides encoding same
WO2015042209A1 (en) 2013-09-18 2015-03-26 The Procter & Gamble Company Laundry care compositions containing thiophene azo carboxylate dyes
CA2921432A1 (en) 2013-09-18 2015-03-26 The Procter & Gamble Company Laundry care composition comprising carboxylate dye
US9834682B2 (en) 2013-09-18 2017-12-05 Milliken & Company Laundry care composition comprising carboxylate dye
CA2921433A1 (en) 2013-09-18 2015-03-26 The Procter & Gamble Company Laundry care composition comprising carboxylate dye
EP2857485A1 (en) 2013-10-07 2015-04-08 WeylChem Switzerland AG Multi-compartment pouch comprising alkanolamine-free cleaning compositions, washing process and use for washing and cleaning of textiles and dishes
EP2857487A1 (en) 2013-10-07 2015-04-08 WeylChem Switzerland AG Multi-compartment pouch comprising cleaning compositions, washing process and use for washing and cleaning of textiles and dishes
EP2857486A1 (en) 2013-10-07 2015-04-08 WeylChem Switzerland AG Multi-compartment pouch comprising cleaning compositions, washing process and use for washing and cleaning of textiles and dishes
ES2723948T3 (en) 2013-12-13 2019-09-04 Danisco Us Inc Serine proteases from Bacillus species
EP3910057A1 (en) 2013-12-13 2021-11-17 Danisco US Inc. Serine proteases of the bacillus gibsonii-clade
MX2016007759A (en) 2013-12-16 2016-08-19 Du Pont Use of poly alpha-1,3-glucan ethers as viscosity modifiers.
EP3083705B1 (en) 2013-12-18 2020-09-30 DuPont Industrial Biosciences USA, LLC Cationic poly alpha-1,3-glucan ethers
WO2015112338A1 (en) 2014-01-22 2015-07-30 The Procter & Gamble Company Method of treating textile fabrics
EP3097173B1 (en) 2014-01-22 2020-12-23 The Procter and Gamble Company Fabric treatment composition
WO2015112340A1 (en) 2014-01-22 2015-07-30 The Procter & Gamble Company Method of treating textile fabrics
WO2015112341A1 (en) 2014-01-22 2015-07-30 The Procter & Gamble Company Fabric treatment composition
CA2841024C (en) 2014-01-30 2017-03-07 The Procter & Gamble Company Unit dose article
CN105992796A (en) 2014-02-14 2016-10-05 纳幕尔杜邦公司 Poly-alpha-1,3-1,6-glucans for viscosity modification
EP3110779A1 (en) 2014-02-25 2017-01-04 The Procter & Gamble Company A process for making renewable surfactant intermediates and surfactants from fats and oils and products thereof
US10752562B2 (en) 2014-02-25 2020-08-25 The Procter & Gamble Company Process for making renewable surfactant intermediates and surfactants from fats and oils and products thereof
EP2915873A1 (en) 2014-03-06 2015-09-09 The Procter and Gamble Company Dishwashing composition
EP2915872A1 (en) 2014-03-06 2015-09-09 The Procter and Gamble Company Dishwashing composition
MX2016011467A (en) 2014-03-11 2016-11-16 Du Pont Oxidized poly alpha-1,3-glucan as detergent builder.
