US20020192792A1 - Laccase mutants - Google Patents

Laccase mutants Download PDF

Info

Publication number
US20020192792A1
US20020192792A1 US09/869,877 US86987701A US2002192792A1 US 20020192792 A1 US20020192792 A1 US 20020192792A1 US 86987701 A US86987701 A US 86987701A US 2002192792 A1 US2002192792 A1 US 2002192792A1
Authority
US
United States
Prior art keywords
gly
leu
ala
val
pro
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
US09/869,877
Other languages
English (en)
Inventor
Palle Schneider
Steffen Danielsen
Allan Svendsen
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
Individual
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=26068820&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20020192792(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Individual filed Critical Individual
Assigned to NOVOZYMES A/S reassignment NOVOZYMES A/S ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DANIELSEN, STEFFEN, SVENDSEN, ALLAN, SCHNEIDER, PALLE
Publication of US20020192792A1 publication Critical patent/US20020192792A1/en
Abandoned legal-status Critical Current

Links

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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
    • A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
    • A21D2/24—Organic nitrogen compounds
    • A21D2/26—Proteins
    • A21D2/267—Microbial proteins
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
    • 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
    • C11D3/38627—Preparations containing enzymes, e.g. protease or amylase containing lipase
    • 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
    • C11D3/38645—Preparations containing enzymes, e.g. protease or amylase containing cellulase
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0061—Laccase (1.10.3.2)
    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
    • C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
    • C12Y110/03002—Laccase (1.10.3.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21062—Subtilisin (3.4.21.62)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/911—Microorganisms using fungi

