WO1994006905A1 - Serine-proteases de type subtilisine mutees - Google Patents

Serine-proteases de type subtilisine mutees Download PDF

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Publication number
WO1994006905A1
WO1994006905A1 PCT/EP1993/002492 EP9302492W WO9406905A1 WO 1994006905 A1 WO1994006905 A1 WO 1994006905A1 EP 9302492 W EP9302492 W EP 9302492W WO 9406905 A1 WO9406905 A1 WO 9406905A1
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WO
WIPO (PCT)
Prior art keywords
protease
subtilisin
proteases
mutated
sequence
Prior art date
Application number
PCT/EP1993/002492
Other languages
German (de)
English (en)
Inventor
Andrea SÄTTLER
Detlev Riesner
Susanne Kanka
Karl-Heinz Maurer
Original Assignee
Cognis Gesellschaft Für Bio- Und Umwelt-Technologie Gmbh
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 Cognis Gesellschaft Für Bio- Und Umwelt-Technologie Gmbh filed Critical Cognis Gesellschaft Für Bio- Und Umwelt-Technologie Gmbh
Priority to JP6507790A priority Critical patent/JPH08501447A/ja
Priority to EP93920727A priority patent/EP0662126A1/fr
Publication of WO1994006905A1 publication Critical patent/WO1994006905A1/fr

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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