CN106170546A (en) 2014-03-21 2016-11-30 丹尼斯科美国公司 The serine protease of bacillus
JP6275864B2 (en) 2014-03-27 2018-02-07 ザ プロクター アンド ギャンブル カンパニー Cleaning composition containing polyetheramine
US20150275143A1 (en) 2014-03-27 2015-10-01 The Procter & Gamble Company Cleaning compositions containing a polyetheramine
WO2015148890A1 (en) 2014-03-27 2015-10-01 The Procter & Gamble Company Cleaning compositions containing a polyetheramine
EP2924106A1 (en) 2014-03-28 2015-09-30 The Procter and Gamble Company Water soluble unit dose article
EP2924105A1 (en) 2014-03-28 2015-09-30 The Procter and Gamble Company Water soluble unit dose article
EP2940116B1 (en) 2014-04-30 2018-10-17 The Procter and Gamble Company Detergent
WO2015171592A1 (en) 2014-05-06 2015-11-12 Milliken & Company Laundry care compositions
EP3152288A1 (en) 2014-06-06 2017-04-12 The Procter & Gamble Company Detergent composition comprising polyalkyleneimine polymers
US20170121696A1 (en) * 2014-06-12 2017-05-04 Novozymes A/S Oxidation stable alpha-amylase variants
US9714403B2 (en) 2014-06-19 2017-07-25 E I Du Pont De Nemours And Company Compositions containing one or more poly alpha-1,3-glucan ether compounds
EP3158043B1 (en) 2014-06-19 2021-03-10 Nutrition & Biosciences USA 4, Inc. Compositions containing one or more poly alpha-1,3-glucan ether compounds
US9617502B2 (en) 2014-09-15 2017-04-11 The Procter & Gamble Company Detergent compositions containing salts of polyetheramines and polymeric acid
WO2016048674A1 (en) 2014-09-25 2016-03-31 The Procter & Gamble Company Cleaning compositions containing a polyetheramine
US20160090552A1 (en) 2014-09-25 2016-03-31 The Procter & Gamble Company Detergent compositions containing a polyetheramine and an anionic soil release polymer
US9388368B2 (en) 2014-09-26 2016-07-12 The Procter & Gamble Company Cleaning compositions containing a polyetheramine
DK3207129T3 (en) 2014-10-17 2020-02-24 Danisco Us Inc SERIN PROTEAS OF THE BACILLUS ART
WO2016069544A1 (en) 2014-10-27 2016-05-06 Danisco Us Inc. Serine proteases
US20180010074A1 (en) 2014-10-27 2018-01-11 Danisco Us Inc. Serine proteases of bacillus species
WO2016069569A2 (en) 2014-10-27 2016-05-06 Danisco Us Inc. Serine proteases
US20170335306A1 (en) 2014-10-27 2017-11-23 Danisco Us Inc. Serine proteases
EP3212662B1 (en) 2014-10-27 2020-04-08 Danisco US Inc. Serine proteases
AR102683A1 (en) 2014-11-17 2017-03-15 Procter & Gamble BENEFIT AGENT SUPPLY COMPOSITIONS
EP3026099B1 (en) 2014-11-26 2021-02-17 The Procter and Gamble Company Cleaning pouch
ES2690336T3 (en) 2014-11-26 2018-11-20 The Procter & Gamble Company Cleaning bag
ES2690335T3 (en) 2014-11-26 2018-11-20 The Procter & Gamble Company Cleaning bag
PL3026102T3 (en) 2014-11-26 2019-06-28 The Procter & Gamble Company Cleaning pouch
PL3034588T3 (en) 2014-12-17 2019-09-30 The Procter And Gamble Company Detergent composition
EP3034592A1 (en) 2014-12-17 2016-06-22 The Procter and Gamble Company Method of automatic dishwashing
EP3034596B2 (en) 2014-12-17 2021-11-10 The Procter & Gamble Company Detergent composition
EP3034589A1 (en) 2014-12-17 2016-06-22 The Procter and Gamble Company Detergent composition
EP3034591A1 (en) 2014-12-17 2016-06-22 The Procter and Gamble Company Method of automatic dishwashing
EP3034590A1 (en) 2014-12-17 2016-06-22 The Procter and Gamble Company Method of automatic dishwashing
EP3034597A1 (en) 2014-12-17 2016-06-22 The Procter and Gamble Company Detergent composition
ES2668504T3 (en) 2014-12-22 2018-05-18 The Procter & Gamble Company Process for recycling detergent bags
CN108064306B (en) 2014-12-23 2022-11-01 营养与生物科学美国4公司 Enzymatically produced cellulose
PL3050953T3 (en) 2015-02-02 2019-07-31 The Procter And Gamble Company Detergent composition