Definitions

  • the invention relates to laccase mutants with improved stability properties.
  • Laccase is a polyphenol oxidase (EC 1.10.3.2) which catalyses the oxidation of a variety It of inorganic and aromatic compounds, particularly phenols, with the concomitant reduction of molecular oxygen to water.
  • laccases are able to catalyze the oxidation of a variety of inorganic and aromatic compounds
  • laccases have been suggested in many industrial applications such as lignin modification, paper strengthening, dye transfer inhibition in detergents, phenol polymerization, hair colouring, and waste water treatment.
  • a major problem with the use of laccases is their poor stability against oxidative attack from e.g. radicals formed from the oxidation of mediators (also referred to as “enhancing agents”).
  • the invention provides variants of a Coprinus laccase and of Coprinus-like laccases with improved oxidative stability as compared to the parent Coprinus laccase or Coprinus-like laccase.
  • the invention relates to DNA encoding such variants and to the use of the variants for various industrial purposes.
  • Coprinus cinereus laccase comprising the amino acid sequence shown in SEQ ID No. 1: 100%;
  • Polyporus pinsitus (I) laccase comprising the amino acid sequence shown in SEQ ID No. 2: 74.4%;
  • Polyporus pinsitus (II) laccase comprising the amino acid sequence shown in SEQ ID No. 3: 73.8%;
  • Phlebia radiata laccase comprising the amino acid sequence shown in SEQ ID No. 4: 69.9%;
  • Rhizoctonia solani (I) laccase comprising the amino acid sequence shown in SEQ ID No. 5: 64.8%;
  • Rhizoctonia solani (II) laccase comprising the amino acid sequence shown in SEQ ID No.6: 63.0%;
  • Rhizoctonia solani (III) laccase comprising the amino acid sequence shown in SEQ ID No. 7: 61.0%;
  • Rhizoctonia solani (IV) laccase comprising the amino acid sequence shown in SEQ ID No. 8: 59.7%;
  • Myceliophthora thermophila laccase comprising the amino acid sequence shown in SEQ ID No. 10:56.5%.
  • Coprinus-like laccase is intended to indicate a laccase which, on the amino acid level, displays a homology of at least 50% and less than 100% to the Coprinus cinereus laccase SEQ ID NO.1, or at least 55% and less than 100% to the Coprinus cinereus laccase SEQ ID NO.1, or at least 60% and less than 100% to the Coprinus cinereus laccase SEQ ID NO.1, or at least 65% and less than 100% to the Coprinus cinereus laccase SEQ ID NO.1, or at least 70% and less than 100% to the Coprinus cinereus laccase SEQ ID NO.1, or at least 75% and less than 100% to the Coprinus cinereus laccase SEQ ID NO.
  • derived from is intended not only to indicate a laccase produced or producible by a strain of the organism in question, but also a laccase encoded by a DNA sequence isolated from such strain and produced in a host organism containing said DNA sequence.
  • the term is intended to indicate a laccase which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the laccase in question.
  • the Coprinus laccase consists of the 539 amino acids derived from Coprinus cinereus laccase IFO 8371 as disclosed in SEQ ID No.1.
  • the three-dimensional structure is believed to be representative for the structure of any Coprinus-like laccase.
  • the laccase structure is made up of three plastocyanin-like domains. These three domains all have a similar beta-barrel fold.
  • type 1 copper ion is coordinated by two histidines and one cysteine.
  • type 3 copper consists of two type 3 copper atoms (pair of copper atoms) bound to a total of 6 histidine ligands.
  • Q19 in SEQ ID No. 1 is an A1 in the crystallized protein
  • Q243 in SEQ ID No. 1 is an E225 in the crystallized protein.
  • Coprinus laccase structure may be identified in other Coprinus-like laccases on the basis of a model (or solved) structure of the relevant Coprinus-like laccase or simply on the basis of an alignment between the amino acid sequence of the Coprinus-like laccase in question with that of the Coprinus laccase used herein for identifying the amino acid residues of the respective structural elements.
  • Coprinus laccase variants of the invention which are defined by modification of specific amino acid residues of the parent Coprinus laccase, it will be understood that variants of Coprinus-like laccases modified in an equivalent position (as determined from the best possible amino acid sequence alignment between the respective sequences) are intended to be covered as well.
  • the laccase mutants of the present invention may be designed by constructing a variant of a parent Coprinus laccase, which variant has laccase activity and improved stability as compared to the parent laccase, which method comprises:
  • the laccase mutants of the invention may also be designed by constructing a variant of a parent Coprinus-like laccase, which variant has laccase activity and improved stability as compared to the parent laccase, which method comprises:
  • step i) of the methods of the invention may be performed by use of any suitable computer programme capable of analysing and/or comparing amino acid sequences.
  • the structural part which is identified in step i) of the methods of the invention may be composed of one amino acid residue. However, normally the structural part comprises more than one amino acid residue, typically constituting one of the above mentioned parts of the Coprinus structure such as one of the copper centres.
  • the laccase variants of the invention have improved oxidative stability compared to the un-modified parent laccases. Improved oxidative stability means that the laccase variants of the invention have improved tolerance towards oxidative chemical compounds, such as radicals formed from laccase mediated oxidation of radical precursor compounds.
  • the radical precursor compounds may preferably be mediators or “enhancing agents”, such as those described in EP 705327 (compounds containing N—OH, N—O and NR—OH groups), WO9501426 (compounds containing two aromatic rings etc.), WO 96/10079 (methylsyringate type of compounds) and/or WO 99/57360 (N-hydroxyacetanilide type of compounds).
  • laccase variants may be obtained by:
  • the modification of an amino acid residue or structural part is typically accomplished by suitable modifications of a DNA sequence encoding the parent enzyme in question.
  • modified as used in the methods according to the invention is intended to have the following meaning: When used in relation to an amino acid residue the term is intended to mean replacement of the amino acid residue in question with another amino acid residue.
  • the term is intended to mean: replacement of one or more amino acid residues of said structural part with other amino acid residues, or addition of one or more amino acid residues to said part, or deletion of one or more amino acid residues of said structural part.
  • the construction of the variant of interest is accomplished by cultivating a microorganism comprising a DNA sequence encoding the variant under conditions which are conducive for producing the variant, and optionally subsequently recovering the variant from the resulting culture broth. This is described in detail further below.
  • Preferred positions for mutations are the following: CcL: MtL: F21 V52 H91 G121 F112 F141 H133 — H153 Y177 Y176 H206 H230 M260 H309 P336 F335 T365 Y347 I380 S349 I382 Y375 V406 Y416 — F449 — E455 A506 F456 W507 Y490 W543
  • CcL Coprinus cinereus laccase comprising the amino acid sequence shown in SEQ ID No. 1;
  • MtL Myceliophthora thermophila laccase comprising the amino acid sequence shown in SEQ ID No. 10.
  • a variant of a parent Coprinus laccase which comprises one or more of the following substitutions in SEQ ID No. 1:
  • F21 A I, L, N, R, S, Q;
  • F335 A I, L, N, R, S, Q;
  • F456 A I, L, N, R, S, Q;
  • a variant of a parent Myceliophthora thermophila laccase which comprises a mutation in a position corresponding to at least one of the following positions in SEQ ID No. 10:
  • H206 A I, L, N, R, S, Q;
  • T365 A I, L, N, R, S, Q;
  • V406A I, L, N, R, S, Q;
  • laccase variants of the invention may be added to and thus become a component of a detergent composition.
  • the detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.
  • the invention provides a detergent additive comprising the laccase variants of the invention.
  • the detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase.
  • enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an
  • the properties of the chosen enzyme(s) should be compatible with the selected detergent, (i.e. pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.
  • Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included.
  • the protease may be a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease.
  • alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).
  • Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.
  • Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.
  • Preferred commercially available protease enzymes include AlcalaseTM, SavinaseTM, PrimaseTM, EverlaseTM, EsperaseTM, and KannaseTM (Novozymes A/S), MaxataseTM, MaxacalTM, MaxapemTM, ProperaseTM, PurafectTM, Purafect OxPTM, FN2TM, and FN3TM (Genencor International Inc.).
  • Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa ( T. lanuginosus ) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.
  • lipase variants such as those described in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97104079 and WO 97/07202.
  • Preferred commercially available lipase enzymes include LipolaseTM, Lipolase UltraTM and LipoprimeTM (Novozymes A/S).
  • Amylases [0115] Amylases:
  • Suitable amylases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, ⁇ -amylases obtained from Bacillus, e.g. a special strain of B. licheniformis , described in more detail in GB 1,296,839.
  • Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.
  • amylases are DuramylTM, TermamylTM, FungamylTM and BANTM (Novozymes A/S), RapidaseTM and PurastarTM (Genencor International Inc.).
  • Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178, 5,776,757 and WO 89/09259.
  • cellulases are the alkaline or neutral cellulases having colour care benefits.
  • Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940.
  • Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, U.S. Pat. Nos. 5,457,046, 5,686,593, 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.
  • cellulases include CelluzymeTM, and CarezymeTM (Novozymes A/S), ClazinaseTM, and Puradax HATM (Genencor International Inc.), and KAC-500(B) TM (Kao Corporation).
  • Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g. from C. cinereus , and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.
  • the detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes.
  • a detergent additive of the invention i.e. a separate additive or a combined additive, can be formulated e.g. as a granulate, a liquid, a slurry, etc.
  • Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.
  • Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat. Nos. 4 , 106 , 991 and 4,661,452 and may optionally be coated by methods known in the art.
  • waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
  • Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods.
  • Protected enzymes may be prepared according to the method disclosed in EP 238,216.
  • the detergent composition of the invention may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid.
  • a liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or non-aqueous.
  • the detergent composition comprises one or more surfactants, which may be non-ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic.
  • the surfactants are typically present at a level of from 0.1% to 60% by weight.
  • the detergent When included therein the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
  • an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
  • the detergent When included therein the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides”).
  • a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides”).
  • glucamides N-acyl N-alkyl derivatives of glucosamine
  • the detergent may contain 0-65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenyisuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
  • a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenyisuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
  • the detergent may comprise one or more polymers.
  • examples are carboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
  • the detergent may contain a bleaching system which may comprise a H 2 O 2 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.
  • a bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone type.
  • the enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708.
  • a polyol such as propylene glycol or glycerol
  • a sugar or sugar alcohol lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid
  • the detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
  • fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
  • any enzyme, in particular the laccase variants of the invention may be added in an amount corresponding to 0.01-100 mg of enzyme protein per liter of wash liquor, preferably 0.05-10 mg of enzyme protein per liter of wash liquor, more preferably 0.1-5 mg of enzyme protein per liter of wash liquor, and most preferably 0.1-1 mg of enzyme protein per liter of wash liquor.
  • laccase variants of the invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202 which is hereby incorporated as reference.
  • the DNA sequence encoding a parent laccase may be isolated from any cell or microorganism producing the laccase 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 laccase to be studied.
  • homologous, labelled oligonucleotide probes may be synthesized and used to identify laccase-encoding clones from a genomic library prepared from the organism in question.
  • a labelled oligonucleotide probe containing sequences homologous to a known laccase gene could be used as a probe to identify laccase-encoding clones, using hybridization and washing conditions of lower stringency.
  • a method for identifying laccase-encoding clones involves inserting cDNA into an expression vector, such as a plasmid, transforming laccase-negative fungi with the resulting cDNA library, and then plating the transformed fungi onto agar containing a substrate for laccase, thereby allowing clones expressing the laccase to be identified.
  • an expression vector such as a plasmid, transforming laccase-negative fungi with the resulting cDNA library
  • the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method.
  • oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.
  • the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, the fragments corresponding to various parts of the entire DNA sequence), in accordance with standard techniques.
  • the DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers.
  • oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligonucleotide synthesis.
  • a single-stranded gap of DNA bridging the laccase-encoding sequence, is created in a vector carrying the laccase gene.
  • the synthetic nucleotide, bearing the desired mutation is annealed to a homologous portion of the single-stranded DNA. The remaining gap is then filled in with T7 DNA polymerase and the construct is ligated using T4 ligase.
  • the random mutagenesis of a DNA sequence encoding a parent laccase may conveniently be performed by use of any method known in the art.
  • the random mutagenesis may be performed by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the random mutagenesis may be performed by use of any combination of these mutagenizing agents.
  • the mutagenizing agent may, e.g., be one which induces transitions, transversions, inversions, scrambling, deletions, and/or insertions.
  • Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues.
  • UV ultraviolet
  • MNNG N-methyl-N′-nitro-N-nitrosoguanidine
  • EMS ethyl methane sulphonate
  • sodium bisulphite formic acid
  • nucleotide analogues examples include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide ana
  • the mutagenesis is typically performed by incubating the DNA sequence encoding the parent enzyme to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions for the mutagenesis to take place, and selecting for mutated DNA having the desired properties.
  • the oligonucleotide may be doped or spiked with the three non-parent nucleotides during the synthesis of the oligonucleotide at the positions which are to be changed.
  • the doping or spiking may be done so that codons for unwanted amino acids are avoided.
  • the doped or spiked oligonucleotide can be incorporated into the DNA encoding the laccase enzyme by any published technique, using e.g. PCR, LCR or any DNA polymerase and ligase.
  • PCR-generated mutagenesis When PCR-generated mutagenesis is used, either a chemically treated or non-treated gene encoding a parent laccase enzyme is subjected to PCR under conditions that increase the misincorporation of nucleotides (Deshler 1992; Leung et al., Technique, Vol.1, 1989, pp. 11-15).
  • a mutator strain of E. coli (Fowler et al., Molec. Gen. Genet., 133, 1974, pp. 179-191), S. cereviseae or any other microbial organism may be used for the random mutagenesis of the DNA encoding the laccase enzyme by e.g. transforming a plasmid containing the parent enzyme into the mutator strain, growing the mutator strain with the plasmid and isolating the mutated plasmid from the mutator strain. The mutated plasmid may subsequently be transformed into the expression organism.
