EP1082443A1 - Variants de serine-proteases ayant des deletions et des substitutions d'acides amines - Google Patents

Variants de serine-proteases ayant des deletions et des substitutions d'acides amines

Info

Publication number
EP1082443A1
EP1082443A1 EP99907806A EP99907806A EP1082443A1 EP 1082443 A1 EP1082443 A1 EP 1082443A1 EP 99907806 A EP99907806 A EP 99907806A EP 99907806 A EP99907806 A EP 99907806A EP 1082443 A1 EP1082443 A1 EP 1082443A1
Authority
EP
European Patent Office
Prior art keywords
gly
leu
ala
asn
val
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.)
Withdrawn
Application number
EP99907806A
Other languages
German (de)
English (en)
Inventor
Donn Nelton Rubingh
Elizabeth Ellen Sikorski
Paul Elliott Correa
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.)
Procter and Gamble Co
Original Assignee
Procter and Gamble Co
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
Application filed by Procter and Gamble Co filed Critical Procter and Gamble Co
Publication of EP1082443A1 publication Critical patent/EP1082443A1/fr
Withdrawn legal-status Critical Current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)
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/0208—Tissues; Wipes; Patches
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/86—Products or compounds obtained by genetic engineering
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/10—Washing or bathing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair

Definitions

  • mutation refers to alterations in gene sequences and amino acid sequences produced by those gene sequences. Mutations may be deletions, substitutions, or additions of amino acid residues to the wild-type protein sequence.
  • subtilisin BPN' As used herein, the term "variant" means a protein, herein specifically a protease, having an amino acid sequence which differs from that of the wild-type protease. As referred to herein, while the variants of the present invention are not limited to those of subtilisin BPN', all amino acid numbering is with reference to the amino acid sequence for subtilisin BPN' which is represented by SEQ ID NO:l . The amino acid sequence for subtilisin BPN' is further described by Wells, J.A., E. Ferrari, D.J. Henner, D.A. Estell, and E.Y. Chen, Nucleic Acids Research. Vol. II, 7911 - 7925 (1983), incorporated herein by reference.
  • the present inventors have discovered an epitope region in serine proteases which corresponds to positions 70 - 84 of subtilisin BPN'.
  • the present inventors have further discovered that one or more amino acid deletions and / or substitutions in the amino acid sequence of a wild-type serine protease provides variants which evoke a deceased allergenic and / or immune response relative to the corresponding wild-type serine protease.
  • a variant may be designated by referring to the deleted amino acid positions which characterize the variant.
  • a variant of a serine protease having deletions at positions 70, 75, 76, 77, 78, 79, 80, 81, and 82 may be designated as ⁇ 70, 75 - 82.
  • substitutions may be indicated by providing the wild-type amino acid residue, followed by the position number, followed by the substituted amino acid residue to be substituted. Wherein the substituted amino acid residue may be any natural amino acid, the symbol "*" is provided. Multiple substitutions comprising a variant are separated by the symbol "+".
  • a substitution of valine for glycine at position 70 is designated either Gly70Val or G70V.
  • Preferred serine proteases for mutation include subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin 309, proteinase K, and thermitase.
  • the most preferred serine protease for mutation is subtilisin BPN'.
  • Substitutions in the epitope region are made by replacing the wild-type amino acid residue with another natural amino acid residue such as one given in Table I.
  • ASN 76 ASN 77 ILE 79 GLY 80 LEU 82 GLY 83
  • ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82
  • ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
  • Table 11 exemplifies non-limiting preferred double mutation variants having one deletions and one or more substitutions:
  • Fermentation is as follows. Bacillus subtilis cells (PG632) containing the variant of interest are grown to mid-log phase in one liter of LB broth containing 10 g/L glucose, and inoculated into a Biostat C fermentor (Braun Biotech, Inc., Allentown, PA) in a total volume of 9 liters.
  • the fermentation medium contains yeast extract, casein hydrosylate, soluble - partially hydrolyzed starch (Maltrin M-250), antifoam, buffers, and trace minerals (see "Biology of Bacilli: Applications to Industry", Doi, R. H. and M. McGloughlin, eds. (1992)).
  • the broth is kept at a constant pH of 7.5 during the fermentation run. Kanamycin (50 ⁇ g/mL) is added for antibiotic selection of the mutagenized plasmid.
  • the cells are grown for 18 hours at 37 °C to an ⁇ ⁇ Q of about 60 and the product harvested.
  • protease activity of a variant of the present invention may be assayed by methods which are well-known in the art. Two such methods are set forth herein below: Skin Flake Activity Method
  • the variants of the present invention may be used in a variety of detergent compositions where high sudsing and good cleansing activity is desired.
  • the variants can be used with various conventional ingredients to provide fully-formulated hard-surface cleaners, dishwashing compositions, fabric laundering compositions, and the like.
  • Such compositions can be in the form of liquids, granules, bars, and the like.
  • Such compositions can be formulated as "concentrated" detergents which contain as much as from about 30% to about 60% by weight of surfactants.
  • 2,2,4-trimethyl-l,3-pentanediol and 2-ethyl-l,3-hexanediol When used, such solvents are typically present at levels of from about 0.5% to about 15%, more preferably from about 3% to about 11 ).
  • dishwashing compositions comprise one or more variants of the present invention.
  • “dishwashing composition” refers to all forms of compositions for cleaning dishes including, but not limited to, granular and liquid forms. Dishwashing compositions of the present invention are illustrated by the following examples.
  • Liquid fabric cleaning compositions of the present invention are illustrated by the following examples. 78
  • the present variants are particularly suited for use in personal care compositions such as, for example, leave-on and rinse-off hair conditioners, shampoos, leave-on and rinse-off acne compositions, facial milks and conditioners, shower gels, soaps, foaming and non-foaming facial cleansers, cosmetics, hand, facial, and body lotions and moisturizers, leave-on facial moisturizers, cosmetic and cleansing wipes, oral care compositions, and contact lens care compositions.
  • the present personal care compositions comprise one or more variants of the present invention and a personal care carrier.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Birds (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Dermatology (AREA)
  • Detergent Compositions (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Cosmetics (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Cette invention se rapporte à des variants de sérine-protéases ayant une immunogénicité inférieure à celle des protéases du type sauvage correspondantes. Cette invention se rapporte plus particulièrement à des variants ayant une séquence d'acides aminés modifiée par rapport à une séquence d'acides aminés de type sauvage, cette séquence d'acides aminés modifiée comportant une délétion et, éventuellement, une substitution d'une ou de plusieurs positions spécifiquement identifiées correspondant à la subtilisine BPN'. Cette invention se rapporte en outre à des gènes mutants codant ces variants et à des compositions de produits de nettoyage et de produits de soins personnels comprenant ces variants.
EP99907806A 1998-03-26 1999-03-25 Variants de serine-proteases ayant des deletions et des substitutions d'acides amines Withdrawn EP1082443A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US7947798P 1998-03-26 1998-03-26
US79477P 1998-03-26
PCT/IB1999/000520 WO1999049057A1 (fr) 1998-03-26 1999-03-25 Variants de serine-proteases ayant des deletions et des substitutions d'acides amines

