WO1991002802A1 - Hybrid plasmids for the production of subtilisin carlsberg in bacillus - Google Patents
Hybrid plasmids for the production of subtilisin carlsberg in bacillus Download PDFInfo
- Publication number
- WO1991002802A1 WO1991002802A1 PCT/EP1990/001347 EP9001347W WO9102802A1 WO 1991002802 A1 WO1991002802 A1 WO 1991002802A1 EP 9001347 W EP9001347 W EP 9001347W WO 9102802 A1 WO9102802 A1 WO 9102802A1
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- WIPO (PCT)
- Prior art keywords
- bacillus
- protease
- plasmid
- subtilisin carlsberg
- dna
- Prior art date
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- 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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- 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
Definitions
- the invention relates to new hybrid plasmids which make it possible to express the alkaline protease subtilisin Carlsberg and related proteolytic enzymes in increased amounts in Bacillus.
- proteases are known which are formed by microorganisms, in particular bacteria and fungi, and are removed from the cell by active transport, so that they collect in the culture medium. Numerous enzymes of this type are already being used industrially in processes that involve the degradation of proteins. Important applications are, for example, detergents and cleaning agents or also animal foods.
- German patent application DE 35 27 913 describes hybrid plasmids for microorganisms of the genus Bacillus which contain a double-stranded DNA which has the following structure: promoter which is recognized by the RNA polymerase of microorganisms of the genus Bacillus, ribosome binding site , Start codon, structural gene which codes for subtilisin Carlsberg or its parts with proteolytic activity or variants with proteolytic activity including their leader sequences, stop codon.
- German patent application P 38 21 491 describes that plasmids which produce an even higher protease expression can be obtained by dividing the piece of DNA which is in pC50 between two relatively adjacent Aval sites and a part of the promoter is removed by cutting with Aval and ligating.
- the invention therefore relates to hybrid plasmids for generation. of protease in Bacillus, consisting of a Bacillus-common basic plasmid and a DNA sequence which codes for a protease of the Subti ⁇ lisin Carlsberg type, characterized in that a DNA sequence is inserted as protease sequence which is for a protease of the Subtilisin Carlsberg type is coded and upstream is limited by the Aval interface closest to the structure, downstream by the closest neighboring Sstl interface.
- the cut between the SstI interface and the Aval interface closest to this interface is preferred in an output plasmid such as pC50.
- a particularly preferred starting material is a plasmid pC50 according to DE 35 27 913.
- the restriction endonuclease SstI cuts at base pair 1539 behind the protease gene or behind its transcriptional terminator.
- the starting point is plasmids which contain the structural gene from Subtilisin Carlsberg or an enzyme similar to Subtilisin Carlsberg.
- Subtilisin Carlsberg An enzyme similar to Subtilisin Carlsberg is understood here to mean those enzymes which differ from Subtilisin Carlsberg in up to 1% of the amino acids and which also have protease activity in a comparable manner. have their circumference, the structural genes having to have the interfaces mentioned.
- the preferred starting material is a plasmid pC50, produced from a pBC16 portion and the structural gene of Subtilisin Carlsberg from Bacillus licheniformis DSM641 or from Bacillus licheniformis DSM 5440.
- the corresponding starting plasmid is called pC50.
- the Aval / SstI fragment from pC50 (FIG. 1) comprising subtilisin Carlsberg protease genes was cloned into the multiple cloning site of the E. coli vector pUC19 (1).
- pC50 was cleaved with Aval and SstI and pUC19 ( Figure 2) with Xmal and SstI.
- pC50 is additionally cleaved with PvuII, which leaves the Aval / SstI fragment carrying the protease gene part intact, but cuts the pBC16 vector part twice.
- the enzymes were extracted twice with half the volume of phenol, based on the DNA solution, then mixed in a quantitative ratio of 1: 1, the mixture was extracted 5 times with the same volume of ether and then with twice the volume of ethanol 20 Incubated for minutes at -20 ° C and the DNA pelleted by centrifugation. After drying, the DNA was dissolved in a suitable buffer. The DNA concentration was adjusted to 20 ⁇ g / ml and T4 DNA ligase was added in a concentration of 1 U / ⁇ g of the DNA used. The ligase reaction was carried out at 6 ° C. for about 18 h.
