EP1196547A2 - Conjugues de protease comprenant des regions epitope protegees steriquement - Google Patents

Conjugues de protease comprenant des regions epitope protegees steriquement

Info

Publication number
EP1196547A2
EP1196547A2 EP00945317A EP00945317A EP1196547A2 EP 1196547 A2 EP1196547 A2 EP 1196547A2 EP 00945317 A EP00945317 A EP 00945317A EP 00945317 A EP00945317 A EP 00945317A EP 1196547 A2 EP1196547 A2 EP 1196547A2
Authority
EP
European Patent Office
Prior art keywords
protease
moiety
epitope
group
subtilisin
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Withdrawn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
EP00945317A
Other languages
German (de)
English (en)
Inventor
Donn Nelton Rubingh
David John Weisgerber
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 EP1196547A2 publication Critical patent/EP1196547A2/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Withdrawn legal-status Critical Current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
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • 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
    • 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

Definitions

  • proteases In the cleaning arts, the mostly widely utilized of these proteases are the se ⁇ ne proteases Most of these se ⁇ ne proteases are produced by bacte ⁇ al organisms while some are produced by other organisms, such as fungi See Siezen et al.. "Homology Modelling and Protein Engineering Strategy of Subtilases, the Family of Subtihsin-Like Se ⁇ ne Proteases", Protein Engineering, Vol 4, No 7, pp. 719 - 737 (1991). Unfortunately, the efficacy of the wild-type proteases in their natural environment is frequently not optimized for the artificial environment of a cleaning composition. Specifically, protease characte ⁇ stics such as, for example, thermal stability, pH stability, oxidative stability, and substrate specificity are not necessa ⁇ ly optimized for utilization outside the natural environment of the protease.
  • the epitope protection positions for the first epitope region are selected from 1, 2, 3, 4, 5, 6, 7, 12, 17, 36, 40, 41, 43, 44, 45, 67, 86, 87, 89, 206, 209, 210, 212, 213, 214, 215, and 216 corresponding to subtilisin BPN';
  • protease conjugates of the present invention have decreased immunogenicity relative to the parent protease. Accordingly, such protease conjugates are suitable for use in several types of compositions including, but not limited to, laundry, dish, hard surface, skin care, hair care, beauty care, oral care, and contact lens compositions.
  • variable means a protein having an amino acid sequence which differs from that of the corresponding wild-type protein
  • prote B for use as parent ammo acid sequences herein are disclosed in EP 251 ,446, assigned to Genencor International. Inc., published January 7, 1988, as characte ⁇ zed by the wild-type subtilisin BPN " ammo acid sequence with mutations at one or more of the following positions: Tyr21, Thr22.
  • the protease moieties herein have three epitope regions: a first epitope region, a second epitope region, and a third epitope region.
  • the first epitope region occurs at positions 70 - 84 corresponding to subtilisin BPN';
  • the second epitope region occurs at positions 103 - 126 corresponding to subtilisin BPN', and the third epitope region occurs at positions 220 - 246 corresponding to subtilisin BPN'
  • the epitope protection positions for the second epitope region are 25, 26, 27, 46, 47, 48, 49, 50, 51, 52, 53, 54, 91, 99, 100, 101, 102, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 140, 141, 144, and 145 corresponding to subtilisin BPN.