  • the DNA sequence to be mutagenized may conveniently be present in a genomic or cDNA library prepared from an organism expressing the parent laccase enzyme.
  • the DNA sequence may be present on a suitable vector such as a plasmid or a bacteriophage, which as such may be incubated with or otherwise exposed to the mutagenizing agent.
  • the DNA to be mutagenized may also be present in a host cell either by being integrated in the genome of said cell or by being present on a vector harboured in the cell.
  • the DNA to be mutagenized may be in isolated form. It will be understood that the DNA sequence to be subjected to random mutagenesis is preferably a cDNA or a genomic DNA sequence.
  • the mutated DNA is expressed by culturing a suitable host cell carrying the DNA sequence under conditions allowing expression to take place.
  • the host cell used for this purpose may be one which has been transformed with the mutated DNA sequence, optionally present on a vector, or one which was carried the DNA sequence encoding the parent enzyme during the mutagenesis treatment.
  • suitable host cells are fungal hosts such as Aspergillus niger or Aspergillus oryzae.
  • the mutated DNA sequence may further comprise a DNA sequence encoding functions permitting expression of the mutated DNA sequence.
  • the random mutagenesis may advantageously be localized to a part of the parent laccase in question. This may, e.g., be advantageous when certain regions of the enzyme have been identified to be of particular importance for a given property of the enzyme, and when modified are expected to result in a variant having improved properties. Such regions may normally be identified when the tertiary structure of the parent enzyme has been elucidated and related to the function of the enzyme.
  • the localized random mutagenesis is conveniently performed by use of PCR-generated mutagenesis techniques as described above or any other suitable technique known in the art.
  • the DNA sequence encoding the part of the DNA sequence to be modified may be isolated, e.g. by being inserted into a suitable vector, and said part may subsequently be subjected to mutagenesis by use of any of the mutagenesis methods discussed above.
  • a microorganism capable of expressing the mutated laccase enzyme of interest is incubated on a suitable medium and under suitable conditions for the enzyme to be secreted, the medium being provided with a double filter comprising a first protein-binding filter and on top of that a second filter exhibiting a low protein binding capability.
  • the microorganism is located on the second filter.
  • the first filter comprising enzymes secreted from the microorganisms is separated from the second filter comprising the microorganisms.
  • the first filter is subjected to screening for the desired enzymatic activity and the corresponding microbial colonies present on the second filter are identified.
  • the filter used for binding the enzymatic activity may be any protein binding filter e.g. nylon or nitrocellulose.
  • the top filter carrying the colonies of the expression organism may be any filter that has no or low affinity for binding proteins e.g. cellulose acetate or DuraporeTM.
  • the filter may be pretreated with any of the conditions to be used for screening or may be treated during the detection of enzymatic activity.
  • the enzymatic activity may be detected by a dye, fluorescence, precipitation, pH indicator, IR-absorbance or any other known technique for detection of enzymatic activity.
  • the detecting compound may be immobilized by any immobilizing agent, e.g., agarose, agar, gelatine, polyacrylamide, starch, filter paper, cloth; or any combination of immobilizing agents.
  • immobilizing agent e.g., agarose, agar, gelatine, polyacrylamide, starch, filter paper, cloth; or any combination of immobilizing agents.
  • the laccase activity may be measured using 10-(2-hydroxyethyl)-phenoxazine (HEPO) as substrate.
  • HEPO 10-(2-hydroxyethyl)-phenoxazine
  • HEPO was synthesized using the same procedure as described for 10-(2-hydroxyethyl)-phenothiazine, (G. Cauquil in Bulletin de la Society Chemique de France, 1960, p. 1049).
  • oxygen laccases E.C. 1.10.3.2
  • Coprinus cinereus laccase was measured using 0.4 mM HEPO in 50 mM sodium acetate, pH 5.0, 0.05% TWEEN-20 at 30° C. The absorbance at 528 nm was followed for 200 s and the rate calculated from the linear part of the progress curve.
  • the Myceliophthora thermophila laccase was measured using 0.4 mM HEPO in 25 mM Tris-HCl, pH 7.5, 0.05% Tween-20 at 30° C. The absorbance at 528 nm was followed for 200 s and the rate calculated from the linear part of the progress curve.
  • the Polyporus pinsitus laccase was measured using 0.4 mM HEPO in 50 mM MES-NaOH, pH 5.5. The absorbance at 528 nm was followed for 200 sec. and the rate calculated from the linear part of the progress curve.
  • the stability against oxidation by radicals (oxidative stability) of Coprinus variants or Coprinus-like variants may be measured as described in the following.
  • the enzyme is diluted in 100 mM phosphate pH 5 or 6 (which is closest to the pH optimum for the enzyme with methylsyringate as substrate) to a concentration of 0.1 mg enzyme protein per ml.
  • sample and reference are stored at room temperature (approx. 25° C.) for 20 hours. After dilution residual activity of sample and reference is determined by the LACU or LAMU assays using syringaldazine as substrate.
  • Conditions for some fungal laccases are: Laccase from Incubation Assay Polyporus/Trametes pH 5 LACU Coprinus cinereus pH 6 LAMU Myceliophthora thermophila pH 6 LAMU Rhizoctonia solani pH 6 LAMU
  • LACU Laccase Activity
  • Laccase activity may be determined from the oxidation of syringaldazin under aerobic conditions.
  • the violet colour produced is photometered at 530 nm.
  • the analytical conditions are 19 mM syringaldazin, 23 mM acetate buffer, pH 5.5, 30° C., 1 min. reaction time.
  • laccase unit is the amount of enzyme that catalyses the conversion of 1.0 ⁇ mole syringaldazin per minute at these conditions.
  • LAMU Laccase Activity
  • Laccase activity may be determined from the oxidation of syringaldazin under aerobic conditions.
  • the violet colour produced is photometered at 530 nm.
  • the analytical conditions are 19 mM syringaldazin, 23 mM Tris/maleate buffer, pH 7.5, 30° C., 1 min. reaction time.
  • laccase unit is the amount of enzyme that catalyses the conversion of 1.0 ⁇ mole syringaldazin per minute at these conditions.
  • 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 a laccase 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 a laccase variant of the invention, especially in a fungal host are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic 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, polyadenylation sequences operably connected to the DNA sequence encoding the laccase variant of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
  • the vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.
  • the vector may also comprise a selectable marker, e.g. a gene, the product of which complements a defect in the host cell, such as 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 one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance.
  • the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, e.g. as described in WO 91/17243.
  • the cell of the invention is advantageously used as a host cell in the recombinant production of a laccase variant of the invention.
  • the cell may be transformed with the DNA construct of the invention encoding the variant, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g. by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.
  • the cell of the invention may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g. a fungal cell.
  • 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 a laccase 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 laccase 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 laccase 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 laccase variants of this invention possesses valuable properties allowing for various industrial applications, in particular lignin modification, paper strengthening, dye transfer inhibition in detergents, phenol polymerization, hair dyeing, bleaching of textiles (in particular bleaching of denim as described in WO 96/12845 and WO 96112846) and waste water treatment.
  • Any detergent composition normally used for enzymes may be used, e.g., the detergent compositions disclosed in WO 95/01426.
US09/869,877 2000-04-28 2001-04-30 Laccase mutants Abandoned US20020192792A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DKPA200000707 2000-04-28
DKPA200000707 2000-04-28
DKPA200100327 2001-02-28
DKPA200100327 2001-02-28