Definitions

  • the invention relates to mutations in the structural gene of subtilisin proteins that lead to new enzymes with a changed amino acid sequence.
  • subtilisin proteases are widespread in nature and are widely used for numerous technical applications. For many of these applications, it is desirable to improve the stability properties of the enzymes in relation to the applications required in each case. A summary of such considerations is given by J. A. Wells and D. A. Estell in TIBS (Trends in Biochemical Sciences), 13, page 291 ff (1988). Another review article that quotes virtually all known sequences and structures of subtilisin-like serine proteases was by R. R. Siezen et al. in Protein Engineering, Vol. 4 (7), pages 719 to 737 (1991). In the area of detergents, the stability against the irreversible inactivation at elevated temperatures plays a role in the proteases.
  • the invention therefore relates to a subtilisin-like serine protease, produced by mutating the structural gene of a wild strain and expressing the mutated structural gene in a production strain, characterized in that after the BPN 'count in position 194 of the protease, a glutamic acid residue and, if desired a proline residue is present in position 188.
  • the numbering of the positions to be exchanged in the proteases mutated according to the invention relates in each case to the protease BPN'-.
  • the person skilled in the art will place the amino acid sequence of this protease under the numbered sequence of the protease BPN 'in such a way that maximum agreement is achieved. In individual cases, this may require omissions or insertions of one or more amino acids.
  • the type of numbering is described in the already cited W089 / 6279.
  • the person skilled in the art can start from a large number of subtilisin-like serine proteases.
  • subtilisin-like serine proteases are, for example, Subtilisin BPN 1 -, Subtilisin Carlsberg or also the Subtilisins from Bacillus lentus. It is clear to the person skilled in the art that in order to generate clones which produce new, mutated enzymes, he does not start from the enzyme itself, but from the structural gene coding therefor and subjects it to mutagenesis and then uses it again in a production strain.
  • proteases which carry the amino acid .alanine in position 194 (all positions according to BPN 'count), which is then exchanged according to the invention for glutamic acid.
  • those proteases are assumed which carry the amino acid alanine in 194 and the amino acid serine in 188, an exchange A194E and S188P being carried out.
  • Suitable starting materials for a mutation of this type are, for example, proteases of the Subtilisin Carlsberg type and its variants.
  • a subtilisin Carlsberg variant with a mutation N158S and S161N (BPN 'count) is particularly preferred.
  • the structural gene of such a protease is described in European patent application EP 214435.
  • a protease from Bacillus lentus is used.
  • a protease which is described in WO91 / 02792, FIG. 29, is also particularly suitable.
  • amino acid serine in position 182 corresponds to serine 188 after BPN 'counting. The difference here is due to the fact that the protein described therein contains some deletions from BPN '.
  • protease mutants are produced which, after subtilisin BPN 'count, contain the following amino acid sequence: 188P-189F-190S-191S-192V-193G-194E-195E-196L-197E-198V-199M. It is particularly preferred in the sense of the invention that this sequence is inserted into a protease, as described in WO91 / 02792, between positions 181 and 194.
  • the new protease mutants according to the invention show improved stability.
  • the half-lives of the autoproteolytic degradation are extended over a wide temperature range, which increases the heat and storage stability at the same time.
  • the prolongation of the half-life is also achieved under non-physiological conditions, for example in the presence of complexing agents.
  • the proteases according to the invention can be produced with the aid of numerous methods.
  • the methods of random in vitro mutagenesis (RIM) and directed mutagenesis are preferred.
  • the modified structural genes produced in this way can be expressed and produced in a conventional manner in known expression systems.
  • W091 / 02792. For expression in a production strain and for the aforementioned applications W089 / 06279, EP 260299, EP 130756, EP 246678, EP 247647, EP 251 446.
  • mutants according to the invention not only show increased stability of the protease when used in detergents and cleaning agents, but they can also be produced on an industrial scale during purification without additional cooling of the process medium.
  • the starting point was the structural gene according to European patent application EP 214 435 and the expression vector pC51 mentioned there.
  • This sequence was first provided with the restriction site Nar1 at position 597 of the gene sequence of pC51 by T after G exchange by directed mutation, which did not result in an amino acid exchange, the new plasmid being called pASl.
  • Position 597 corresponds to the amino acid alanine in position 200 after BPN 'count.
  • Restriction of pASl with Narl and Pstl results in a 142 bp gene fragment which contains the region of the subtilisin sequence which codes for the weak calcium binding site of the molecule.
  • This double-stranded gene fragment was assembled in vitro from synthetic oligonucleotides and referred to as a gene cassette.
  • Nucleotide sequence GCT CCA TTC TCC AGC GTC GGA GAA GAG CTT GAA GTC ATG Amino acid sequence: A P F S S V G E E L E V M Position: 187 188 189 190 191 192 193 194 195 196 197 198 199
  • the mutagenic oligonucleotide III is marked.
  • the amino acid sequence is given in the one-letter code, selected positions are numbered.
  • the original wild-type nucleotide was indicated above or below the exchange at mutated positions outside the oligonucleotide III.
  • oligonucleotide molecules III During the synthesis of oligonucleotide molecules III, all four phosphoramidite solutions were combined with the three remaining phosphoramidites contaminated.
  • concentration of a doping phosphoride in the solution was 2% of the concentration of the wild-type nucleotide. This doping was suitable for introducing an average of 1-2 nucleotide exchanges per synthesized molecule at any position via the oligonucleotide III sequence.
  • All other oligonucleotides (I, II, IV, V, VI) were largely synthesized with wild-type sequences, directed nucleotide exchanges were inserted at a few positions, by which the amino acid sequence was not changed, but suitable restriction sites were inserted.