  • the epitope protection positions for the second epitope region are 27, 47, 48, 50, 52, 102, 127, 128, 130, 131, 132, 134, 138, and 141 corresponding to subtilisin BPN'.
  • a /?NA assay (DelMar et al.. Analytical Biochemistry, Vol. 99, pp. 316 - 320 (1979)) is used to determine the active protease concentration for fractions collected du ⁇ ng gradient elution. This assay measures the rate at which /j-nitroamlme is released as the protease hydrolyzes the soluble synthetic substrate, succinyl-alanme-alanine-prolme-phenylalanine-/?- nitroanilme (sAAPF-pNA). The rate of production of yellow color from the hydrolysis reaction is measured at 410 nm on a specfrophotometer and is proportional to the active protease moiety concentration. In addition, absorbance measurements at 280 nm are used to determine the total protein concentration. The active protease/total-protem ratio gives the protease pu ⁇ ty, and is used to identify fractions to be pooled for the stock solution.
  • a va ⁇ ant of subtilisin BPN' with a substitution of leucme for tyrosine at position 217 and a substitution of cysteme for phenylalanme at position 17 is prepared.
  • a concenfration of approximately 2 mg / mL m phosphate buffer (pH 5.5) of the variant is achieved.
  • the pH is then raised to 7.5 with dilute sodium hydroxide.
  • the va ⁇ ant is mixed with the dimethyl polyethylene glycol maleimide at a 25: 1 activated polymer to va ⁇ ant excess. After one hour of mixing at ambient temperature, the pH of the mixture is adjusted to 5.5 with dilute phosphoric acid and filtered through a molecular weight cut-off ultrafilter to remove excess polymer.
  • a va ⁇ ant of subtilisin BPN' with a substitution of leucme for tyrosine at position 217 and a substitution of cysteme for serme at position 214 is prepared.
  • a 20 mL solution of the va ⁇ ant is prepared at a concenfration of approximately 1 mg / mL in 0.01 M KH 2 P0 4 buffer (pH 8.6).
  • Oxygen is gently bubbled through the solution at ambient temperature for approximately one hour to form the desired protease conjugate dimer.
  • the resulting protease conjugate is obtained from the solution by standard methods.
  • the present cleaning compositions further comp ⁇ se a cleaning composition earner compnsmg one or more cleaning composition mate ⁇ als compatible with the protease conjugate
  • cleaning composition matenal means any material selected for the particular type of cleaning composition desired and the form of the product (e g , liquid, granule, bar, spray, stick, paste, gel), which mate ⁇ als are also compatible with the protease conjugate used m the composition
  • the specific selection of cleaning composition materials is readily made by conside ⁇ ng the surface matenal to be cleaned, the desired form of the composition for the cleaning condition du ⁇ ng use (e g , through the wash detergent use).
  • Enzyme stabilizers may also be used in the cleaning compositions.
  • Such enzyme stabilizers include propylene glycol (preferably from about 1% to about 10%), sodium formate (preferably from about 0.1% to about 1%) and calcium formate (preferably from about 0.1% to about 1%)
  • the surfactant component when present, may comp ⁇ se as little as 0.1% of the compositions herem, but typically the compositions will contain from about 0.25% to about 10%. more preferably from about 1% to about 5% of surfactant.
  • dishwashing compositions compnse one or more vanants of the present invention.
  • “dishwashing composition” refers to all forms of compositions for cleaning dishes including, but not limited to, granular and liquid forms.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Detergent Compositions (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Cosmetics (AREA)