Publications (1)

Publication Number Publication Date
US20020192792A1 true US20020192792A1 (en) 2002-12-19

Family

ID=26068820

Family Applications (6)

Application Number Title Priority Date Filing Date
US09/869,877 Abandoned US20020192792A1 (en) 2000-04-28 2001-04-30 Laccase mutants
US10/258,783 Expired - Lifetime US7226770B2 (en) 2000-04-28 2001-04-30 Lipolytic enzyme variant
US09/957,806 Abandoned US20050181446A1 (en) 2000-04-28 2001-09-21 Protein variants having modified immunogenicity
US12/699,979 Abandoned US20110045572A1 (en) 2000-04-28 2010-02-04 Protein variants having modified immunogenicity
US13/936,300 Abandoned US20140011259A1 (en) 2000-04-28 2013-07-08 Protein Variants Having Modified Immunogenicity
US14/514,652 Abandoned US20150037872A1 (en) 2000-04-28 2014-10-15 Protein Variants Having Modified Immunogenicty

Family Applications After (5)

Application Number Title Priority Date Filing Date
US10/258,783 Expired - Lifetime US7226770B2 (en) 2000-04-28 2001-04-30 Lipolytic enzyme variant
US09/957,806 Abandoned US20050181446A1 (en) 2000-04-28 2001-09-21 Protein variants having modified immunogenicity
US12/699,979 Abandoned US20110045572A1 (en) 2000-04-28 2010-02-04 Protein variants having modified immunogenicity
US13/936,300 Abandoned US20140011259A1 (en) 2000-04-28 2013-07-08 Protein Variants Having Modified Immunogenicity
US14/514,652 Abandoned US20150037872A1 (en) 2000-04-28 2014-10-15 Protein Variants Having Modified Immunogenicty

Country Status (8)

Country Link
US (6) US20020192792A1 (de)
EP (7) EP2236611A1 (de)
AT (1) ATE471377T1 (de)
AU (3) AU2001254620A1 (de)
CA (1) CA2406621A1 (de)
DE (1) DE60142415D1 (de)
ES (1) ES2588756T3 (de)
WO (3) WO2001083761A1 (de)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114703212A (zh) * 2022-03-01 2022-07-05 东华大学 一种运用特定区段随机突变法改造漆酶的方法及漆酶菌株lac123