  • Figure 1 shows the nucleotides of the wild type sequence above or below the sequence actually synthesized.
  • the upper and lower strands of the cassette fragment were assembled from oligonucleotides with wild-type sequence and the mutated oligonucleotides and the strands were hybridized. ⁇ Opmol / oligonucleotide was used for the hybridization.
  • the resulting synthetic fragments were trimmed with Pstl and Narl in order to convert any multimers into the monomers.
  • the synthetic cassette fragments were purified from the free oligonucleotides by gel elution.
  • 0.05 ⁇ g of the eluted cassette fragment was cloned into 0.5 ⁇ g Pstl, Narl, Smal-cut, dephosphorylated and purified pUC19 vector.
  • the entire ligation product was transformed into Escherichia coli XL-1 and the transformants were amplified in four 250 ml cultures.
  • the plasmids which were 50% replication products of the upper, mutagenic and 50% replication products of the lower wild-type strand were prepared from the amplified Escherichia coli cultures. 40 ⁇ g of the plasmid preparation were cut with Xhol, Pstl and Narl and the mutagenic, amplified cassette fragments were separated by gel electrophoresis.
  • Wild-type fragments were cut up by this treatment and are no longer contained in the eluate.
  • 0.08 ⁇ g of the eluted mutagenic cassette fragments were cloned into 1.7 ⁇ g of the expression vector pASl and transformed into Bacillus subtilis strain DB104 in aliquots of 0.25 ⁇ g / batch.
  • This strain is deficient in the genomic and alkaline proteases. It only expresses the plasmid-encoded enzyme variants (Doi et al. (1986), Trends in Biotechnologie 4, pp. 232 - 235). All in all 25,000 clones per batch were generated in this way.
  • the resulting mutant bank was called RIM2.
  • the stabilized protease variants were increased by temperature gradient gel electrophoresis to thermostability, i. e. increased autoproteolysis stability and increased structural stability, examined, for the screening of the mutant library.
  • the method of W089 / 06279, page 24 can also be used.
  • the Pstl-Narl fragments of stabilized protease variant genes were cloned into the Escherichia coli vector pUC19 for the purpose of easier sequencing. Fourteen clones are selected from the mutant library, which express proteases with increased temperature stability.
  • the plasmid to be sequenced were prepared from the Bacillus clone of the RIM2 bank and cut with Pstl and Narl.
  • the cassette fragment to be cloned is cut out.
  • 0.006 ⁇ g of the cassette fragment was ligated into 0.055 ⁇ g of the Escherichia coli vector pUC19 and the ligate was transformed into Escherichia coli XL-1.
  • the transformants were amplified and the plasmids of a clone prepared from a 4 ml culture. These plasmids were used for a conventional sequencing reaction in which the entire Pstl-Narl cassette was sequenced.
  • the A194E and the S188P exchange were found.
  • Example 1 shows the nucleotide sequence and the amino acid sequence of the A194E, S188P clone.
  • the A194E exchange is responsible for the increased thermostability.
  • directed mutagenesis Various methods of directed mutagenesis are available for the introduction of the stabilizing glutamate residue at the corresponding positions of related subtilisin genes and for the introduction of the sequence according to claim 3.
  • a preferred method because of its high efficiency is the cassette mutagenesis described by Wells et al, Gene 34, 315-323, (1985) analogously to Example 1.
  • the gene fragment to be mutated, the ends with restriction sites suitable for cloning, is composed in vitro of synthetic oligonucleotides.
  • the desired nucleotide exchanges are introduced during the oligonucleotide synthesis. With each exchange in oligonucleotides, which form the upper strand, the corresponding complementary exchanges are also introduced in the lower strand, so that no mismatches occur after hybridization to the double-stranded fragment.
  • the synthetic cassette is cloned into the target vector cut with the same restriction enzymes via the terminal restriction sites.
  • the cut target vector is separated from the cut wild-type gene fragment by gel elution.
  • the Pstl described in Example 1 can be used.
  • Narl cassette can be used, which covers the entire area of the weak calcium binding site. The following exchanges must be made: Q191S, Y192V, G195E, D197E, I198V, V199M.
  • the stabilizing exchange A194E and the S188P exchange are introduced. Since it is directed mutagenesis, the synthetic Pstl-Narl fragment with the introduced mutations can be cloned directly (ie without amplification in Escherichia coli, as necessary for Rando mutagenesis) into a Bacillus vector and into a suitable one Bacillus expression strain can be transformed.
  • TGGE Vertical temperature gradient gel electrophoresis
  • This method is characterized by a temperature gradient which is applied to the gel perpendicular to the separation direction of the sample by means of a thermostatic plate.
  • the 0.8 mm thick 10% polyacrylamide gel (20 cm x 20 cm) was polymerized on gel-yellow film and placed on the thermostatic plate made of Teflon-coated aluminum.
  • the plate was cooled with the help of coolable water thermostats (Julabo) on one side and heated on the other side, so that a linear temperature gradient could develop over the entire width.
  • the sample was placed in a 13 cm long, 0.3 cm wide application bag, which was perpendicular to the course of the temperature gradient.
  • the temperature gradient was applied (corner temperature selection depending on the application, but not higher than 80 ° C.
  • the corner temperatures for those TGGEs which show the stabilization by the A194E exchange were 50 ° C. and 70 ° C.
  • the separation with an applied temperature gradient was carried out at 350 V and a limited current of 110 mA for 30 minutes.
  • Acetic acid, pH 5.0 was used and 0.1 M CaCl2 in the gel and electrode buffer were also used.
  • the subtilisin Carlsberg variant according to EP 214 435 with an S188P mutation of the enzyme and with an S188P and A194E mutation of the enzyme were compared with one another by the method mentioned.
  • the two mutants can be produced by directed or by random in vitro mutagenesis.
  • the structural genes were cloned into expression vectors by generally known methods and expressed in a protease-free, generally accessible strain. Cultures of the cloned protease-free strain were used and, after a fermentation period of 24 hours, 20 ml of culture supernatant were obtained and concentrated.