- the DNA was analyzed by the SDS-NaCl method by Guerry et al. (4), with subsequent cesium chloride density gradient centrifugation.
- the bacillus vectors pUBUO (5; FIG. 4) and pBC16 (6; FIG. 5) were used as vectors for the cloning of the Aval / Sstl protease gene fragment. These two plasmids are largely homologous and differ essentially only in the resistance determinant (kanamycin for pUBUO and tetracycline for pBC16).
- both pH9 and pUBUO and pBC16 were cleaved with BamHI and EcoRI, the latter being only incompletely EcoRI cleavage, since two EcoRI cleavage sites are present in pBC16, of which the cleavage site is in position 1 how the corresponding EcoRI site in pUBUO should be used for the insertion of the EcoRI / BamHI-P300 protease fragment.
- BamHI and EcoRI sites in pH9 originate from the multiple cloning site of pUC19. Even in the case of pH9, the EcoRI cleavage must be carried out incompletely, since an additional EcoRI cleavage site exists in the protease gene.
- pH9 to pUBUO or pBC16 in a quantitative ratio of 2: 1 was used for the ligation.
- the total DNA concentration was 200 ⁇ g / ml.
- the transformants were placed on full medium agar plates with 15 ⁇ g / ml tetracycline for ligation with pBC16 as vector, and 10 ⁇ g / ml Kanamycin selected for ligation with pUBUO as vector.
- an immunological method was used which is based on a method by Broo e and Gilbert (8, 9). This method was already used in the original cloning of the subtilisin Carlsberg protease gene in the published patent application DE 3527 913 AI.
- Immunologically positive clones were identified by means of restriction analysis of rapid lysates and the pBC16 derivative was designated as pC51 (FIG. 6) and the pUBHO derivative as pH70 (FIG. 7).
- Bacillus subtilis SB202 pC51 and Bacillus subtilis SB202 pH70 were compared at 37 ° C to Bacillus subtilis SB202 in shake flasks in a complex medium suitable for protease production (9.1 g / 1 KH 2 P0, 11.8 g / 1 K 2 HP0 4 9/1 gS ⁇ 4x7H2 ⁇ , 0.5 g / 1 MnS ⁇ 4x2H 0, 0.2 g / 1 CaCl2 H2 ⁇ , 3 g / 1 soy flour, 18 g / 1 casein acc.
- a complex medium suitable for protease production 9.1 g / 1 KH 2 P0, 11.8 g / 1 K 2 HP0 4 9/1 gS ⁇ 4x7H2 ⁇ , 0.5 g / 1 MnS ⁇ 4x2H 0, 0.2 g / 1 CaCl2 H2 ⁇ , 3 g / 1 soy flour, 18 g / 1 casein acc.
- Bacillus subtilis SB202 1 Bacillus subtilis SB202 1
- Bacillus subtilis SB202pH70 60-80
- plasmid DNA was processed (10) and protoplasts from Bacillus licheniformis DSM5440 were hereby trans- formed.
- the transformants were selected on DM3 agar (11) with 15 ⁇ g / ml tetracycline for the selection of pC51 and on mannitol agar (12) with 10 ⁇ g / ml neoycin for the selection of pH 70.
- Colonies that were grown were inoculated onto calcium caseinate agar (Merck) with 15 ⁇ g / ml tetracycline (pC51) or 10 ⁇ g / ml kanamycin (pH70). Transformants which, after growth, had a lysis yard larger than E312 on these plates were checked for their agreement with the plasmid DNAs used for the transformation after restriction enzyme analysis of rapid lysates.
