FI105483B - Förfarande för ökning av produktionen av ett intressant endogent enzym i en alkalofil Bacillus-värdcell - Google Patents

Förfarande för ökning av produktionen av ett intressant endogent enzym i en alkalofil Bacillus-värdcell Download PDF

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FI105483B
FI105483B FI884904A FI884904A FI105483B FI 105483 B FI105483 B FI 105483B FI 884904 A FI884904 A FI 884904A FI 884904 A FI884904 A FI 884904A FI 105483 B FI105483 B FI 105483B
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bacillus
host cell
dna
enzyme
protease
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FI884904A
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FI884904A (fi
FI884904A0 (fi
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Eekelen Christiaan A G Van
Der Laan Johannes C Van
Leonardus J S M Mulleners
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Genencor Int
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • 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

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  • Health & Medical Sciences (AREA)
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  • Mycology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Claims (13)

1. Förfarande för ökning av produktionen av ett intres-sant endogent enzym i en alkalofil Bacillus-värdcell, kannetecknat därav, att man i närvaro av ett fusogen vid alkaliskt pH, kombinerar en protoplast av nämnda värdcell och en plasmidkonstruktion, vilken innehäller en DNA-sekvens, som kodar för nämnda enzym, ett replikationssystem, funktionellt i nämnda värdcell, och en selektionsmarkörgen, varvid nämnda DNA blir inkorporerad i nämnda värdcell, väljer ut värdceller, vilka stabilt innehäller nämnda DNA och odlar dessa värdceller under förhällanden, vilka gynnar syntesen av nämnda intressant enzym.
2. Förfarande enligt patentkravet 1, kännetecknat därav, att man förbehandlar nämnda Bacillus-celler med lysozym i ett f i I ··' alkaliskt medium vid ca. 20 °C - ca. 37 °C för att bilda '' protoplaster, • · » avskiljer protoplasterna frän lysozymet, • · I · · • · · • · kombinerar dessa protoplaster med nämnda plasmidkonstruk- • · tion i närvaro av ett fusogen vid ca. 20 °C - ca. 37 °C, • · « « · • · /···. utspäder fusogenet och regenererar nämnda Bacillus-cel- • · ler, och odlar de regenererade Bacillus-cellerna under selektiva förhällanden. « 1 · , « i * t · • · 27 105483
3. Förfarande enligt patentkravet l eller 2, kännetecknat därav, att nämnda plasmidkonstruktion innehäller i transkriptionsriktningen en transkriptions- och transla-tionsinitierande regulatorregion, en DNA-sekvens, som kodar för nämnda enzym och en transkriptions- och trans-lationsterminerande regulatorregion, varvid nämnda regu-latorregioner är funktionella i nämnda värdcell.
4. Förfarande enligt nägot av de ovannämnda patentkraven, kännetecknat därav, att en otransformerad stam av nämnda värcell är en alkalofil Bacillus av vildtyp eller en mutant därav, vilken producerar nämnda enzym, varvid den endogena produktionen av enzymet ökar i värdcellen.
5. Förfarande enligt nägot av patentkraven 1-3, kännetecknat därav, att ett den otransformerade stammen av nämnda värdcell är en mutantstam av en Bacillus av vildtyp, vilken inte producerar det intressanta enzymet.
6. Förfarande enligt nägot av de ovannämnda patentkraven, kännetecknat därav, att Bacillus av vildtyp är Bacillus novo arten PB92.
7. Förfarande enligt nägot av de ovannämnda patentkraven, kännetecknat .därav, att nämnda endogena enzym är ett V · serinproteas. • · · • *· *
8. Förfarande enligt patentkravet 7, kännetecknat därav, :Y: att nämnda serinproteas är ett högalkaliskt serinproteas.
..···. 9. Förfarande enligt nägot av de ovannämnda patentkraven, • · ,···. kännetecknat därav, att nämnda enzym är ett högalkaliskt i · . ' serinproteas med ätminstone 70 %:s homologi tili aminosy- rasekvensen: X « ' ; I - I M M • I • I I • · 28 105483 H2N-A-Q-S-V-P-W-G-I-S-R-V-Q-A-P-A-A-H-N-R-G-L-T-G-S-G-V-K-V-A-V-L-D-T-G-I-S-T-H-P-D-L-N-I-R-G-G-A-S-F-V-P-G-E-P-S-T-Q-D-G-N-G-H-G-T-H-V-A-G-T-I-A-A-L-N-N-S-I-G-V-L-G-V-A-P-N-A-E-L-Y-A-V-K-V-L-G-A-S-G-S-G-S-V-S-S-I-A-Q-G-L-E-W-A-G-N-N-G-M-H-V-A-N-L-S-L-G-S-P-S-P-S-A-T-L-E-Q-A-V-N-S-A-T-S-R-G-V-L-V-V-A-A-S-G-N-S-G-A-G-S-I-S-Y-P-A-R-Y-A-N-A-M-A-V-G-A· T-D-Q-N-N-N-R-A-S-F-S-Q-Y-G-A-G-L-D-I-V-A-P-O-V-N-V-Q-S-T-Y-P-G-S-T-Y-A-S-L-N-G-T-S-M-A-T-P-H-V-A-G-A-A-A-L-V-K-Q-K.-N-P-S-W-S-N-V-Q-I-R-N-H-L-K-N-T-A-T-S-L-G-8-T-N-L-Y-G-S-G-L-V-N-A-E-A-A-T-R-COOH.
10. Förfarande enligt nägot av de ovannämnda patentkra-ven, kännetecknat därav, att nämnda DNA är stabilt integ-rerade i värdcellens genom.
11. Förfarande enligt patentkravet 10, kännetecknat därav, att ätminstone tvä kopior av nämnda DNA är stabilt integrerade i värdcellens genom.
12. Förfarande enligt patentkravet 10, kännetecknat därav, att tvä kopior av proteasgenen är tandemformigt ställda i värdcellens kromosom separerade av plasmidsek-venser.
13. Förfarande enligt nägot av de ovan nämnda patentkra- ven, kännetecknat därav, att markörgenen är en antibio- ·;* tisk resistensgen. »··· • · • · · • · · • · • · • · · • · · • · • « · * · • · « « * • · · • · » f * V · « ·
FI884904A 1987-02-27 1988-10-24 Förfarande för ökning av produktionen av ett intressant endogent enzym i en alkalofil Bacillus-värdcell FI105483B (sv)