Publications (1)

Publication Number Publication Date
EP1082443A1 true EP1082443A1 (fr) 2001-03-14

Family

ID=22150818

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99907806A Withdrawn EP1082443A1 (fr) 1998-03-26 1999-03-25 Variants de serine-proteases ayant des deletions et des substitutions d'acides amines

Country Status (9)

Country Link
EP (1) EP1082443A1 (fr)
JP (1) JP2002518993A (fr)
KR (1) KR20010052223A (fr)
CN (1) CN1303437A (fr)
AR (1) AR023021A1 (fr)
AU (1) AU750336B2 (fr)
BR (1) BR9909147A (fr)
CA (1) CA2324422A1 (fr)
WO (1) WO1999049057A1 (fr)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU5928100A (en) * 1999-07-22 2001-02-13 Procter & Gamble Company, The Subtilisin protease variants having amino acid deletions and substitutions in defined epitope regions
US6803222B2 (en) 2000-11-22 2004-10-12 Kao Corporation Alkaline proteases
DE10360805A1 (de) 2003-12-23 2005-07-28 Henkel Kgaa Neue Alkalische Protease und Wasch- und Reinigungsmittel, enthaltend diese neue Alkalische Protease
DE102007003143A1 (de) 2007-01-16 2008-07-17 Henkel Kgaa Neue Alkalische Protease aus Bacillus gibsonii und Wasch- und Reinigungsmittel enthaltend diese neue Alkalische Protease
DE102007032111B4 (de) 2007-07-09 2017-07-20 Henkel Ag & Co. Kgaa Neue Proteasen und Wasch- und Reinigungsmittel enthaltend diese Proteasen
DE102007036756A1 (de) 2007-08-03 2009-02-05 Henkel Ag & Co. Kgaa Neue Proteasen und Wasch- und Reinigungsmittel, enthaltend diese neuen Proteasen
DE102007051092A1 (de) 2007-10-24 2009-04-30 Henkel Ag & Co. Kgaa Subtilisin aus Becillus pumilus und Wasch- und Reinigungsmittel enthaltend dieses neue Subtilisin
DK2421973T3 (en) * 2009-04-24 2018-07-30 Danisco Us Inc PROTEAS WITH MODIFIED PRO AREAS
RU2639534C2 (ru) * 2009-09-25 2017-12-21 Новозимс А/С Применение вариантов протеазы
WO2013076269A1 (fr) * 2011-11-25 2013-05-30 Novozymes A/S Variants de subtilase et polynucléotides codants pour ceux-ci
CN102839165B (zh) * 2012-09-26 2014-12-10 金普诺安生物科技（苏州）有限公司 基因突变型重组蛋白酶k及其工业化生产方法
EP4291647A1 (fr) * 2021-02-12 2023-12-20 Fresenius Kabi iPSUM S.r.l. Variants de subtilisine et leur utilisation

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0916732B1 (fr) * 1987-04-06 2007-08-22 Novozymes A/S Stabilisation de protéines par mise au point des interactions électrostatiques aux sites de liaison d'ions métalliques
ES2227508T3 (es) * 1989-06-26 2005-04-01 Unilever N.V. Composiciones detergentes enzimaticas.
WO1992010755A1 (fr) * 1990-12-05 1992-06-25 Novo Nordisk A/S Proteines presentant des epitopes modifies et procedes de preparation de ces proteines
US5567601A (en) * 1993-06-01 1996-10-22 University Of Maryland Subtilisin mutants lacking a primary calcium binding site
WO1999049056A1 (fr) * 1998-03-26 1999-09-30 The Procter & Gamble Company Variants de serine-proteases ayant des substitutions d'acides amines

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9949057A1 *

Also Published As

Publication number Publication date
JP2002518993A (ja) 2002-07-02
CN1303437A (zh) 2001-07-11
CA2324422A1 (fr) 1999-09-30
BR9909147A (pt) 2000-12-05
AU750336B2 (en) 2002-07-18
AR023021A1 (es) 2002-09-04
KR20010052223A (ko) 2001-06-25
WO1999049057A1 (fr) 1999-09-30
AU2742699A (en) 1999-10-18

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