Families Citing this family (95)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK6488D0 (da) * 1988-01-07 1988-01-07 Novo Industri As Enzymer
US6682924B1 (en) * 1995-05-05 2004-01-27 Novozymes A/S Protease variants and compositions
US20120165241A1 (en) * 1995-05-05 2012-06-28 Unilever Plc Subtilase Variants
US6936289B2 (en) 1995-06-07 2005-08-30 Danisco A/S Method of improving the properties of a flour dough, a flour dough improving composition and improved food products
ATE231186T1 (de) 1998-07-21 2003-02-15 Danisco Lebensmittel
US6686164B1 (en) * 1998-10-30 2004-02-03 Novozymes A/S Low allergenic protein variants
JP2003504046A (ja) * 1999-07-09 2003-02-04 ノボザイムス アクティーゼルスカブ グルコアミラーゼ変異体
US20020192792A1 (en) 2000-04-28 2002-12-19 Palle Schneider Laccase mutants
AU7235901A (en) 2000-07-06 2002-01-21 Novozymes As Method of preparing a dough or a baked product made from a dough, with addition of lipolytic enzymes
DK1352057T3 (da) * 2001-01-10 2009-07-20 Novozymes As Upolytisk enzymvariant
DE10121463A1 (de) * 2001-05-02 2003-02-27 Henkel Kgaa Neue Alkalische Protease-Varianten und Wasch- und Reinigungsmittel enthaltend diese neuen Alkalischen Protease-Varianten
BR0209154A (pt) 2001-05-18 2004-07-20 Danisco Processo de preparação de uma massa com uma enzima
DK200101090A (da) * 2001-07-12 2001-08-16 Novozymes As Subtilase variants
US20030119066A1 (en) * 2001-10-05 2003-06-26 Novozymes A/S Diagnostic kit for detecting immunogenic response and method of screening
DE10153792A1 (de) * 2001-10-31 2003-05-22 Henkel Kgaa Neue Alkalische Protease-Varianten und Wasch- und Reinigungsmittel enthaltend diese neuen Alkalischen Protease-Varianten
EP1523553B1 (de) * 2002-01-16 2009-12-02 Genencor International, Inc. Mehrfachsubstituierte proteasevarianten
WO2003062380A2 (en) * 2002-01-16 2003-07-31 Genencor International, Inc. Multiply-substituted protease variants
WO2003060112A1 (en) 2002-01-16 2003-07-24 Novozymes A/S Lipolytic enzyme variants and method for their production
CN101177675B (zh) 2002-02-08 2014-05-21 诺维信公司 肌醇六磷酸酶变体
US7511015B2 (en) * 2002-02-19 2009-03-31 The United States Of America As Represented By The Department Of Health And Human Services Modified defensins and their use
CN1668737A (zh) * 2002-05-16 2005-09-14 阿尔克-阿贝洛有限公司 重组Bet.V.1过敏原突变体及其方法和制备
US20060228791A1 (en) * 2002-06-26 2006-10-12 Novozymes A/S Subtilases and subtilase variants having altered immunogenicity
US7888093B2 (en) 2002-11-06 2011-02-15 Novozymes A/S Subtilase variants
ATE389731T1 (de) * 2002-12-12 2008-04-15 Novozymes As Verfahren zur auswahl eines lipolytischen enzyms
DE10260903A1 (de) * 2002-12-20 2004-07-08 Henkel Kgaa Neue Perhydrolasen
US20050196766A1 (en) 2003-12-24 2005-09-08 Soe Jorn B. Proteins
MXPA05007654A (es) 2003-01-17 2005-09-30 Danisco Metodo.
US7955814B2 (en) 2003-01-17 2011-06-07 Danisco A/S Method
US7294499B2 (en) 2003-01-30 2007-11-13 Novozymes A/S Subtilases
US20060205628A1 (en) * 2003-02-18 2006-09-14 Novozymes A/S Detergent compositions
WO2004078960A1 (en) 2003-02-26 2004-09-16 Genencor International, Inc. Amylases producing an altered immunogenic response and methods of making and using the same
EP2500423B1 (de) 2003-02-26 2015-06-17 Danisco US Inc. Amylasen, die eine veränderte immunogene Reaktion bewirken, und Verfahren zu ihrer Herstellung und Verwendung
WO2004096844A2 (en) * 2003-04-25 2004-11-11 Novozymes A/S Group 1 mite polypeptide variants
DK2270140T3 (en) * 2003-05-09 2016-03-14 Novozymes As Lipolytic enzyme variants
WO2004111216A2 (en) * 2003-06-19 2004-12-23 Novozymes A/S Phospholipase variants
EP1639107B1 (de) 2003-06-19 2013-08-14 Novozymes A/S Verbesserte proteasen und methoden zu deren herstellung
WO2004111221A1 (en) 2003-06-19 2004-12-23 Novozymes A/S Proteases
ES2358092T3 (es) 2003-10-10 2011-05-05 Novozymes A/S Variantes de proteasa.
US7718408B2 (en) 2003-12-24 2010-05-18 Danisco A/S Method
GB0716126D0 (en) 2007-08-17 2007-09-26 Danisco Process
US7906307B2 (en) 2003-12-24 2011-03-15 Danisco A/S Variant lipid acyltransferases and methods of making
GB0405637D0 (en) 2004-03-12 2004-04-21 Danisco Protein
US20090060933A1 (en) * 2004-06-14 2009-03-05 Estell David A Proteases producing an altered immunogenic response and methods of making and using the same
MXPA06014649A (es) 2004-06-21 2007-03-12 Novozymes As Proteasas.
GB0416035D0 (en) * 2004-07-16 2004-08-18 Danisco Protein
DK1776455T3 (da) 2004-07-16 2015-06-22 Dupont Nutrition Biosci Aps Lipolytisk enzym, anvendelser deraf i fødevareindustrien
AR050895A1 (es) 2004-10-04 2006-11-29 Novozymes As Polipeptidos que tienen actividad de fitasa y polinucleotidos que los codifican
CN103224918B (zh) 2004-10-04 2015-08-26 诺维信公司 具有肌醇六磷酸酶活性的多肽和编码它的多核苷酸
WO2006042558A2 (en) * 2004-10-22 2006-04-27 Novozymes A/S Group 2 mite polypeptide variants
AU2006239562B2 (en) * 2005-04-28 2010-12-23 Unilever Plc Compositions comprising tripeptides inhibiting ace
EP1874128A1 (de) * 2005-04-28 2008-01-09 Hindustan Unilever Limited Peptide mit ace-hemmwirkung
JP5113044B2 (ja) 2005-06-13 2013-01-09 ノボザイムス アクティーゼルスカブ デガミングされた脂肪酸アルキルエステルの生成
US20070161531A1 (en) 2005-07-08 2007-07-12 Novozymes A/S Subtilase variants
US20070148311A1 (en) * 2005-12-22 2007-06-28 Bunge Oils, Inc. Phytosterol esterification product and method of make same
EP2371949B1 (de) * 2006-01-23 2017-04-19 Novozymes A/S Lipase Varianten
CA2635942A1 (en) * 2006-01-23 2007-08-02 The Procter & Gamble Company Detergent compositions
JP2009523901A (ja) 2006-01-23 2009-06-25 ザ プロクター アンド ギャンブル カンパニー 洗剤組成物
WO2007107573A1 (en) 2006-03-22 2007-09-27 Novozymes A/S Use of polypeptides having antimicrobial activity
BRPI0709732B1 (pt) 2006-04-04 2017-06-06 Novozymes As fitase, sequência de ácidos nucleicos isolada, construção de ácido nucleico, vetor de expressão recombinante, microrganismo recombinante, método para produzir a fitase, composição, método para melhorar o valor nutritivo de uma ração para animal, processo para reduzir os níveis de fitato no esterco de animal, método para o tratamento de proteínas de legume, e, uso da fitase ou da composição na ração para animal
EP2455462B1 (de) * 2006-12-21 2016-03-16 Novozymes A/S Lipasevarianten zur pharmazeutischen Verwendung
US8221743B2 (en) 2006-12-22 2012-07-17 Novozymes A/S Use of polypeptides against diseases caused by protozoans
BRPI0720801A2 (pt) 2007-01-25 2014-09-16 Dupont Nutrition Biosci Aps Produção de um lipídio aciltranfersase a partir de células transformadas de bacillus licheniformis.
FI20075059A0 (fi) * 2007-01-29 2007-01-29 Valtion Teknillinen Allergeeniä sitovat monoklonaaliset IgE-vasta-aineet ja hypoallergeenit:tyypin l lgE:n ja allergeenin immunokompleksivuorovaikutus
US8460905B2 (en) * 2007-09-11 2013-06-11 Bunge Oils, Inc. Enzymatic degumming utilizing a mixture of PLA and PLC phospholipases with reduced reaction time
US8956853B2 (en) * 2007-01-30 2015-02-17 Bunge Oils, Inc. Enzymatic degumming utilizing a mixture of PLA and PLC phospholipases
EP2129781B1 (de) 2007-03-26 2014-01-22 Novozymes A/S Hafnia-phytase
US8241876B2 (en) 2008-01-07 2012-08-14 Bunge Oils, Inc. Generation of triacylglycerols from gums
DK2254996T3 (da) 2008-02-29 2013-10-07 Dsm Ip Assets Bv Lipaser med høj specificitet for kortkædede fedtsyrer og anvendelser deraf
JP5647976B2 (ja) 2008-06-06 2015-01-07 ダニスコ・ユーエス・インク 変異体微生物プロテアーゼを含む組成物及び方法
EP2650364B1 (de) 2008-09-26 2015-05-20 Novozymes A/S Hafnia-Phytasevarianten
EP2362902B1 (de) * 2008-11-11 2012-10-24 Danisco US, Inc., Genencor Division Zusammensetzungen und verfahren mit einer subtilisinvariante
GB0915572D0 (en) 2009-09-07 2009-10-07 Reckitt Benckiser Nv Detergent composition
BR112012006497A2 (pt) * 2009-09-25 2015-09-08 Novozymes As uso de uma variante de subtilisina, composição de lavagem de pratos, e, uso de uma composição.
CN102666824A (zh) 2009-12-21 2012-09-12 丹尼斯科美国公司 能够改进经脂肪酶处理的脂质基污渍的清洁的表面活性剂
BR112013003725B1 (pt) * 2010-08-17 2020-05-26 Council Of Scintific & Industrial Research Método para obtenção de enzima lacase a partir de arthrographis sp
MX2014007446A (es) * 2011-12-20 2014-08-01 Novozymes As Variantes de subtilasa y polinucleotidos que las codifican.
US10180434B2 (en) 2012-03-13 2019-01-15 Array Bridge Inc. Antibody array used for the analysis of the three-dimensional structure of protein therapeutics and its production
CN104736700B (zh) 2012-10-12 2019-02-01 丹尼斯科美国公司 包含脂解酶变体的组合物和方法
WO2014067933A1 (en) 2012-10-31 2014-05-08 C-Lecta Gmbh Bioactive carrier preparation for enhanced safety in care products and food
CA2923991C (en) * 2013-10-11 2021-07-06 Mauna Kea Technologies Method for characterizing images acquired through a video medical device
WO2015144936A1 (en) * 2014-03-28 2015-10-01 Novozymes A/S Resolubilization of protein crystals at low ph
KR101655276B1 (ko) * 2014-10-23 2016-09-08 대한민국(환경부 국립생물자원관장) 단풍잎돼지풀 추출물을 유효성분으로 함유하는 항산화 및 피부 미백용 화장료 조성물
US10781428B2 (en) 2014-12-02 2020-09-22 Novozymes A/S Laccase variants and polynucleotides encoding same
EP3234125B1 (de) 2014-12-19 2020-04-15 Novozymes A/S Proteasevarianten
WO2016097350A1 (en) * 2014-12-19 2016-06-23 Novozymes A/S Protease variants and polynucleotides encoding same
US11291214B2 (en) * 2015-04-06 2022-04-05 Dupont Nutrition Biosciences Aps Proteases for high protein fermented milk products
CA2995771A1 (en) * 2015-09-25 2017-03-30 Imcyse Sa Improved methods and compounds for eliminating immune responses to therapeutic agents
BR112018071466A2 (pt) 2016-04-19 2019-03-19 Imcyse Sa novos peptídeos de ligação a cd1d imunogênicos
CN108239626B (zh) * 2016-12-27 2022-10-04 丰益(上海)生物技术研发中心有限公司 一种高酯化活力的脂肪酶突变体
CN109280091A (zh) * 2018-11-27 2019-01-29 苏州卫生职业技术学院 一种苦瓜多糖的生物酶提取工艺
CN109912707B (zh) * 2019-03-07 2022-03-11 辽宁师范大学 可作为肿瘤诊断标记物的七鳃鳗免疫蛋白lip突变体
CN111979208B (zh) * 2019-05-23 2023-01-10 弈柯莱生物科技(上海)股份有限公司 一种l-谷氨酸脱氢酶突变体及其应用
CN110684751B (zh) * 2019-10-23 2021-06-25 江南大学 一种催化能力提高的淀粉分支酶突变体
US20220049230A1 (en) 2020-08-13 2022-02-17 Novozymes A/S Phytase variants and polynucleotides encoding same
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

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5998353A (en) * 1996-12-19 1999-12-07 Novo Nordisk A/S Laccase mutants

Family Cites Families (88)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1585105A (en) 1976-04-29 1981-02-25 Unilever Ltd Emulsions
GB1590432A (en) 1976-07-07 1981-06-03 Novo Industri As Process for the production of an enzyme granulate and the enzyme granuate thus produced
JPS57189638A (en) 1981-05-20 1982-11-22 Yakult Honsha Co Ltd Production of liquid fermented milk
DK289083A (da) 1983-06-23 1984-12-24 Novo Industri As Lipase, fremgangsmaade til fremstilling deraf og anvendelse deraf
DK263584D0 (da) 1984-05-29 1984-05-29 Novo Industri As Enzymholdige granulater anvendt som detergentadditiver
DE3423699C1 (de) 1984-06-27 1986-01-16 Maggi AG, Kempttal Saucenverbesserer in Tuben
GB8525012D0 (en) 1985-10-10 1985-11-13 Cpc International Inc Carbohydrate refining process
US4810414A (en) 1986-08-29 1989-03-07 Novo Industri A/S Enzymatic detergent additive
EP0305216B1 (de) 1987-08-28 1995-08-02 Novo Nordisk A/S Rekombinante Humicola-Lipase und Verfahren zur Herstellung von rekombinanten Humicola-Lipasen
EP0319064B1 (de) 1987-12-03 1992-05-13 Unilever N.V. Verfahren zur Herstellung einer Wasser-in-Öl-Emulsion
DK6488D0 (da) * 1988-01-07 1988-01-07 Novo Industri As Enzymer
JP2709736B2 (ja) 1988-08-11 1998-02-04 昭和産業株式会社 油脂の精製方法
JP2794574B2 (ja) 1988-08-11 1998-09-10 昭和産業株式会社 リゾレシチンの製造方法
JPH02160984A (ja) 1988-12-09 1990-06-20 Novo Ind As 古紙の脱インキ方法
JPH02160997A (ja) 1988-12-13 1990-06-20 Jujo Paper Co Ltd ピッチトラブル防止法
JP2877439B2 (ja) 1989-05-17 1999-03-31 協和醗酵工業株式会社 卵の改質方法
US5665587A (en) * 1989-06-26 1997-09-09 Novo Nordisk A/S Modified subtilisins and detergent compositions containing same
DE3922748B4 (de) 1989-07-11 2006-01-05 Röhm GmbH & Co. KG Enzymatisches Weichverfahren
ATE97555T1 (de) 1989-09-29 1993-12-15 Unilever Nv Getrocknetes lyso-phospholipoprotein enthaltendes nahrungsmittel.
EP0495064B1 (de) * 1990-08-13 2001-07-11 "BIOMAY" Produktions- und Handelsgesellschaft m.b.H. Birkenpollenallergen p14 zur diagnose und therapie von allergischen krankheiten
AT401180B (de) * 1990-08-13 1996-07-25 Biomay Biotech Prod Für das baumpollenallergen p14 codierende rekombinante dna-moleküle, daraus hergestellte und abgeleitete polypeptide und deren verwendung
US5892013A (en) * 1990-09-13 1999-04-06 Novo Nordisk A/S Lipase variants
AU657278B2 (en) * 1990-09-13 1995-03-09 Novo Nordisk A/S Lipase variants
US5869438A (en) 1990-09-13 1999-02-09 Novo Nordisk A/S Lipase variants
JPH0687751B2 (ja) 1990-09-26 1994-11-09 辻製油株式会社 高濃度のリゾホスファチジルコリンを含むリゾレシチンを採取する方法
BR9106919A (pt) 1990-09-28 1993-08-17 Procter & Gamble Tensoativos de polihidroxi amida de acido graxo para melhorar o desempenho de enzima
DK249990D0 (da) 1990-10-17 1990-10-17 Novo Nordisk As Fremgangsmaade til enzymatisk pulpbehandling
CA2331936C (en) * 1990-12-05 2007-07-31 Novozymes A/S Proteins with changed epitopes and methods for the production thereof
JPH04240286A (ja) 1991-01-25 1992-08-27 Novo Nordisk As 耐熱性リパーゼによるピッチトラブル防止法
DE4109826A1 (de) 1991-03-26 1992-11-05 Roehm Gmbh Enzymatisch unterstuetze aescher- und beizverfahren
DK0585285T3 (da) 1991-05-01 1999-05-10 Novo Nordisk As Stabiliserede enzymer
DE4115938A1 (de) 1991-05-16 1992-11-19 Metallgesellschaft Ag Enzymatisches verfahren zur verminderung des gehaltes an phosphorhaltigen bestandteilen in pflanzlichen und tierischen oelen
ATE326572T1 (de) 1991-12-20 2006-06-15 Novozymes As Entfernung von hydrophoben estern aus textilien
DK0567662T3 (da) 1992-04-25 1997-08-25 Nestle Sa Fremgangsmåde til aromatisering af en mælkechokolade
DK104592D0 (da) 1992-08-21 1992-08-21 Novo Nordisk As Fremgangsmaade
DK0628256T3 (da) 1993-06-11 1998-03-09 Nestle Sa Middel til termisk stabilisering af proteiner og produkt opnået hermed
US6436690B1 (en) * 1993-09-15 2002-08-20 The Procter & Gamble Company BPN′ variants having decreased adsorption and increased hydrolysis wherein one or more loop regions are substituted
DE4339556C1 (de) 1993-11-19 1995-02-02 Metallgesellschaft Ag Verfahren zum Entschleimen von Pflanzenöl mittels Enzymen
AU3741995A (en) 1994-10-26 1996-05-23 Novo Nordisk A/S Enzymatic detergent composition
GB2296011B (en) 1994-12-13 1999-06-16 Solvay Novel fusarium isolate and lipases, cutinases and enzyme compositions derived therefrom
US6475765B1 (en) * 1995-03-09 2002-11-05 Procter & Gamble Company Subtilisin DY variants having decreased adsorption and increased hydrolysis
IL117350A0 (en) * 1995-03-09 1996-07-23 Procter & Gamble Proteinase k variants having decreased adsorption and increased hydrolysis
US5837517A (en) * 1995-05-05 1998-11-17 Novo Nordisk A/S Protease variants and compositions
US6682924B1 (en) * 1995-05-05 2004-01-27 Novozymes A/S Protease variants and compositions
JP4307549B2 (ja) 1995-07-14 2009-08-05 ノボザイムス アクティーゼルスカブ 脂肪分解活性を有する修飾された酵素
JP4068142B2 (ja) 1995-08-11 2008-03-26 ノボザイムス アクティーゼルスカブ 新規の脂肪分解酵素
ZA967411B (en) * 1995-09-01 1997-04-16 Novo Nordisk Biotech Inc Blue copper oxidase mutants with enhanced activity
WO1997027276A2 (en) 1996-01-24 1997-07-31 Novo Nordisk Biotech, Inc. Nucleic acids encoding polypeptides having absidia lipase activity
US5804201A (en) * 1996-03-11 1998-09-08 The Rockefeller University Immunomodulatory peptides of vespid antigen 5
EP0897423B1 (de) 1996-04-25 2004-10-13 Novozymes A/S Alkalisches, lipolytisches enzym
CA2254781A1 (en) 1996-05-15 1997-11-20 The Procter & Gamble Company Detergent compositions comprising specific lipolytic enzyme and a specific surfactant system
JP3791058B2 (ja) 1996-07-30 2006-06-28 日本油脂株式会社 リゾレシチンの製造方法
WO1998008939A1 (en) 1996-08-27 1998-03-05 Novo Nordisk A/S Novel lipolytic enzymes
JPH10155493A (ja) 1996-10-04 1998-06-16 Sankyo Co Ltd アスペルギルス属由来のホスホリパーゼａ１をコードする遺伝子
CN1136311C (zh) * 1996-11-04 2004-01-28 诺沃奇梅兹有限公司 枯草杆菌酶变异体和组合物
DK0869167T4 (da) 1996-12-09 2010-03-08 Novozymes As Reduktion af phosphor-indeholdende bestanddele i spiseolier; som omfatter en stor mængde ikke-hydrerbart phosphor, ved anvendelse af en phospholipase, en phospholipase fra en trådsvamp, der har en phospholipase A og/eller B aktivitet
WO1998027198A1 (en) * 1996-12-19 1998-06-25 Novo Nordisk A/S Laccase mutants
DE19701348A1 (de) 1997-01-16 1998-07-23 Roehm Gmbh Protein mit Phospholipaseaktivität
EP1017794A1 (de) * 1997-02-06 2000-07-12 Novo Nordisk A/S Polypeptid-polymer-konjugate mit hinzugefügten oder entfernten bindungsgruppen
AU5983398A (en) * 1997-02-28 1998-09-18 Novo Nordisk A/S Laccase mutants
JP3853464B2 (ja) 1997-04-08 2006-12-06 辻製油株式会社 植物性リゾレシチンの製造法
ATE331793T1 (de) 1997-04-09 2006-07-15 Danisco Verwendung von lipase zur verbesserung von teigen und backwaren
CN1272137A (zh) * 1997-08-29 2000-11-01 诺沃挪第克公司 蛋白酶变体及组合物
CN100593036C (zh) * 1997-08-29 2010-03-03 诺沃奇梅兹有限公司 蛋白酶变体及组合物
MA25044A1 (fr) * 1997-10-23 2000-10-01 Procter & Gamble Compositions de lavage contenant des variants de proteases multisubstituees.
US6780629B2 (en) * 1997-11-21 2004-08-24 Novozymes A/S Subtilase enzymes
EP1032655B1 (de) * 1997-11-21 2005-06-29 Novozymes A/S Protease-varianten und zusammensetzungen
US6773907B2 (en) * 1997-11-21 2004-08-10 Peter Kamp Hansen Subtilase enzymes
WO1999034826A1 (en) * 1998-01-09 1999-07-15 Circassia Limited Methods and compositions for desensitisation
CA2319437C (en) * 1998-01-31 2009-06-16 University Of Arkansas Methods and reagents for decreasing allergic reactions
AU3247699A (en) * 1998-02-17 1999-09-06 Novo Nordisk A/S Lipase variant
DK1062341T3 (da) * 1998-03-16 2008-11-03 Alk Abello As Muterede rekombinante allergener
US6835550B1 (en) * 1998-04-15 2004-12-28 Genencor International, Inc. Mutant proteins having lower allergenic response in humans and methods for constructing, identifying and producing such proteins
US6461849B1 (en) * 1998-10-13 2002-10-08 Novozymes, A/S Modified polypeptide
US6376450B1 (en) * 1998-10-23 2002-04-23 Chanchal Kumar Ghosh Cleaning compositions containing multiply-substituted protease variants
US6686164B1 (en) * 1998-10-30 2004-02-03 Novozymes A/S Low allergenic protein variants
ES2220114T3 (es) * 1998-10-30 2004-12-01 Novozymes A/S Variantes de proteinas poco alergenicas.
NZ511340A (en) 1998-11-27 2003-07-25 Novozymes As Lipolytic enzyme variants
EP1033405A3 (de) * 1999-02-25 2001-08-01 Ceres Incorporated DNS-fragmente mit bestimmter Sequenz und die dadurch kodierte Polypeptide
WO2001037147A2 (en) * 1999-11-03 2001-05-25 Algonomics Nv Apparatus and method for structure-based prediction of amino acid sequences
US6777218B1 (en) * 2000-03-14 2004-08-17 Novozymes A/S Subtilase enzymes having an improved wash performance on egg stains
AU4981101A (en) * 2000-04-03 2001-10-15 Maxygen Inc Subtilisin variants
US20020192792A1 (en) 2000-04-28 2002-12-19 Palle Schneider Laccase mutants
US7109016B2 (en) * 2000-08-21 2006-09-19 Novozymes A/S Subtilase enzymes
US6893855B2 (en) * 2000-10-13 2005-05-17 Novozymes A/S Subtilase variants
US7888093B2 (en) * 2002-11-06 2011-02-15 Novozymes A/S Subtilase variants
EP1623029B1 (de) * 2003-05-07 2008-09-03 Novozymes A/S Enzymvarianten von subtilisin (subtilasen)
US20070161531A1 (en) * 2005-07-08 2007-07-12 Novozymes A/S Subtilase variants

