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WO1999034002A2 - Proteases provenant d'organismes a gram positif - Google Patents

Proteases provenant d'organismes a gram positif Download PDF

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Publication number
WO1999034002A2
WO1999034002A2 PCT/US1998/027018 US9827018W WO9934002A2 WO 1999034002 A2 WO1999034002 A2 WO 1999034002A2 US 9827018 W US9827018 W US 9827018W WO 9934002 A2 WO9934002 A2 WO 9934002A2
Authority
WO
WIPO (PCT)
Prior art keywords
metalloprotease
nucleic acid
seq
gram positive
microorganism
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.)
Ceased
Application number
PCT/US1998/027018
Other languages
English (en)
Other versions
WO1999034002A3 (fr
Inventor
David A. Estell
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.)
Danisco US Inc
Original Assignee
Genencor International Inc
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 Genencor International Inc filed Critical Genencor International Inc
Priority to AU19299/99A priority Critical patent/AU1929999A/en
Priority to US09/555,062 priority patent/US6528255B1/en
Priority to EP98964106A priority patent/EP1042489A2/fr
Publication of WO1999034002A2 publication Critical patent/WO1999034002A2/fr
Publication of WO1999034002A3 publication Critical patent/WO1999034002A3/fr
Anticipated expiration legal-status Critical
Priority to US10/285,074 priority patent/US7078216B2/en
Ceased 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
    • 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
    • C11D3/38663—Stabilised liquid enzyme compositions
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • 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)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)

Definitions

  • Gram positive microorganisms such as members of the group Bacillus
  • Gram positive bacteria have been used for large-scale industrial fermentation due, in part, to their ability to secrete their fermentation products into the culture media.
  • secreted proteins are exported across a cell membrane and a cell wall, and then are subsequently released into the external media usually maintaining their native conformation.
  • proteases found in microorganisms are based on their catalytic mechanism which results in four groups: serine proteases, metalloproteases, cysteine proteases, and aspartic proteases. These categories can be distinguished by their sensitivity to various inhibitors. For example, serine proteases are inhibited by phenylmethylsulfonylfluoride (PMSF) and diisopropylfluorophosphate (DIFP); metalloproteases by chelating agents; cysteine proteases by iodoacetamide and heavy metals and aspartic proteases by pepstatin.
  • PMSF phenylmethylsulfonylfluoride
  • DIFP diisopropylfluorophosphate
  • metalloproteases by chelating agents
  • cysteine proteases by iodoacetamide and heavy metals and aspartic proteases by pepstatin.
  • Metalloproteases form the most diverse of the catalytic types of proteases.
  • Family m40 includes bacterial enzymes such as the hippurate hydrolase from Campylobacter jejuni (HipO)and the hydantoin utilization protein C (HyuC) from Pseudomonas sp. SUMMARY OF THE INVENTION
  • the present invention is based upon Applicant's discovery of this new metalloprotease, MP (YhaA), which in addition to providing a new and useful protease and methods of detecting DNA encoding other such proteases in a gram positive microorganism, provides several advantages which may facilitate optimization and/or modification of strains of gram positive microorganisms, such as Bacillus, for expression of desired, e.g. heterologous, proteins. Such optimizations, as described below in detail, allow the construction of strains that can have decreased proteolytic degradation of desired expression products.
  • MP YhaA
  • the present invention encompasses the naturally occurring MP encoded by nucleic acid found in a Bacillus species as well as the nucleic acid and amino acid molecules having the sequences disclosed in SEQ ID NOS: 1 and 2.
  • the gram positive microorganism is a Bacillus.
  • the Bacillus is preferably selected from the group consisting of Bacillus subtilis, Bacillus stearothermophilus, Bacillus licheniformis and Bacillus amyloliquefaciens.
  • the invention further provides for a metalloprotease that has at least 80%, preferably at least 90%, most preferably 95% homology with the amino acid sequence of SEQ ID NO: 2.
  • Bacillus subtilis MP in Figures 1A-1F (SEQ ID NOS:1 and 2). Due to the degeneracy of
  • the present invention encompasses any nucleic acid sequence that encodes the Bacillus subtilis MP amino acid sequence.
  • the present invention provides expression vectors and host cells comprising a nucleic acid encoding a gram positive MP.
  • the present invention also provides methods of making the gram positive MP.
  • the present invention encompasses novel amino acid variations of gram positive MP amino acid sequences disclosed herein that have proteolytic activity.
  • Naturally occurring gram positive MP as well as proteolytically active amino acid variations or derivatives thereof have application in the textile industry, in cleaning compositions and in animal feed.
  • the production of desired heterologous proteins or polypeptides in gram positive microorganisms may be hindered by the presence of one or more proteases which degrade the produced heterologous protein or polypeptide.
  • One advantage of the present invention is that it provides methods and expression systems which can be used to prevent that degradation, thereby enhancing yields of the desired heterologous protein or polypeptide.
  • Bacillus includes ail members known to those of skill in the art, including but not limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus and B. thunngiensis.
  • a B. subtilis MP gene of the present invention or its specific RNA, or a fragment thereof, such as a probe or primer may be isolated and labeled and then used in hybridization assays to detect a gram positive MP gene.
  • a B. subtilis MP gene of the present invention or its specific RNA, or a fragment thereof, such as a probe or primer may be isolated and labeled and then used in hybridization assays to detect a gram positive MP gene.
  • Those DNA fragments sharing substantial sequence similarity to the probe will hybridize under stringent conditions.
  • One method for mutating a nucleic acid encoding a gram positive metalloprotease is to clone the nucleic acid or part thereof, modify the nucleic acid by site directed mutagenesis and reintroduce the mutated nucleic acid into the cell on a plasmid.
  • the mutated gene can be introduced into the chromosome.
  • the parent host cell the result is that the naturally occurring nucleic acid and the mutated nucleic acid are located in tandem on the chromosome.
  • the modified sequence is left in the chromosome having thereby effectively introduced the mutation into the chromosomal gene for progeny of the parent host cell.
  • Codons preferred by a particular gram positive host cell can be selected, for example, to increase the rate of expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from a naturally occurring sequence.
  • MP can be formulated into known powdered and liquid detergents having pH between 6.5 and 12.0 at levels of about .01 to about 5% (preferably .1 % to .5%) by weight.
  • These detergent cleaning compositions can also include other enzymes such as known proteases, amylases, cellulases, lipases or endoglycosidases, as well as builders and stabilizers.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Detergent Compositions (AREA)