EP3050951A1 (en) 2015-02-02 2016-08-03 The Procter and Gamble Company Method of dishwashing
EP3050954A1 (en) 2015-02-02 2016-08-03 The Procter and Gamble Company New use of sulfonated polymers
EP3050950B1 (en) 2015-02-02 2018-09-19 The Procter and Gamble Company New use of sulfonated polymers
EP3050948B1 (en) 2015-02-02 2018-09-19 The Procter and Gamble Company New use of complexing agent
EP3050947A1 (en) 2015-02-02 2016-08-03 The Procter and Gamble Company Detergent pack
EP3050952A1 (en) 2015-02-02 2016-08-03 The Procter and Gamble Company Method of dishwashing
EP3050955B2 (en) 2015-02-02 2023-11-08 The Procter & Gamble Company Detergent pack
EP3088506B1 (en) 2015-04-29 2018-05-23 The Procter and Gamble Company Detergent composition
CN107548415A (en) 2015-04-29 2018-01-05 宝洁公司 The method of laundering of textile fabrics
JP6545822B2 (en) 2015-04-29 2019-07-17 ザ プロクター アンド ギャンブル カンパニーＴｈｅ Ｐｒｏｃｔｅｒ ＆ Ｇａｍｂｌｅ Ｃｏｍｐａｎｙ Cloth processing method
CN112143591A (en) 2015-04-29 2020-12-29 宝洁公司 Method for treating fabric
HUE043043T2 (en) 2015-04-29 2019-07-29 Procter & Gamble Method of treating a fabric
CN111718806B (en) 2015-05-04 2022-01-04 美利肯公司 Leuco triphenylmethane colorants as bluing agents in laundry care compositions
CN107750275A (en) 2015-05-08 2018-03-02 诺维信公司 Alpha-amylase variants and the polynucleotides for encoding them
AU2016259703B2 (en) 2015-05-08 2021-12-23 Novozymes A/S Alpha-amylase variants and polynucleotides encoding same
RU2733987C2 (en) 2015-05-13 2020-10-09 ДАНИСКО ЮЭс ИНК. Versions of protease of aprl and application thereof
EP3101102B2 (en) 2015-06-05 2023-12-13 The Procter & Gamble Company Compacted liquid laundry detergent composition
EP3101107B1 (en) 2015-06-05 2019-04-24 The Procter and Gamble Company Compacted liquid laundry detergent composition
EP3101103B1 (en) 2015-06-05 2019-04-24 The Procter and Gamble Company Compacted liquid laundry detergent composition
ES2666186T3 (en) 2015-06-05 2018-05-03 The Procter & Gamble Company Compacted liquid detergent composition for laundry
DK3307427T3 (en) 2015-06-09 2023-11-06 Danisco Us Inc OSMOTIC BURST CAPSULES
WO2016201040A1 (en) 2015-06-09 2016-12-15 Danisco Us Inc. Water-triggered enzyme suspension
WO2016201069A1 (en) 2015-06-09 2016-12-15 Danisco Us Inc Low-density enzyme-containing particles
US11499146B2 (en) 2015-06-17 2022-11-15 Danisco Us Inc. Bacillus gibsonii-clade serine proteases
EP3124586A1 (en) 2015-07-29 2017-02-01 The Procter and Gamble Company Process for reducing malodour in a pack
EP3124587B1 (en) 2015-07-29 2019-03-20 The Procter and Gamble Company Multi-phase unit-dose cleaning product
EP3371308B1 (en) 2015-11-05 2022-05-11 Danisco US Inc. Paenibacillus sp. mannanases
CN108603183B (en) 2015-11-05 2023-11-03 丹尼斯科美国公司 Paenibacillus species and bacillus species mannanases
EP3374488B1 (en) 2015-11-13 2020-10-14 DuPont Industrial Biosciences USA, LLC Glucan fiber compositions for use in laundry care and fabric care
WO2017083229A1 (en) 2015-11-13 2017-05-18 E. I. Du Pont De Nemours And Company Glucan fiber compositions for use in laundry care and fabric care
JP7045313B2 (en) 2015-11-13 2022-03-31 ニュートリション・アンド・バイオサイエンシーズ・ユーエスエー・フォー，インコーポレイテッド Glucan fiber composition for use in laundry care and textile care
JP2019502779A (en) 2015-11-26 2019-01-31 ザ プロクター アンド ギャンブル カンパニー Liquid detergent composition containing protease and encapsulated lipase
EP3178917A1 (en) 2015-12-08 2017-06-14 The Procter and Gamble Company Cleaning pouch
EP3387124B1 (en) 2015-12-09 2021-05-19 Danisco US Inc. Alpha-amylase combinatorial variants
US20180362946A1 (en) 2015-12-18 2018-12-20 Danisco Us Inc. Polypeptides with endoglucanase activity and uses thereof
MX2018007485A (en) * 2015-12-30 2018-08-01 Novozymes As Enzyme variants and polynucleotides encoding the same.