Abstract

L'invention concerne des conjugués de subtilisine protéase comprenant une fraction de protéase et une ou plusieurs fractions d'addition. Chaque fraction d'addition est liée par covalence à une position épitope protégée de la fraction de protéase. Ces conjugués de protéase présentent une immunogénicité réduite par rapport à une protéase apparentée. L'invention concerne également des compositions de nettoyage et de soins personnels comprenant ces conjugués de protéase.
EP00945317A 1999-07-22 2000-07-11 Conjugues de protease comprenant des regions epitope protegees steriquement Withdrawn EP1196547A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US14497999P 1999-07-22 1999-07-22
US144979P 1999-07-22
PCT/US2000/018854 WO2001007577A2 (fr) 1999-07-22 2000-07-11 Conjugues de protease comprenant des regions epitope protegees steriquement

Publications (1)

Publication Number Publication Date
EP1196547A2 true EP1196547A2 (fr) 2002-04-17

Family

ID=22511045

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00945317A Withdrawn EP1196547A2 (fr) 1999-07-22 2000-07-11 Conjugues de protease comprenant des regions epitope protegees steriquement

Country Status (10)

Country Link
EP (1) EP1196547A2 (fr)
JP (1) JP2003505069A (fr)
KR (1) KR20020021396A (fr)
CN (1) CN1372593A (fr)
AU (1) AU777550B2 (fr)
BR (1) BR0012692A (fr)
CA (1) CA2379723A1 (fr)
CZ (1) CZ2002211A3 (fr)
MX (1) MXPA02000837A (fr)
WO (1) WO2001007577A2 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MXPA05007151A (es) 2002-12-31 2005-09-21 Nektar Therapeutics Al Corp Polimeros terminados en maleimida hidroliticamente estables.
US7432331B2 (en) 2002-12-31 2008-10-07 Nektar Therapeutics Al, Corporation Hydrolytically stable maleimide-terminated polymers
EP2105493B1 (fr) 2008-03-25 2014-05-14 Diversey, Inc. Procédé de lubrification solide utilisant des lubrifiants à base d'huile
EP2105494B1 (fr) 2008-03-25 2019-05-08 Diversey, Inc. Procédé de lubrification d'une courroie de transporteur
RU2560978C2 (ru) 2008-11-11 2015-08-20 ДАНИСКО ЮЭс ИНК. Протеазы, содержащие одну или несколько комбинируемых мутаций
US20100192985A1 (en) * 2008-11-11 2010-08-05 Wolfgang Aehle Compositions and methods comprising serine protease variants

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4844897A (en) * 1985-09-13 1989-07-04 Hiroshi Maeda Anti-tumor protease preparations
JPH02219571A (ja) * 1989-02-20 1990-09-03 Kanebo Ltd 修飾プロテアーゼ及びその製造法
FI932561L (fi) * 1990-12-05 1993-06-04 Novo Nordisk As Proteiner med foeraendrade epitoper och foerfaranden foer deras framstaellning
FI972443A7 (fi) * 1994-12-07 1997-06-09 Novozymes As Polypeptidi, jonka allergeenisuus on vähentynyt
US6946280B1 (en) * 1996-03-29 2005-09-20 Genencor International, Inc. Enzyme multimer and process of producing same
CN1246146A (zh) * 1996-11-26 2000-03-01 金克克国际有限公司 化学修饰的酶
ATE375387T1 (de) * 1997-06-25 2007-10-15 Novozymes As Modifiziertes polypeptid
US6395532B1 (en) * 1998-01-23 2002-05-28 Genencor International, Inc. Modified enzymes and their use for peptide synthesis
US6495136B1 (en) * 1998-03-26 2002-12-17 The Procter & Gamble Company Proteases having modified amino acid sequences conjugated to addition moieties
WO2000028007A2 (fr) * 1998-11-10 2000-05-18 Genencor International, Inc. Serines hydrolases mutantes chimiquement modifiees presentant une activite catalytique et une selectivite chirale ameliorees
AU2200100A (en) * 1998-12-21 2000-07-12 Genencor International, Inc. Chemically modified enzymes with multiple charged variants

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0107577A2 *

Also Published As

Publication number Publication date
WO2001007577A3 (fr) 2001-08-30
CZ2002211A3 (cs) 2002-05-15
CA2379723A1 (fr) 2001-02-01
MXPA02000837A (es) 2002-07-30
AU5928300A (en) 2001-02-13
AU777550B2 (en) 2004-10-21
BR0012692A (pt) 2002-04-09
CN1372593A (zh) 2002-10-02
KR20020021396A (ko) 2002-03-20
JP2003505069A (ja) 2003-02-12
WO2001007577A2 (fr) 2001-02-01

Similar Documents

Publication Publication Date Title
US6495136B1 (en) Proteases having modified amino acid sequences conjugated to addition moieties
US6586223B1 (en) Subtilisin protease variants having amino acid substitutions in defined epitope regions
EP1206526A2 (fr) Variants de protease subtilisine possedant des suppressions et des substitutions d'acides amines dans certaines regions de sites antigeniques
US6566115B1 (en) Protease conjugates having sterically protected clip sites
US6946128B1 (en) Protease conjugates having sterically protected epitope regions
AU777550B2 (en) Protease conjugates having sterically protected epitope regions
EP1196546A2 (fr) Proteases de subtisiline a reticulation intermoleculaire a antigenicite reduite
MXPA00009385A (en) Protease conjugates
CZ20003396A3 (cs) Proteázový konjugát a prostředek osobní hygieny

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20020125

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

RIN1 Information on inventor provided before grant (corrected)

Inventor name: WEISGERBER, DAVID, JOHN

Inventor name: RUBINGH, DONN, NELTON

Inventor name: CORREA, PAUL, ELLIOTT

17Q First examination report despatched

Effective date: 20040521

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20060112