- Example 3 Expression of pC51 / pH70 in Bacillus lichenifo ⁇ is DSM 5440
- Bacillus liche ⁇ iformus DSM5440 which contains the plasmids pC50, pC51 or pH70, was shaken in a flask in a complex medium suitable for protease production (2.4 g / 1 H2PO, 1 g / 1 MgS ⁇ 4x7H2 ⁇ , 0.5 g / 1 MnS ⁇ 4x2H2 ⁇ , 0, 2 g / 1 CaCl2x2H 2 0.3 g / 1 soy flour, 12 g / 1 casein acc. To Hammarsten, 120 g / 1 amylase-treated potato starch) at 39 ° C and 34 ° C. Samples were taken at various times to determine protease activity.
- a complex medium suitable for protease production 2.4 g / 1 H2PO, 1 g / 1 MgS ⁇ 4x7H2 ⁇ , 0.5 g / 1 MnS ⁇ 4x2H2 ⁇ , 0, 2 g / 1 CaCl2
- protease measurements were carried out using N-CBZ-valine-p-nitrophenyl ester as the substrate, the substrate being cleaved into N-CBZ amino acid and p-nitrophenol * .
- the formation of p-nitrophenol was measured as a measure of the activity of the protease at 340 nm in the photometer (13).
- Strain Relative Protease Activity (%)
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Abstract
Description
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Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DEP3928046.2 | 1989-08-25 | ||
DE19893928046 DE3928046A1 (en) | 1989-08-25 | 1989-08-25 | HYBRID PLASMIDE FOR THE PRODUCTION OF SUBTILISIN CARLSBERG IN BACILLUS |
Publications (1)
Publication Number | Publication Date |
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WO1991002802A1 true WO1991002802A1 (en) | 1991-03-07 |
Family
ID=6387817
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP1990/001347 WO1991002802A1 (en) | 1989-08-25 | 1990-08-16 | Hybrid plasmids for the production of subtilisin carlsberg in bacillus |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP0495790A1 (en) |
DE (1) | DE3928046A1 (en) |
WO (1) | WO1991002802A1 (en) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0214435A2 (en) * | 1985-08-03 | 1987-03-18 | Henkel Kommanditgesellschaft auf Aktien | Alkaline protease, method for the production of hybrid vectors and genetically transformed microorganisms |
EP0348814A1 (en) * | 1988-06-25 | 1990-01-03 | Henkel Kommanditgesellschaft auf Aktien | Hybrid plasmid for producing subtilisin Carlsberg in bacillus |
EP0351717A2 (en) * | 1988-07-21 | 1990-01-24 | Henkel Kommanditgesellschaft auf Aktien | Plasmid for production of alpha-amylase in Bacillus |
-
1989
- 1989-08-25 DE DE19893928046 patent/DE3928046A1/en not_active Withdrawn
-
1990
- 1990-08-16 WO PCT/EP1990/001347 patent/WO1991002802A1/en not_active Application Discontinuation
- 1990-08-16 EP EP19900912849 patent/EP0495790A1/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0214435A2 (en) * | 1985-08-03 | 1987-03-18 | Henkel Kommanditgesellschaft auf Aktien | Alkaline protease, method for the production of hybrid vectors and genetically transformed microorganisms |
EP0348814A1 (en) * | 1988-06-25 | 1990-01-03 | Henkel Kommanditgesellschaft auf Aktien | Hybrid plasmid for producing subtilisin Carlsberg in bacillus |
EP0351717A2 (en) * | 1988-07-21 | 1990-01-24 | Henkel Kommanditgesellschaft auf Aktien | Plasmid for production of alpha-amylase in Bacillus |
Non-Patent Citations (1)
Title |
---|
Nucleic Acids Research, Band 13, Nr. 24, Dezember 1985, IRL Press Limited, (OXFORD, GB), M. JACOBS et al.: "Cloning, Sequencing and Expression of Subtilisin Carlsberg from Bacillus Licheniformis", seiten 8913-8926, siehe seite 8916, zeile 12 - seite 8918, zeile 23 * |
Also Published As
Publication number | Publication date |
---|---|
EP0495790A1 (en) | 1992-07-29 |
DE3928046A1 (en) | 1991-02-28 |
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