DK179660B1 (en) * 2016-04-08 2019-03-13 Novozymes A/S Stabilized Alpha-Amylase Variants and use of the same
EP3228687B1 (en) 2016-04-08 2019-05-22 The Procter and Gamble Company Dishwashing cleaning composition
EP3228686B1 (en) 2016-04-08 2021-10-27 The Procter & Gamble Company Automatic dishwashing
WO2017176501A1 (en) 2016-04-08 2017-10-12 The Procter & Gamble Company Automatic dishwashing cleaning composition
ES2802454T3 (en) 2016-04-08 2021-01-19 Procter & Gamble Dishwasher cleaning composition
CN105802940B (en) * 2016-04-18 2019-04-16 广西大学 A kind of bacillus licheniformis high-temperatureα-amylase mutant and its application
EP3241889B1 (en) 2016-05-03 2019-03-20 The Procter and Gamble Company Cleaning composition
EP3452569A1 (en) 2016-05-03 2019-03-13 The Procter and Gamble Company Automatic dishwashing detergent composition
EP3241891B1 (en) 2016-05-03 2019-04-03 The Procter and Gamble Company Automatic dishwashing detergent composition
EP3241890B1 (en) 2016-05-03 2019-06-26 The Procter and Gamble Company Automatic dishwashing detergent composition
JP2019518440A (en) 2016-05-03 2019-07-04 ダニスコ・ユーエス・インク Protease variant and use thereof
US20190136218A1 (en) 2016-05-05 2019-05-09 Danisco Us Inc Protease variants and uses thereof
MX2018014768A (en) 2016-05-31 2019-03-06 Danisco Us Inc Protease variants and uses thereof.
EP4151726A1 (en) 2016-06-17 2023-03-22 Danisco US Inc Protease variants and uses thereof
EP3257931A1 (en) 2016-06-17 2017-12-20 The Procter and Gamble Company Detergent composition
EP3266860B1 (en) 2016-07-08 2020-04-08 The Procter and Gamble Company Process for making a particle
EP3275989A1 (en) 2016-07-26 2018-01-31 The Procter and Gamble Company Automatic dishwashing detergent composition
EP3275987A1 (en) 2016-07-26 2018-01-31 The Procter and Gamble Company Automatic dishwashing detergent composition
EP3275986B1 (en) 2016-07-26 2020-07-08 The Procter and Gamble Company Automatic dishwashing detergent composition
EP3275985A1 (en) 2016-07-26 2018-01-31 The Procter and Gamble Company Automatic dishwashing detergent composition
EP3275988B1 (en) 2016-07-26 2020-07-08 The Procter and Gamble Company Automatic dishwashing detergent composition
EP3290503A3 (en) 2016-09-01 2018-05-30 The Procter & Gamble Company Automatic dishwashing cleaning composition
EP3312265A1 (en) 2016-10-18 2018-04-25 The Procter and Gamble Company Detergent composition
BR112019006576A2 (en) 2016-11-01 2019-07-02 Milliken & Co leuco dyes as bleaching agents in laundry care compositions
MX2019005120A (en) 2016-11-01 2019-06-20 Procter & Gamble Leuco colorants as bluing agents in laundry care compositions.
CN109890949B (en) 2016-11-01 2021-10-01 宝洁公司 Leuco colorants as bluing agents in laundry care compositions, packages, kits and methods thereof
US20180119056A1 (en) 2016-11-03 2018-05-03 Milliken & Company Leuco Triphenylmethane Colorants As Bluing Agents in Laundry Care Compositions
US20190264138A1 (en) 2016-11-07 2019-08-29 Danisco Us Inc. Laundry detergent composition
CN110088261B (en) 2016-12-02 2022-05-06 宝洁公司 Cleaning compositions comprising enzymes
WO2018102479A1 (en) 2016-12-02 2018-06-07 The Procter & Gamble Company Cleaning compositions including enzymes
US10550443B2 (en) 2016-12-02 2020-02-04 The Procter & Gamble Company Cleaning compositions including enzymes
US20200392477A1 (en) 2016-12-21 2020-12-17 Danisco Us Inc. Protease variants and uses thereof
WO2018118950A1 (en) 2016-12-21 2018-06-28 Danisco Us Inc. Bacillus gibsonii-clade serine proteases
EP3339410A1 (en) 2016-12-22 2018-06-27 The Procter & Gamble Company Automatic dishwashing composition
EP3339423A1 (en) 2016-12-22 2018-06-27 The Procter & Gamble Company Automatic dishwashing detergent composition
EP3583210B1 (en) 2017-03-15 2021-07-07 Danisco US Inc. Trypsin-like serine proteases and uses thereof
JP2020515269A (en) 2017-03-31 2020-05-28 ダニスコ・ユーエス・インク α-amylase combination mutant
EP3601515A1 (en) 2017-03-31 2020-02-05 Danisco US Inc. Delayed release enzyme formulations for bleach-containing detergents
EP3415592A1 (en) 2017-06-15 2018-12-19 The Procter & Gamble Company Water-soluble unit dose article comprising a solid laundry detergent composition
EP3418364A1 (en) 2017-06-19 2018-12-26 The Procter & Gamble Company Automatic dishwashing cleaning composition
EP3418365A1 (en) 2017-06-19 2018-12-26 The Procter & Gamble Company Automatic dishwashing cleaning composition
EP3418366A1 (en) 2017-06-19 2018-12-26 The Procter & Gamble Company Automatic dishwashing cleaning composition
BR112019027976A2 (en) 2017-06-30 2020-07-07 Danisco Us Inc. low agglomeration particles, containing enzymes
EP3441450A1 (en) 2017-08-11 2019-02-13 The Procter & Gamble Company Automatic dishwashing composition
MX2020001606A (en) 2017-08-18 2020-08-03 Danisco Us Inc Alpha-amylase variants.