Priority Applications (1)

Application Number Priority Date Filing Date Title
FI925413A FI106726B (sv) 1987-02-27 1992-11-27 Transformation av för högalkaliska proteaser kodande gener i Bacillus-bakterier och vid förfarandet använda vektorer, DNA-sekvenser och prober

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP87200358 1987-02-27
EP87200358 1987-02-27
NL8800007 1988-02-26
PCT/NL1988/000007 WO1988006624A2 (en) 1987-02-27 1988-02-26 Molecular cloning and expression of genes encoding proteolytic enzymes

Publications (3)

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FI884904A FI884904A (fi) 1988-10-24
FI884904A0 FI884904A0 (fi) 1988-10-24
FI105483B true FI105483B (sv) 2000-08-31

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Application Number Title Priority Date Filing Date
FI884904A FI105483B (sv) 1987-02-27 1988-10-24 Förfarande för ökning av produktionen av ett intressant endogent enzym i en alkalofil Bacillus-värdcell
FI925413A FI106726B (sv) 1987-02-27 1992-11-27 Transformation av för högalkaliska proteaser kodande gener i Bacillus-bakterier och vid förfarandet använda vektorer, DNA-sekvenser och prober
FI20001152A FI119028B (sv) 1987-02-27 2000-05-15 Förfarande för att transformera alkalofila Bacillus-stammar

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Application Number Title Priority Date Filing Date
FI925413A FI106726B (sv) 1987-02-27 1992-11-27 Transformation av för högalkaliska proteaser kodande gener i Bacillus-bakterier och vid förfarandet använda vektorer, DNA-sekvenser och prober
FI20001152A FI119028B (sv) 1987-02-27 2000-05-15 Förfarande för att transformera alkalofila Bacillus-stammar

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US (1) US5217878A (sv)
JP (1) JP3026567B2 (sv)
KR (1) KR0127560B1 (sv)
CN (1) CN88101681A (sv)
AT (2) ATE181110T1 (sv)
AU (1) AU620026B2 (sv)
BG (1) BG49049A3 (sv)
BR (1) BR8805647A (sv)
CA (1) CA1339100C (sv)
DE (2) DE3856339T2 (sv)
DK (2) DK175738B1 (sv)
FI (3) FI105483B (sv)
HU (1) HU213220B (sv)
LT (1) LT4000B (sv)
LV (1) LV10307B (sv)
NO (1) NO884423L (sv)
PT (1) PT86864B (sv)
RU (1) RU2023723C1 (sv)
WO (1) WO1988006624A2 (sv)

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ATE180016T1 (de) 1999-05-15
CN88101681A (zh) 1988-11-09
DK175852B1 (da) 2005-04-04
PT86864B (pt) 1992-05-29
NO884423D0 (no) 1988-10-05
JPH01502876A (ja) 1989-10-05
FI119028B (sv) 2008-06-30
DK175738B1 (da) 2005-02-07
KR0127560B1 (ko) 1997-12-29
FI925413A (fi) 1992-11-27
KR890700665A (ko) 1989-04-26
LV10307A (lv) 1994-10-20
DK200401262A (da) 2004-08-20
FI20001152A (sv) 2000-05-15
HU213220B (en) 1997-03-28
LV10307B (en) 1995-08-20
DK593988A (da) 1988-10-26
BR8805647A (pt) 1989-10-31
FI106726B (sv) 2001-03-30
AU620026B2 (en) 1992-02-13
FI884904A (fi) 1988-10-24
WO1988006624A2 (en) 1988-09-07
AU1398088A (en) 1988-09-26
ATE181110T1 (de) 1999-06-15
JP3026567B2 (ja) 2000-03-27
DE3856331T2 (de) 1999-10-14
WO1988006624A3 (en) 1988-11-17
CA1339100C (en) 1997-07-29
PT86864A (pt) 1988-03-01
NO884423L (no) 1988-10-05
RU2023723C1 (ru) 1994-11-30
DE3856339D1 (de) 1999-07-15
BG49049A3 (en) 1991-07-15
FI884904A0 (fi) 1988-10-24
US5217878A (en) 1993-06-08
LT4000B (en) 1996-06-25
DE3856339T2 (de) 1999-11-04
DK593988D0 (da) 1988-10-26
LTIP1820A (en) 1995-08-25
DE3856331D1 (de) 1999-06-17
FI925413A0 (fi) 1992-11-27

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