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5998353A (en) * 1996-12-19 1999-12-07 Novo Nordisk A/S Laccase mutants

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114703212A (zh) * 2022-03-01 2022-07-05 东华大学 一种运用特定区段随机突变法改造漆酶的方法及漆酶菌株lac123

Also Published As

Publication number Publication date
WO2001083559A2 (en) 2001-11-08
ATE471377T1 (de) 2010-07-15
AU2001254623A1 (en) 2001-11-12
WO2001083761A1 (en) 2001-11-08
US20150037872A1 (en) 2015-02-05
WO2001083770A3 (en) 2002-05-02
WO2001083770A2 (en) 2001-11-08
EP1555322A1 (de) 2005-07-20
AU2001254620A1 (en) 2001-11-12
EP2258852A1 (de) 2010-12-08
US20110045572A1 (en) 2011-02-24
EP1280919A2 (de) 2003-02-05
CA2406621A1 (en) 2001-11-08
EP2236611A1 (de) 2010-10-06
EP2258853A1 (de) 2010-12-08
DE60142415D1 (de) 2010-07-29
EP2258835A1 (de) 2010-12-08
US20030144165A1 (en) 2003-07-31
WO2001083559A3 (en) 2002-06-20
US7226770B2 (en) 2007-06-05
EP1555322B1 (de) 2010-06-16
US20140011259A1 (en) 2014-01-09
EP2258853B1 (de) 2016-06-08
EP1280817A2 (de) 2003-02-05
US20050181446A1 (en) 2005-08-18
ES2588756T3 (es) 2016-11-04
AU2001254622A1 (en) 2001-11-12

Similar Documents

Publication Publication Date Title
US20020192792A1 (en) Laccase mutants
US6309871B1 (en) Polypeptides having alkaline α-amylase activity
EP0675949B1 (de) Rekombinante lipase und alpha-amylase varianten
US6399561B1 (en) Methods and compositions for bleaching a dye in solution
EP1169434B1 (de) Polypeptide mit alkaliner alpha-amylase-aktivität und für diese kodierende nukleinsäuren
KR100787392B1 (ko) 알칼리 α-아밀라제 활성을 가지는 폴리펩티드 및 그것을코드하는 핵산
EP2004789B1 (de) Stabilisierte flüssige enzymzusammensetzung
KR100305140B1 (ko) 프로테아제변이체
US8263368B2 (en) Alpha-amylase mutants
US20100311636A1 (en) Detergent compositions and the use of enzyme combinations therein
WO2002031124A2 (en) Alpha-amylase variant with altered properties
US6110884A (en) Protease variants
US6184015B1 (en) Laccase mutants
US6623948B1 (en) Nucleic acid sequences encoding alkaline alpha-amylases
EP0956344A1 (de) Laccase-varianten aus myceliophtora und scytalidium mit verbesserter stabilität.
EP0972014A1 (de) Laccasemutanten.
US6218170B1 (en) Laccase mutants
US20040091994A1 (en) Alpha-amylase variant with altered properties

Legal Events

Date Code Title Description
AS Assignment

Owner name: NOVOZYMES A/S, DENMARK

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SCHNEIDER, PALLE;DANIELSEN, STEFFEN;SVENDSEN, ALLAN;REEL/FRAME:012713/0494;SIGNING DATES FROM 20010515 TO 20010521

STCB Information on status: application discontinuation

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