EP3456808A1 (en) 2017-09-13 2019-03-20 The Procter & Gamble Company Automatic dishwashing cleaning composition
ES2874024T3 (en) 2017-10-05 2021-11-04 Procter & Gamble Cleaning composition for dishwashing
EP3467085A1 (en) 2017-10-05 2019-04-10 The Procter & Gamble Company Dishwashing cleaning composition
EP3694973A1 (en) 2017-10-12 2020-08-19 The Procter & Gamble Company Leuco colorants as bluing agents in laundry care compositions
EP3694980A1 (en) 2017-10-12 2020-08-19 The Procter and Gamble Company Leuco colorants in combination with a second whitening agent as bluing agents in laundry care compositions
CN111201309A (en) 2017-10-12 2020-05-26 宝洁公司 Leuco colorants as bluing agents in laundry care compositions
TWI715878B (en) 2017-10-12 2021-01-11 美商美力肯及公司 Leuco colorants and compositions
JP7230043B2 (en) 2017-11-01 2023-02-28 ミリケン・アンド・カンパニー Leuco compounds, colorant compounds, and compositions containing them
CN111373039A (en) 2017-11-29 2020-07-03 丹尼斯科美国公司 Subtilisin variants having improved stability
MX2020006518A (en) 2017-12-21 2020-10-28 Danisco Us Inc Enzyme-containing, hot-melt granules comprising a thermotolerant desiccant.
WO2019156670A1 (en) 2018-02-08 2019-08-15 Danisco Us Inc. Thermally-resistant wax matrix particles for enzyme encapsulation
EP3530723B1 (en) 2018-02-21 2023-03-29 The Procter & Gamble Company Automatic dishwashing composition
US20210214703A1 (en) 2018-06-19 2021-07-15 Danisco Us Inc Subtilisin variants
US20210363470A1 (en) 2018-06-19 2021-11-25 Danisco Us Inc Subtilisin variants
CN112805361A (en) 2018-07-31 2021-05-14 丹尼斯科美国公司 Variant alpha-amylases with amino acid substitutions that reduce PKA of generalized acids
US20210189295A1 (en) 2018-08-30 2021-06-24 Danisco Us Inc Enzyme-containing granules
WO2020068486A1 (en) 2018-09-27 2020-04-02 Danisco Us Inc Compositions for medical instrument cleaning
BR112021006967A2 (en) 2018-10-12 2021-07-13 Danisco Us Inc. alpha-amylases with mutations that improve stability in the presence of chelators
WO2020112599A1 (en) 2018-11-28 2020-06-04 Danisco Us Inc Subtilisin variants having improved stability
US11248194B2 (en) 2019-03-14 2022-02-15 The Procter & Gamble Company Cleaning compositions comprising enzymes
CA3127171A1 (en) 2019-03-14 2020-09-17 The Procter & Gamble Company Method for treating cotton
MX2021011121A (en) 2019-03-14 2021-10-14 Procter & Gamble Cleaning compositions comprising enzymes.