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Abstract

L'invention concerne une sérine-protéase de type subtilisine, obtenue par mutation du gène de structure d'une souche sauvage et expression du gène de structure muté, dans une souche de production, et a pour but d'accroître la stabilité à la température de l'enzyme. Ce but est atteint, conformément à l'invention, grâce au fait qu'on insère dans la protéase, en position 194 selon le comptage BPN', un reste d'acide glutamique, et éventuellement, de la proline, en position 188.
PCT/EP1993/002492 1992-09-23 1993-09-15 Serine-proteases de type subtilisine mutees WO1994006905A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP6507790A JPH08501447A (ja) 1992-09-23 1993-09-15 突然変異ズブチリシン様セリンプロテアーゼ
EP93920727A EP0662126A1 (fr) 1992-09-23 1993-09-15 Serine-proteases de type subtilisine mutees

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DEP4231726.6 1992-09-23
DE19924231726 DE4231726A1 (de) 1992-09-23 1992-09-23 Mutierte subtilisinartige Serinproteasen

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WO1994006905A1 true WO1994006905A1 (fr) 1994-03-31

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EP (1) EP0662126A1 (fr)
JP (1) JPH08501447A (fr)
DE (1) DE4231726A1 (fr)
WO (1) WO1994006905A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996034935A2 (fr) * 1995-05-05 1996-11-07 Unilever N.V. Variantes de subtilisine
WO1999049056A1 (fr) * 1998-03-26 1999-09-30 The Procter & Gamble Company Variants de serine-proteases ayant des substitutions d'acides amines
US6908757B1 (en) 1998-03-26 2005-06-21 The Procter & Gamble Company Serine protease variants having amino acid deletions and substitutions

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5647976B2 (ja) * 2008-06-06 2015-01-07 ダニスコ・ユーエス・インク 変異体微生物プロテアーゼを含む組成物及び方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0214435A2 (fr) * 1985-08-03 1987-03-18 Henkel Kommanditgesellschaft auf Aktien Protéase alkaline, procédé pour la préparation de vecteurs hybrides et de micro-organismes transformés génétiquement
WO1989006279A1 (fr) * 1988-01-07 1989-07-13 Novo-Nordisk A/S Genes de subtilisine mutes
WO1989009819A1 (fr) * 1988-04-12 1989-10-19 Genex Corporation Mutations combinantes de stabilisation de subtilisine
EP0516200A1 (fr) * 1991-05-01 1992-12-02 Unilever N.V. Compositions de détergents contenant des enzymes stabilisés
WO1992021760A1 (fr) * 1991-05-29 1992-12-10 Cognis, Inc. Enzymes proteolytiques mutantes tirees de bacillus

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0214435A2 (fr) * 1985-08-03 1987-03-18 Henkel Kommanditgesellschaft auf Aktien Protéase alkaline, procédé pour la préparation de vecteurs hybrides et de micro-organismes transformés génétiquement
WO1989006279A1 (fr) * 1988-01-07 1989-07-13 Novo-Nordisk A/S Genes de subtilisine mutes
WO1989009819A1 (fr) * 1988-04-12 1989-10-19 Genex Corporation Mutations combinantes de stabilisation de subtilisine
EP0516200A1 (fr) * 1991-05-01 1992-12-02 Unilever N.V. Compositions de détergents contenant des enzymes stabilisés
WO1992021760A1 (fr) * 1991-05-29 1992-12-10 Cognis, Inc. Enzymes proteolytiques mutantes tirees de bacillus

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996034935A2 (fr) * 1995-05-05 1996-11-07 Unilever N.V. Variantes de subtilisine
WO1996034935A3 (fr) * 1995-05-05 1997-01-16 Unilever Nv Variantes de subtilisine
WO1999049056A1 (fr) * 1998-03-26 1999-09-30 The Procter & Gamble Company Variants de serine-proteases ayant des substitutions d'acides amines
US6569663B1 (en) 1998-03-26 2003-05-27 The Procter & Gamble Company Serine protease variants having amino acid substitutions
US6908757B1 (en) 1998-03-26 2005-06-21 The Procter & Gamble Company Serine protease variants having amino acid deletions and substitutions

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Publication number Publication date
DE4231726A1 (de) 1994-03-24
JPH08501447A (ja) 1996-02-20
EP0662126A1 (fr) 1995-07-12

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