Abstract

L'invention concerne l'identification d'une nouvelle métalloprotéase dans des micro-organismes à Gram positif. Elle concerne également les séquences d'acides nucléiques et d'acides aminés de cette métalloprotéase; des cellules hôtes présentant une mutation ou une délétion d'une partie ou de la totalité du gène codant la métalloprotéase; des cellules hôtes contenant, de plus, un acide nucléique codant des protéines hétérologues souhaitées, telles que des enzymes; des compositions de nettoyage, des aliments pour animaux et des compositions utilisées pour traiter un textile, qui contiennent cette métalloprotéase.
PCT/US1998/027018 1997-12-30 1998-12-17 Proteases provenant d'organismes a gram positif Ceased WO1999034002A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU19299/99A AU1929999A (en) 1997-12-30 1998-12-17 Proteases from gram positive organisms
US09/555,062 US6528255B1 (en) 1997-12-30 1998-12-17 Proteases from gram positive organisms
EP98964106A EP1042489A2 (fr) 1997-12-30 1998-12-17 Proteases provenant d'organismes a gram positif
US10/285,074 US7078216B2 (en) 1997-12-30 2002-10-31 Proteases from gram positive organisms

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9727466.6A GB9727466D0 (en) 1997-12-30 1997-12-30 Proteases from gram positive organisms
GB9727466.6 1997-12-30

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US09555062 A-371-Of-International 1998-12-17
US10/285,074 Division US7078216B2 (en) 1997-12-30 2002-10-31 Proteases from gram positive organisms

Publications (2)

Publication Number Publication Date
WO1999034002A2 true WO1999034002A2 (fr) 1999-07-08
WO1999034002A3 WO1999034002A3 (fr) 1999-11-04

Family

ID=10824310

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/027018 Ceased WO1999034002A2 (fr) 1997-12-30 1998-12-17 Proteases provenant d'organismes a gram positif

Country Status (4)

Country Link
EP (1) EP1042489A2 (fr)
AU (1) AU1929999A (fr)
GB (1) GB9727466D0 (fr)
WO (1) WO1999034002A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7033817B2 (en) * 1997-12-30 2006-04-25 Genencor International, Inc. Proteases from gram positive organisms

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2003078A1 (fr) * 1988-11-18 1990-05-18 Alan Sloma Deletion de la protease

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7033817B2 (en) * 1997-12-30 2006-04-25 Genencor International, Inc. Proteases from gram positive organisms

Also Published As

Publication number Publication date
WO1999034002A3 (fr) 1999-11-04
AU1929999A (en) 1999-07-19
GB9727466D0 (en) 1998-02-25
EP1042489A2 (fr) 2000-10-11

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