EP3741283A1 (en) 2019-05-22 2020-11-25 The Procter & Gamble Company Automatic dishwashing method
CN114174504A (en) 2019-05-24 2022-03-11 丹尼斯科美国公司 Subtilisin variants and methods of use
WO2020247582A1 (en) 2019-06-06 2020-12-10 Danisco Us Inc Methods and compositions for cleaning
EP3760699A1 (en) 2019-07-02 2021-01-06 The Procter & Gamble Company Automatic dishwashing detergent composition
US20220403359A1 (en) 2019-10-24 2022-12-22 Danisco Us Inc Variant maltopentaose/maltohexaose-forming alpha-amylases
EP3835396A1 (en) 2019-12-09 2021-06-16 The Procter & Gamble Company A detergent composition comprising a polymer
CN111471668B (en) * 2020-02-28 2022-05-24 浙江工业大学 Nitrilase mutant and application thereof in preparation of 1-cyanocyclohexylacetic acid
DE102020205400A1 (en) 2020-04-29 2021-11-04 Henkel Ag & Co. Kgaa Highly alkaline laundry detergent with protease
DE102020205381A1 (en) 2020-04-29 2021-11-04 Henkel Ag & Co. Kgaa Highly alkaline laundry detergent with protease
EP4162016A1 (en) 2020-06-05 2023-04-12 The Procter & Gamble Company Detergent compositions containing a branched surfactant
CN116323935A (en) 2020-08-27 2023-06-23 丹尼斯科美国公司 Enzymes and enzyme compositions for cleaning
EP3974504B1 (en) 2020-09-29 2023-07-26 The Procter & Gamble Company Automatic dishwashing cleaning composition
WO2022093189A1 (en) 2020-10-27 2022-05-05 Milliken & Company Compositions comprising leuco compounds and colorants
MX2023004262A (en) 2020-10-29 2023-04-26 Procter & Gamble Cleaning compositions containing alginase enzymes.
CN116472331A (en) 2020-11-17 2023-07-21 宝洁公司 Automatic dishwashing composition
EP4006131A1 (en) 2020-11-30 2022-06-01 The Procter & Gamble Company Method of laundering fabric
WO2022128620A1 (en) 2020-12-14 2022-06-23 Henkel Ag & Co. Kgaa Method for cleaning a food processor that is driven by an electric motor
DE102021213462A1 (en) 2021-11-30 2023-06-01 Henkel Ag & Co. Kgaa Method for cleaning a food processor operated by an electric motor
EP4011256A1 (en) 2020-12-14 2022-06-15 Henkel AG & Co. KGaA Method for cleaning an electric motorised kitchen appliance
EP4012011A1 (en) 2020-12-14 2022-06-15 Henkel AG & Co. KGaA Cleaning agent, particularly for a kitchen appliance
WO2022129166A1 (en) 2020-12-15 2022-06-23 Novozymes A/S Mutated host cells with reduced cell motility
JP2023551014A (en) 2020-12-23 2023-12-06 ビーエーエスエフ ソシエタス・ヨーロピア Amphiphilic alkoxylated polyamines and their uses
CN116997642A (en) 2021-01-29 2023-11-03 丹尼斯科美国公司 Cleaning compositions and methods relating thereto
WO2022197512A1 (en) 2021-03-15 2022-09-22 The Procter & Gamble Company Cleaning compositions containing polypeptide variants
EP4314222A1 (en) 2021-03-26 2024-02-07 Novozymes A/S Detergent composition with reduced polymer content
WO2022235720A1 (en) 2021-05-05 2022-11-10 The Procter & Gamble Company Methods for making cleaning compositions and detecting soils
EP4086330A1 (en) 2021-05-06 2022-11-09 The Procter & Gamble Company Surface treatment
EP4108150A1 (en) 2021-06-22 2022-12-28 The Procter & Gamble Company A method of treating dishware in a domestic automatic dishwashing machine
EP4108767A1 (en) 2021-06-22 2022-12-28 The Procter & Gamble Company Cleaning or treatment compositions containing nuclease enzymes
WO2023278297A1 (en) 2021-06-30 2023-01-05 Danisco Us Inc Variant lipases and uses thereof
EP4123006A1 (en) 2021-07-19 2023-01-25 The Procter & Gamble Company Composition comprising spores and pro-perfume materials
EP4123007A1 (en) 2021-07-19 2023-01-25 The Procter & Gamble Company Fabric treatment using bacterial spores
WO2023034486A2 (en) 2021-09-03 2023-03-09 Danisco Us Inc. Laundry compositions for cleaning
WO2023039270A2 (en) 2021-09-13 2023-03-16 Danisco Us Inc. Bioactive-containing granules
WO2023064749A1 (en) 2021-10-14 2023-04-20 The Procter & Gamble Company A fabric and home care product comprising cationic soil release polymer and lipase enzyme
EP4194537A1 (en) 2021-12-08 2023-06-14 The Procter & Gamble Company Laundry treatment cartridge
EP4194536A1 (en) 2021-12-08 2023-06-14 The Procter & Gamble Company Laundry treatment cartridge
WO2023104846A1 (en) 2021-12-10 2023-06-15 Novozymes A/S Improved protein production in recombinant bacteria
WO2023114932A2 (en) 2021-12-16 2023-06-22 Danisco Us Inc. Subtilisin variants and methods of use
WO2023114939A2 (en) 2021-12-16 2023-06-22 Danisco Us Inc. Subtilisin variants and methods of use
WO2023114988A2 (en) 2021-12-16 2023-06-22 Danisco Us Inc. Variant maltopentaose/maltohexaose-forming alpha-amylases
WO2023114936A2 (en) 2021-12-16 2023-06-22 Danisco Us Inc. Subtilisin variants and methods of use
WO2023168234A1 (en) 2022-03-01 2023-09-07 Danisco Us Inc. Enzymes and enzyme compositions for cleaning
EP4273210A1 (en) 2022-05-04 2023-11-08 The Procter & Gamble Company Detergent compositions containing enzymes
EP4273209A1 (en) 2022-05-04 2023-11-08 The Procter & Gamble Company Machine-cleaning compositions containing enzymes
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections
EP4279571A1 (en) 2022-05-19 2023-11-22 The Procter & Gamble Company Laundry composition comprising spores
EP4286499A1 (en) 2022-06-01 2023-12-06 The Procter & Gamble Company Dishwashing detergent composition comprising xylanase and sulphonated carboxylate polymer
EP4286500A1 (en) 2022-06-01 2023-12-06 The Procter & Gamble Company Use of xylanase in a dishwashing process
EP4286501A1 (en) 2022-06-01 2023-12-06 The Procter & Gamble Company Dishwashing detergent composition comprising xylanase and block co-polymer
DE102022205591A1 (en) 2022-06-01 2023-12-07 Henkel Ag & Co. Kgaa DETERGENT AND CLEANING AGENTS WITH IMPROVED ENZYME STABILITY
DE102022205588A1 (en) 2022-06-01 2023-12-07 Henkel Ag & Co. Kgaa DETERGENT AND CLEANING AGENTS WITH IMPROVED ENZYME STABILITY
DE102022205594A1 (en) 2022-06-01 2023-12-07 Henkel Ag & Co. Kgaa PERFORMANCE-IMPROVED AND STORAGE-STABLE PROTEASE VARIANTS
DE102022205593A1 (en) 2022-06-01 2023-12-07 Henkel Ag & Co. Kgaa DETERGENT AND CLEANING AGENTS WITH IMPROVED ENZYME STABILITY
WO2023250301A1 (en) 2022-06-21 2023-12-28 Danisco Us Inc. Methods and compositions for cleaning comprising a polypeptide having thermolysin activity
EP4321604A1 (en) 2022-08-08 2024-02-14 The Procter & Gamble Company A fabric and home care composition comprising surfactant and a polyester
EP4324900A1 (en) 2022-08-17 2024-02-21 Henkel AG & Co. KGaA Detergent composition comprising enzymes
WO2024050346A1 (en) 2022-09-02 2024-03-07 Danisco Us Inc. Detergent compositions and methods related thereto
WO2024050343A1 (en) 2022-09-02 2024-03-07 Danisco Us Inc. Subtilisin variants and methods related thereto
WO2024050339A1 (en) 2022-09-02 2024-03-07 Danisco Us Inc. Mannanase variants and methods of use
EP4339269A1 (en) 2022-09-16 2024-03-20 The Procter & Gamble Company Methods and apparatuses for automatic dishwashing chemical distribution

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6093562A (en) * 1996-02-05 2000-07-25 Novo Nordisk A/S Amylase variants

Family Cites Families (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3912590A (en) 1973-01-03 1975-10-14 Novo Industri As Procedure for liquefying starch
JPS57174089A (en) 1981-04-20 1982-10-26 Novo Industri As Chain dividing enzyme product
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
DK122686D0 (en) 1986-03-17 1986-03-17 Novo Industri As PREPARATION OF PROTEINS
DK311186D0 (en) 1986-06-30 1986-06-30 Novo Industri As ENZYMES
ES2052565T3 (en) 1986-07-09 1994-07-16 Novo Nordisk As A PROCEDURE FOR LIQUIDATING A SUSPENSION OF STARCH OR GRAIN YELLOWS.
DE3909096A1 (en) 1989-03-20 1990-09-27 Garabed Antranikian ALPHA AMYLASE
JP3086249B2 (en) 1989-06-29 2000-09-11 ジェネンコー インターナショナル インコーポレイテッド Mutant α-amylase derived from bacteria having high heat, acid and / or alkali stability
EP0531372B2 (en) 1990-05-09 2004-04-14 Novozymes A/S A cellulase preparation comprising an endoglucanase enzyme
PL310326A1 (en) 1993-02-11 1995-12-11 Genencor Int Novel oxidation-stable mutants of alpha-amylase as well as detergent and starch liquefaction compositions containing them
EP0722490B2 (en) 1993-10-08 2013-10-23 Novozymes A/S Amylase variants
MX196038B (en) * 1994-03-29 2000-04-14 Novo Nordisk As Alkaline bacillus amylase.
DK0772684T3 (en) * 1994-06-17 2005-12-12 Genencor Int Amylolytic enzymes derived from B. Licheniformis alpha-amylase with improved characteristics
KR100511499B1 (en) * 1995-02-03 2005-12-21 노보자임스 에이/에스 A method of designing alpha-amylase mutants with predetermined properties
DE19503621A1 (en) 1995-02-03 1996-08-08 Bosch Gmbh Robert Reciprocating pump
US6440716B1 (en) * 1995-02-03 2002-08-27 Novozymes A/S α-amylase mutants
AR000862A1 (en) 1995-02-03 1997-08-06 Novozymes As VARIANTS OF A MOTHER-AMYLASE, A METHOD TO PRODUCE THE SAME, A DNA STRUCTURE AND A VECTOR OF EXPRESSION, A CELL TRANSFORMED BY SUCH A DNA STRUCTURE AND VECTOR, A DETERGENT ADDITIVE, DETERGENT COMPOSITION, A COMPOSITION FOR AND A COMPOSITION FOR THE ELIMINATION OF
US5736499A (en) * 1995-06-06 1998-04-07 Genencor International, Inc. Mutant A-amylase
EP0851913B1 (en) 1995-08-11 2004-05-19 Novozymes A/S Novel lipolytic enzymes
CN1246455C (en) * 1996-04-30 2006-03-22 诺沃奇梅兹有限公司 Alpha amylase mutants
MY119088A (en) * 1996-11-26 2005-03-31 Sony Corp Information input method, information input sheet, and information input apparatus
US6187576B1 (en) * 1997-10-13 2001-02-13 Novo Nordisk A/S α-amylase mutants
KR19990059284A (en) * 1997-12-30 1999-07-26 서평원 Tone Monitoring and Emulation System at Digital Trunk Connections

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6093562A (en) * 1996-02-05 2000-07-25 Novo Nordisk A/S Amylase variants

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011084695A1 (en) 2009-12-21 2011-07-14 Novozymes, Inc. Methods for producing heterologous polypeptides in thiol-disulfide oxidoreductase-deficient bacterial mutant cells
WO2022074037A3 (en) * 2020-10-07 2022-07-07 Novozymes A/S Alpha-amylase variants

Also Published As

Publication number Publication date
BRPI9813328B1 (en) 2016-04-12
CN1550549A (en) 2004-12-01
JP2001521739A (en) 2001-11-13
US20050084937A1 (en) 2005-04-21
US6204232B1 (en) 2001-03-20
EP1027428B1 (en) 2010-12-01
DE69842027D1 (en) 2011-01-13
CN100593034C (en) 2010-03-03
US20040038368A1 (en) 2004-02-26
JP4426094B2 (en) 2010-03-03
WO1999023211A1 (en) 1999-05-14
ES2515218T3 (en) 2014-10-29
US20160222365A1 (en) 2016-08-04
EP2388267A1 (en) 2011-11-23
US6673589B2 (en) 2004-01-06
CN1278298A (en) 2000-12-27
CN1163597C (en) 2004-08-25
US20140017715A1 (en) 2014-01-16
ATE490311T1 (en) 2010-12-15
US20140248685A1 (en) 2014-09-04
AU9737398A (en) 1999-05-24
EP2386568A1 (en) 2011-11-16
CA2308119C (en) 2014-06-03
EP2386569A1 (en) 2011-11-16
EP1027428A1 (en) 2000-08-16
CA2308119A1 (en) 1999-05-14
BRPI9816290B1 (en) 2016-10-11
EP2386569B1 (en) 2014-08-06
BR9813328A (en) 2000-08-22
US20010039253A1 (en) 2001-11-08
EP2386568B1 (en) 2014-08-06
CA2845178A1 (en) 1999-05-14

Similar Documents

Publication Publication Date Title
EP2386569B1 (en) Alpha-amylase mutants
US9856437B2 (en) Amylase variants
US6197565B1 (en) α-Amylase variants
US20150132823A1 (en) Alpha-Amylase Variants
KR100770721B1 (en) ?-amylase mutants

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION