WO1997013847A1 - Systeme d'expression, vecteur et cellule transformee par ce vecteur - Google Patents
Systeme d'expression, vecteur et cellule transformee par ce vecteur Download PDFInfo
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- WO1997013847A1 WO1997013847A1 PCT/BE1996/000109 BE9600109W WO9713847A1 WO 1997013847 A1 WO1997013847 A1 WO 1997013847A1 BE 9600109 W BE9600109 W BE 9600109W WO 9713847 A1 WO9713847 A1 WO 9713847A1
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- nucleotide sequence
- codes
- expression system
- lipase
- strain
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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
- C12N15/78—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Pseudomonas
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
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- 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
- C11D3/38627—Preparations containing enzymes, e.g. protease or amylase containing lipase
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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)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
Definitions
- the invention relates to an expression system which can be used for the production of an enzyme and more particularly capable of carrying out the extracellular production of a lipase.
- the invention also relates to a vector containing this expression system and a cell transformed with this vector, as well as to methods for preparing the expression system, vectors and transformed cells, and to the use thereof for prepare the enzyme.
- the invention also relates to a method for producing the enzyme and the use of this enzyme, in particular in detergent compositions.
- hydrolases such as proteases, lipases, amylases and / or cellulases
- fatty deposits contain triglycerides supplied, for example, by sebum, various food products (oil, sauce, butter, fat), cosmetic products.
- Lipases hydrolyze triglycerides to form products more soluble in water, mono- and di-glycerides, glyeol and free fatty acids.
- Lipases which can be used in detergent compositions are known, such as in particular lipases naturally produced by strains of Pseudomonas, for example the lipase produced by a strain of Pseudomonas stutzeri (British patent application 1372034), the lipase produced by a strain of Pseudomonas mendocina (European patent application 0 571 982), the lipase produced by a strain of Pseudomonas alcaligenes (US patent 5,063,160), and the lipase produced by a strain of Pseudomonas pseudoalcaligenes (European patent application 0 218 272).
- the lipases produced naturally by these strains of Pseudomonas are not efficiently secreted and industrially profitable,
- patent application WO 91/00908 describes a process for the production of a Pseudomonas cepacia lipase by a host strain.
- This Host strain contains a DNA molecule which includes the nucleotide sequence which codes for a modulator and the nucleotide sequence which codes for the lipase of Pseudomonas cepacia. This process seems to be limited to the lipase of Pseudomonas cepacia. It has also been proposed, in patent application WO 94/02617, to transform a strain of Pseudomonas pseudoalcaligenes to produce a lipase.
- This transformation involves the cloning of a DNA sequence which codes for a lipase of Pseudomonas pseudoalcaligenes and of a DNA sequence which codes for the modulator of this same lipase. This transformation is therefore limited to the lipase of Pseudomonas pseudoalcaligenes.
- the main object of the present invention is to provide an expression system making it possible to obtain an enzyme, and in particular a Pseudomonas lipase, in large quantities in the culture medium where a transformed strain containing this expression system is cultivated.
- the present invention also aims to provide a vector, and in particular a plasmid, making it possible to obtain an enzyme, and in particular a lipase from Pseudomonas, having characteristics and properties as close as possible to those of the native (wild) enzyme. ).
- the present invention also aims to provide a transformed strain of bacteria, and more particularly a strain of Pseudomonas wisconsinensis, strain transformed by the vector described above, which expresses and secretes in its culture medium the enzyme, and in particular a lipase, with a high level of productivity.
- the secretion of the enzyme is substantially extracellular.
- the present invention also aims to provide a method for producing an enzyme, and in particular a lipase, by a transformed strain comprising the expression system described above, in particular a strain of bacteria, and in particular of Pseudomonas, which produces a large amount of enzyme extracellularly. 7/13847 PC17BE96 / 00109
- the present invention also aims to provide an enzyme, and in particular a lipase, produced by a transformed strain comprising the expression system described above.
- the strain can be a strain of Pseudomonas and, more particularly, of Pseudomonas wisconsinensis, such as in particular the strain of Pseudomonas wisconsinensis T 92.677 / 1.
- the present invention also aims to provide a DNA molecule comprising the nucleotide sequence which codes for a modulator and in particular for the lipase modulator of Pseudomonas wisconsinensis.
- the present invention also aims to provide a DNA molecule comprising the nucleotide sequence which codes for the GPW protein.
- the GPW protein is part of the lipase operon of Pseudomonas wisconsinensis.
- the present invention also aims to provide a process for the preparation of the expression systems, vectors, transformed strains and DNA molecules described above.
- the invention relates to an expression system which can be used for the production of an enzyme, characterized in that it comprises at least:
- nucleotide sequence which codes for a secretion signal - the nucleotide sequence which codes for a mature enzyme
- promoter is meant the transcription promoter (s).
- the promoter which consists of a DNA sequence showing strong transcription activity, such as sequences known as “enhancers” or “upstream activating sequences”.
- the promoter contains transcription control sequences which influence the expression of the coding DNA.
- the promoter nucleotide sequence of the invention is from a bacterium. Generally, it comes from a strain of Bacillus or a strain of Pseudomonas. Preferably, it comes from a Pseudomonas strain, such as in particular the Pseudomonas strains belonging to the RNA-I group (BERGE Y'S MANUAL OF SYSTEMATIC BACTERIOLOGY, 1984, Vol. 1, pages 159-162). Particularly preferably, it comes from / 13847 PC17BE96 / 00109
- the nucleotide sequence of the promoter of the invention comes from a protein, and in particular from an enzyme. Generally, it comes from a hydrolase. Preferably, it comes from a lipase. In a particularly preferred way, it comes from a Pseudomonas lipase. Good results have been obtained when it comes from a lipase of a strain of Pseudomonas wisconsinensis. Excellent results have been obtained when it comes from a lipase of the Pseudomonas wisconsinensis T 92.677 / 1 strain (LMG P-15151).
- nucleotide sequence which codes for a secretion signal of a protein is meant a DNA sequence corresponding to an amino acid sequence which is naturally operably linked to the amino terminus of the mature sequence of the protein.
- the part of the structural gene that codes for the signal peptide is called the nucleotide sequence that codes for the secretion signal.
- the nucleotide sequence which codes for the secretion signal can come from any enzyme which is produced extracellularly by a microorganism. Any nucleotide sequence which codes for a functional secretion signal in the selected transformed strain may be suitable.
- the expression system of the invention comprises a nucleotide sequence which codes for a secretion signal which comes from a bacterium.
- a bacterium Generally, it comes from a strain of Bacillus or a strain of Pseudomonas.
- a Pseudomonas strain such as in particular the Pseudomonas strains belonging to the RNA-I group (BERGEY'S MANUAL OF SYSTEMATIC BACTERIOLOGY, 1984, Vol. 1, pages 159-162).
- it comes from a strain of Pseudomonas wisconsinensis. Excellent results have been obtained when it comes from the strain of Pseudomonas wisconsinensis T 92.677 / 1 (LMG P-15151).
- the expression system of the invention comprises a nucleotide sequence which codes for a secretion signal from a protein, and in particular from an enzyme.
- it comprises a nucleotide sequence which codes for a secretory signal of a hydrolase, and preferably of a 7/13847 PC17BE96 / 00109
- lipase in a particularly preferred manner, it comprises a nucleotide sequence which codes for a secretion signal from a Pseudomonas lipase. Good results have been obtained with a Pseudomonas wisconsinensis lipase secretion signal sequence. Excellent results have been obtained with the nucleotide sequence which codes for the lipase secretion signal of Pseudomonas wisconsinensis T 92.677 / 1, ie the nucleotide sequence (SEQ LD NO: 4) which signal for secretion of Pseudomonas wisconsinensis T 92.677 / 1 lipase or a modified sequence derived therefrom.
- the nucleotide sequence which codes for a mature enzyme is meant the part of the sequence which is translated into an active enzyme, ie the coding sequence of the enzyme which corresponds to the structural gene of the enzyme without the sequence of the secretory signal.
- the part of the structural gene that codes for the enzyme is called the nucleotide sequence that codes for the mature enzyme.
- the expression system of the invention comprises a nucleotide sequence which codes for an enzyme which comes from a bacterium. Generally, it comes from a strain of Bacillus or a strain of Pseudomonas.
- a Pseudomonas strain such as in particular the Pseudomonas strains belonging to the RNA-I group (BERGEY'S MANUAL OF SYSTEMATIC BACTERIOLOGY, 1984, Vol. 1, pages 159-162).
- a strain of Pseudomonas wisconsinensis Excellent results have been obtained when it comes from the strain of Pseudomonas wisconsinensis T 92.677 / 1 (LMG P-15151).
- the enzyme is a hydrolase.
- the enzyme is a lipase, and preferably a lipase from Pseudomonas.
- the enzyme is a mature lipase from Pseudomonas wisconsinensis.
- Excellent results have been obtained with the nucleotide sequence which codes for the mature lipase of Pseudomonas wisconsinensis T 92.677 / 1, ie the nucleotide sequence (SEQ LD NO: 7) which codes for the mature lipase of Pseudomonas wisconsinensis T 92.677 / 1 or a modified sequence derived therefrom.
- lipase is meant an enzyme which catalyzes the hydrolysis reaction of triglycerides into products more soluble in water, mono- and di-glycerides, glyereol and free fatty acids. This enzyme is classified in the international system under the number EC 3.1.1.3., A lipase is a glycerol ester hydrolase. This definition includes natural enzymes and modified enzymes, such as enzymes whose nucleotide or amino acid sequence has been modified by genetic engineering techniques or by mutagenesis techniques. In a preferred variant of the invention, the nucleotide sequence which codes for the secretion signal comes from the same microorganism as the nucleotide sequence which codes for the mature enzyme.
- they come from a bacterium, and generally from a strain of Bacillus or Pseudomonas.
- they come from a strain of Pseudomonas wisconsinensis. Excellent results have been obtained when they come from a strain of Pseudomonas wisconsinensis T 92.677 / 1.
- the terminator nucleotide sequence is a DNA sequence that acts to complete transcription.
- the terminator nucleotide sequence of the invention is from a bacterium. Generally, it comes from a strain of Bacillus or a strain of Pseudomonas. Preferably, it comes from a Pseudomonas strain, such as in particular the Pseudomonas strains belonging to the RNA-I group (BERGEY'S MANUAL OF SYSTEMATIC BACTERIOLOGY, 1984, Vol. 1, pages 159-162). In a particularly preferred manner, it comes from a strain of Pseudomonas wisconsinensis. Excellent results have been obtained when it comes from the strain of Pseudomonas wisconsinensis T 92.677 / 1 (LMG P-15151).
- the nucleotide sequence of the terminator of the invention comes from a protein, and in particular from an enzyme. Generally, it comes from a hydrolase. Preferably, it comes from a lipase. In a particularly preferred way, it comes from a Pseudomonas lipase. Good results have been obtained when it comes from a lipase of a strain of Pseudomonas wisconsinensis. Excellent results have been obtained when it comes from a lipase of the Pseudomonas wisconsinensis T 92.677 / 1 strain (LMG P-15151).
- the expression system comprises a nucleotide sequence which codes for a modulator.
- module is meant a protein which participates in and helps the expression of an enzyme. In this context, it helps the formation of an active enzyme.
- the expression system comprises at least:
- nucleotide sequence which codes for a secretion signal - the nucleotide sequence which codes for a mature enzyme
- the nucleotide sequence that codes for the modulator comes from a bacterium. Generally, it comes from a strain of Bacillus or a strain of Pseudomonas. Preferably, it comes from a strain of
- Pseudomonas such as in particular the Pseudomonas strains belonging to the RNA-I group (BERGEY'S MANUAL OF SYSTEMATIC BACTERIOLOGY, 1984, Vol. 1, pages 159-162).
- Pseudomonas wisconsinensis In a particularly preferred manner, it comes from a strain of Pseudomonas wisconsinensis. Excellent results have been obtained when it comes from the strain of Pseudomonas wisconsinensis T 92.677 / 1 (LMG P-15151).
- the nucleotide sequence that codes for the modulator comes from the operon of an enzyme. Generally, it comes from the operon of a hydrolase. Preferably, it comes from the operon of a lipase. In a particularly preferred way, it comes from the operon of a lipase of Pseudomonas, and in particular of Pseudomonas wisconsinensis. Good results have been obtained when it comes from the operon of a lipase of the Pseudomonas wisconsinensis T 92.677 / 1 strain (LMG P-15151).
- nucleotide sequence (SEQ LD NO: 1) which codes for the modulator from the lipase operon of Pseudomonas wisconsinensis T 92.677 / 1 or a modified sequence derived therefrom.
- amino acid sequence and the nucleotide sequence (SEQ LD NO: 1) coding for the modulator, as well as its translation into amino acids (SEQ LD NO: 2), is given in FIG. 1 (FIGS. 1a and 1b) .
- “operon” is meant a functional unit of adjacent structural genes dependent on a single promoter.
- the invention also relates to an expression system which comprises at least: - the nucleotide sequence of a promoter, - the nucleotide sequence (SEQ LD NO: 4) which codes for the secretion signal of the lipase of Pseudomonas wisconsinensis T 92.677 / 1, the nucleotide sequence (SEQ LD NO: 7) which codes for the mature lipase of Pseudomonas wisconsinensis T 92.677 / 1,
- SEQ LD NO: 1 the nucleotide sequence which codes for the Pseudomonas wisconsinensis T 92.677 / 1 lipase modulator, and - the nucleotide sequence of a terminator.
- the invention relates to an expression system which comprises at least:
- the sequence (SEQ LD NO: 10) includes:
- SEQ LD NO: 4 the nucleotide sequence which codes for the lipase secretion signal from Pseudomonas wisconsinensis T 92 677/1,
- SEQ LD NO: 7 the nucleotide sequence which codes for the mature lipase of Pseudomonas wisconsinensis T 92.677 / 1,
- SEQ LD NO: 1 the nucleotide sequence which codes for the lipase modulator of Pseudomonas wisconsinensis T 92.677 / 1.
- the nucleotide sequence (SEQ LD NO: 10) is given in Figure 2 ( Figures 2a and 2b).
- the expression system also comprises the nucleotide sequence which codes for a GPW protein.
- the variant of this expression system comprises at least the nucleotide sequence (SEQ LD NO: 12) which codes for the GPW protein originating from the lipase operon of Pseudomonas wisconsinensis T 92.677 / 1 or a modified sequence derived from that -this.
- the GPW protein seems to participate in and help the expression of an enzyme, and, moreover, seems to protect the enzyme from active oxygen.
- the expression system comprises at least: - the nucleotide sequence of a promoter
- nucleotide sequence which codes for a GPW protein - the nucleotide sequence which codes for a secretion signal
- the nucleotide sequence that codes for the GPW protein comes from a bacterium. Generally, it comes from a strain of Bacillus or a strain of Pseudomonas. Preferably, it comes from a Pseudomonas strain, such as in particular the Pseudomonas strains belonging to the RNA-I group (BERGEYS MANUAL OF SYSTEMATIC BACTERIOLOGY, 1984, Vol. 1, pages 159-162). In a particularly preferred manner, it comes from a strain of Pseudomonas wisconsinensis. Excellent results have been obtained when it comes from the strain of Pseudomonas wisconsinensis T 92.677 / 1 (LMG P-15151).
- the nucleotide sequence that codes for the GPW protein comes from the operon of an enzyme. Generally, it comes from the operon of a hydrolase. Preferably, it comes from the operon of a lipase. In a particularly preferred way, it comes from the operon of a lipase of Pseudomonas, and in particular of Pseudomonas wisconsinensis. Good results have been obtained when it comes from the operon of a lipase of the Pseudomonas wisconsinensis T 92.677 / 1 strain (LMG P-15151). Excellent results have been obtained with the nucleotide sequence (SEQ LD NO: 12) which codes for the GPW protein of Pseudomonas wisconsinensis T 92.677 / 1 or a modified sequence derived therefrom.
- the expression system comprises at least:
- nucleotide sequence which codes for a GPW protein - the nucleotide sequence which codes for a secretion signal
- the nucleotide sequence of the promoter is positioned upstream of the nucleotide sequence which codes for the secretion signal.
- the nucleotide sequence that code for the secretion signal is positioned upstream of the nucleotide sequence which codes for the mature enzyme.
- the nucleotide sequence which codes for the mature enzyme is positioned upstream of the nucleotide sequence which codes for the modulator.
- the nucleotide sequence that codes for the modulator is positioned upstream of the terminator nucleotide sequence.
- the nucleotide sequence which codes for the GPW protein is positioned between the nucleotide sequence of the promoter and the nucleotide sequence which codes for the signal secretion. These positions are chosen so that, under appropriate conditions, they allow the expression of the enzyme.
- the sequences included in the expression system are operably linked.
- the promoter nucleotide sequence is operably linked to the nucleotide sequence which codes for the secretion signal
- the nucleotide sequence which codes for the secretion signal is operably linked to the nucleotide sequence which codes for the mature enzyme
- the nucleotide sequence which codes for the mature enzyme is operably linked to the nucleotide sequence which codes for the mature enzyme modulator
- the nucleotide sequence that codes for the modulator is operably linked to the terminator nucleotide sequence.
- the promoter nucleotide sequence is operably linked to the nucleotide sequence which codes for the GPW protein and the nucleotide sequence which codes for the GPW protein is operably linked to the nucleotide sequence which codes for the secretion signal.
- the present invention also relates to a modulator comprising the amino acid sequence of 1 to 352 amino acids (SEQ LD NO: 3) or a modified sequence derived therefrom.
- the modulator of the invention has a molecular weight in the range of about 39 to 40 kDa.
- the modulator has an estimated isoelectric point between about 4.5 and 4.6.
- the present invention also relates to a DNA molecule comprising the nucleotide sequence (SEQ LD NO: 1) which codes for the pseudomonas wisconsinensis T 92.677 / 1 lipase modulator (LMG P-15151) or a modified sequence derived from that -this.
- modified sequence derived from a DNA molecule is meant any DNA molecule obtained by modification of one or more nucleotides of the wild or initial gene.
- the modified sequence derived from the DNA molecule comprises at least 70% homology with the nucleotide sequence of the wild or initial gene, that is to say at least 70% of identical nucleotides and having the same position in the sequence.
- the modified sequence derived from the DNA molecule comprises at least 80% homology with the nucleotide sequence of the wild or initial gene.
- the modified sequence derived from the DNA molecule comprises at least 90% of homology with the nucleotide sequence of the wild or initial gene.
- the present invention also relates to a GPW protein comprising the amino acid sequence of 1 to 199 amino acids (SEQ LD NO.14) or a modified sequence derived therefrom.
- the amino acid sequence (SEQ LD NO.14) and the nucleotide sequence (SEQ LD NO: 12) coding for the protein GPW, as well as its translation into amino acids (SEQ LD NO: 13), is given in Figure 3.
- the GPW protein of the invention has a molecular weight between about 22 and 23 kDa. It has an estimated isoelectric point between about 9.7 and 9.8.
- the GPW protein is able to protect cells from attack by active oxygen and to repair cell membranes damaged by oxygen radicals.
- the nucleotide sequence which codes for the GPW protein has homology with that which codes for a glutathione peroxidase.
- GPW protein can be isolated from a Pseudomonas strain
- GPW protein comes from a Pseudomonas strain belonging to the RNA-I group.
- the GPW protein comes from a strain of Pseudomonas wisconsinensis. Good results have been obtained with the GPW protein from Pseudomonas wisconsinensis T 92.677 / 1 strain and having a nucleotide sequence (SEQ LD NO: 12) or a modified sequence derived therefrom.
- the present invention also relates to a DNA molecule comprising the nucleotide sequence (SEQ LD NO.12) which codes for the GPW protein of Pseudomonas wisconsinensis T 92.677 / 1 (LMG P-15151) or a modified sequence derived therefrom. .
- the invention also relates to a vector containing the expression system, as defined above, and capable of allowing expression of the enzyme.
- the vector according to the invention is a plasmid.
- the plasmid according to the invention is an expression vector or an integration vector
- the plasmid according to the invention is an expression vector.
- Good results have been obtained with the expression vectors pRG930 :: WI12, pRG930 :: WI13, and pRG930 :: WU4.
- the invention also relates to a transformed cell (host cell) containing the vector defined above.
- the transformed cell is chosen so that the nucleotide sequence which codes for the modulator is recognized by this transformed cell, that is to say that it is compatible and functional for this transformed cell.
- the transformed cell is a strain of bacteria.
- the transformed cell is a bacterium chosen from the strains of cherscherichia, Pseudomonas or Bacillus.
- the transformed cell is a strain of Pseudomonas.
- the transformed cell is a strain of Pseudomonas chosen from the strains of Pseudomonas belonging to the RNA-I group. Good results have been obtained with a strain of Pseudomonas wisconsinensis. Excellent results have been obtained with the strain of Pseudomonas wisconsinensis T 92.677 / 1 (LMG P-15151).
- the strain of Pseudomonas wisconsinensis T 92.677 / 1 was deposited in the collection named BELGIAN COORDLNATED COLLECTIONS OF MICROORGANISMS (LMG culture collection, University of Ghent, Laboratory of Microbiology - KL Ledeganckstraat 35, B-9000 Ghent, Belgium) in accordance with the Budapest Treaty under number LMG P-15151 on October 12, 1994.
- the transformed strain is a strain d ⁇ scherichia coli and in particular E. coli HB 101.
- the invention also relates to a method for preparing the expression system. This process includes:
- nucleotide sequence of a promoter of the nucleotide sequence which codes for a secretion signal, of the nucleotide sequence which codes for a mature enzyme, and of the nucleotide sequence of a terminator to from the DNA of one or more microorganisms, and
- the method of preparing the expression system also comprises the isolation of the nucleotide sequence which codes for the modulator from the DNA of a microorganism and the linking of the sequences in an operational manner, so that the positioning of the sequences allow the expression of the enzyme under the action of the modulator.
- the method of preparing the expression system also comprises the isolation of the nucleotide sequence which codes for the GPW protein from the DNA of a microorganism and the binding of the sequences in an operational manner, so that the positioning of the sequences allows the expression of the enzyme.
- the invention also relates to a method for preparing a vector comprising the expression system.
- This method comprises the introduction of the expression system into a vector, this introduction being made in a site chosen so that the positioning of the sequences included in the expression system allow the expression of the enzyme.
- the introduction is made in a chosen site so that the positioning of the sequences allows the expression of the enzyme under the action of the modulator.
- the invention also relates to a process for the preparation of a transformed cell.
- This method involves transforming a cell with a vector containing an expression system under conditions allowing stable introduction of the expression system into the cell.
- the invention also relates to a process for the production of an enzyme. This process involves culturing the transformed cell under appropriate culture conditions and in a culture medium suitable for the production of the enzyme and the recovery of the enzyme obtained
- This process comprises culturing the transformed cell capable of producing the enzyme in an appropriate nutritive medium containing carbon and nitrogen sources and mineral salts under aerobic conditions and harvesting the enzyme thus obtained.
- culture can be solid or liquid
- the culture medium is liquid
- the culture conditions which make it possible to obtain the enzyme of the invention, such as components of the nutritive medium, culture parameters, temperature, pH, aeration, agitation, are well known to those skilled in the art.
- the enzyme harvesting techniques are well known to those skilled in the art and are chosen according to the intended uses of the lipase. Usually, centrifugation, filtration, ultrafiltration, evaporation, microfiltration, crystallization are used. or a combination of one or the other of these techniques such as centrifugation followed by ultrafiltration Examples of such techniques are notably described by R Scriban, Biotechnologie, (Technique et Documentation Lavoisier), 1982, p 267- 276
- the enzyme can then be purified, if necessary and according to the uses envisaged.
- the techniques for purifying enzymes are well known to those skilled in the art, such as precipitation using a salt, such as sulphate. ammonium, or solvent, such as acetone or an alcohol Examples of such techniques are described in particular by R. Scriban, Biotechnologie, (Technique et Documentation Lavoisier), 1982, p 267-276.
- the enzyme can also be dried by atomization or freeze-drying. Examples of such techniques are notably described by R Scriban, Biotechnologie, (Technique et Documentation Lavoisier), 1982, p 267-276
- the enzyme according to the invention has multiple outlets in various industries, such as, for example, the food industries, the pharmaceutical industries or the chemical industries.
- the enzyme such as a hydrolase and in particular a lipase, can in particular be used in detergency
- An example of such use is described in particular in British patent application 1372034 and in European patent application 0 218 272 In this context, it is part of detergency compositions
- FIG. 1 (FIGS. 1 a and 1 b) represents the amino acid sequence
- LD NO.3 of the Pseudomonas wisconsinensis T 92 677/1 lipase modulator and the nucleotide sequence (SEQ LD NO: 1) coding for the lipase modulator of Pseudomonas wisconsinensis, as well as its translation into amino acids (SEQ LD NO: 2).
- Figure 2 shows the nucleotide sequence (SEQ LD NO: 10) including the nucleotide sequence (SEQ LD NO: 4) which codes for the lipase secretion signal of Pseudomonas wisconsinensis T 92.677 / 1 , the nucleotide sequence (SEQ LD NO: 7) which codes for the mature lipase of Pseudomonas wisconsinensis T 92.677 / 1, the intercistronic sequence (SEQ LD NO: 1 1) and the nucleotide sequence (SEQ LD NO: 1) code for the Pseudomonas wisconsinensis T lipase modulator T 92.677 / 1.
- SEQ LD NO: 10 shows the nucleotide sequence (SEQ LD NO: 10) including the nucleotide sequence (SEQ LD NO: 4) which codes for the lipase secretion signal of Pseudomonas wisconsinen
- FIG. 3 represents the amino acid sequence (SEQ LD NO: 14) of the GPW protein and the nucleotide sequence (SEQ LD NO: 12) coding for the GPW protein, as well as its translation into amino acids (SEQ LD NO : 13).
- the present invention is illustrated by the following examples.
- Example 1 Isolation and characterization of the strain of Pseudomonas wisconsinensis T 92.677 / 1
- the strain of Pseudomonas wisconsinensis T 92.677 / 1 was isolated from a soil sample, taken in the United States in the state of Wisconsin.
- 1 g of earth is suspended in 10 ml of demineralized water containing 9 g / l of NaCl. This suspension is diluted 10 times with demineralized water containing 9 g / l of NaCl.
- Medium A contains 10 g / l of tryptone (Difco), 5 g / l of yeast extract, 5 g / l of NaCl, 20 g / l of agar, 2.5 g / l of NaHCO 3 , 7 , 5 g / l of Na 2 CO 3 , 10 g / l of olive oil, 1 g / I of polyvinyl alcohol (25/140) and 0.01 g / l of rhodamine B (Sigma 6626).
- Medium A is prepared as follows.
- An olive oil emulsion is first prepared as follows. 50 ml of distilled water is heated to 80 ° C. 1 g of polyvinyl alcohol is added in small portions to this heated water. Then, 10 g of olive oil are added to the suspension of polyvinyl alcohol. It is then emulsified using an Ultra-turax mixer at 13,500 revolutions per minute (rod 18 GM). The emulsion obtained is sterilized at 121 ° C for 30 minutes.
- An agar is then prepared as follows. 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, 20 g of agar are added in 850 ml of water distilled. The agar suspension obtained is sterilized at 121 ° C for 30 minutes.
- the sterilized olive oil emulsion and the sterilized agar are cooled to 60 ° C, then sterile mixed. Then add the sterilized rhodamine solution to it. Then, 100 ml of sterilized carbonate buffer are added so as to obtain a pH of 9.5.
- the suspension thus obtained is emulsified by means of an Ultra-turax mixer at 13,500 revolutions per minute (rod 18 GM).
- Medium A on which the soil suspension has been spread is incubated for 48 hours at 30 ° C.
- the microorganisms producing a lipase are detected using an ultraviolet light, they are surrounded by a fluorescent halo.
- the microorganisms detected as producing a lipase are cultured on an agar B nutrient medium.
- Medium B contains 10 g / l of tryptone (Difco), 5 g / l of yeast extract, 5 g / l of NaCl, 20 g / l of agar, 2.5 g / l of NaHCO 3 , 7 , 5 g / l Na 2 CO 3 .
- Tryptone, yeast extract, NaCl, agar, which make up medium B are mixed with 900 ml of distilled water, then sterilized at 121 ° C for 30 minutes. The pH is adjusted to 9.5 by the addition of 100 ml of previously sterilized carbonate buffer (containing 25 g / l of NaHCO 3 and 75 g / l of Na2CO 3 ).
- the microorganism has been identified by its biochemical characteristics: Gram negative bacteria, aerobic. No spores are formed.
- the size of the vegetative cells is 0.5-0.7 ⁇ m x 1.5-4.0 ⁇ m.
- the mobility of vegetative cells is positive.
- the lysis test with 3% (w / v) of KOH is positive.
- the catalase test is positive in the presence of 10% (v / v) of hydrogen peroxide.
- the oxidase test is positive in the presence of 1% (w / v) of tetramethyl-1,4-phenylene-diammonium-dichloride.
- the urease test is negative.
- the nitrate reduction test is positive. Comparable tests have in particular been described in European patent application 0 218 272.
- This strain is aerobic, that is to say it develops aerobically. It does not develop enanaerobically, i.e. under an atmosphere of 84% (v / v) N 2 , 8% (v / v) CO 2 , 8% (v / v) H 2 at 37 ° VS .
- the abbreviation% (v / v) represents a percentage expressed in volume by 13847 PC17BE96 / 00109
- the abbreviation% (v / p) represents a percentage expressed by volume by weight.
- the abbreviation% (w / v) represents a percentage expressed by weight per volume.
- the abbreviation% (w / w) represents a percentage expressed in weight by weight.
- This strain is not thermophilic. It shows normal development after incubation on agar medium B at 20 ° C, 30 ° C, 37 ° C and 41 ° C.
- the strain does not produce gas from glucose.
- the strain uses azelate, caprate, citrate, glucose, gluconate, L-arginine, L-histidine, betaine and geraniol.
- the strain does not use adipate, phenylacetate, L-arabinose and maltose. It does not hydrolyze gelatin, starch and esculin.
- the strain belongs to the genus Pseudomonas and to the RNA-I group.
- the biochemical characteristics clearly differentiate the strain of Pseudomonas wisconsinensis, and in particular the strain of Pseudomonas wisconsinensis T 92.677 / 1, of a strain of Pseudomonas mendocina, of a strain of Pseudomonas pseudoalcaligenes, of a strain of Pseudomonas alcaligenes and of a strain of Pseudomonas stutzeri. This is clear from a reading of Table 1 gathering the main biochemical characteristics of these 5 strains.
- the isolated bacterium therefore belongs to the genus Pseudomonas, no known species could be determined
- Example 2 Production of the lipase by the strain of Pseudomonas wisconsinensis T 92 677/1
- the strain of Pseudomonas wisconsinensis T 92.677 / 1 is cultured on a petri dish containing the agar medium B at 30 ° C. for 24 hours. Then, from this culture, a culture is carried out in 25 ml of a liquid medium C.
- Medium C contains 10 g / l of tryptone (Difco), 5 g / l of yeast extract, 10 g / l of NaCl, the pH of the medium is adjusted to 7.0 with NaOH 0, IN, the medium is sterilized at 121 ° C for 30 minutes.
- the culture is carried out at 30 ° C. with orbital stirring at the rate of 200 revolutions per minute with an amplitude of approximately 2.54 cm.
- this culture is introduced into a 20 liter fermenter containing 13 liters of sterilized liquid medium D
- Medium D contains K 2 HPO 4 2.5 g / l, KH 2 PO 4 2.5 g / l, MgSO 4 7H 2 0 1 g / l, (NH4) 2 S04 2 g / l, (NH 2 ) 2 CO 2 g / l, CaCl 2 1 g / l, soy flour 20 g / l, yeast extract 2 g / l, glucose 20 g / l, Mazu ⁇ l antifoam (Mazes Chemicals) 5 g / l.
- the pH is adjusted to 7.4 (with normal phosphoric acid and normal caustic soda) before and after sterilization in the fermenter (30 minutes at 121 ° C).
- the glucose is sterilized separately at pH 4.0 (pH adjusted with normal phosphoric acid) for 30 minutes at 121 ° C.
- the medium is sterilized in the fermenter for 30 minutes at 121 ° C.
- the enzyme activity of the culture thus obtained is measured by the following technique.
- triolein The hydrolysis of triolein is quantified by the neutralization of the fatty acids released under the action of lipase. This measurement is carried out using an automatic titration apparatus, which maintains the pH constant at a set value by the addition of 0.01 N NaOH.
- a lipase unit is defined as the quantity of enzyme which catalyzes the release of one micromole of fatty acid per minute under the standard conditions of the test described below. 10 g of Triolein (Roth 5423.1) and 10 g of gum arabic are mixed
- a dilution buffer is prepared containing 2.34 g / l of NaCl, 2.94 g / l of CaCl 2 -2H 2 0 and 0.61 g / l of Tris (2-amino-2-hydroxymethyl-l, 3 -propanediol).
- An automatic titration apparatus is used, equipped with a burette containing 0.0 IN NaOH, a temperature probe and a pH probe and equipped with a thermostatically controlled reactor.
- the centrifugation supernatant is heated at 40 ° C for 5 minutes. A phase separation is observed. The upper phase is eliminated. 35% (v / v) of acetone is added to the lower phase at 4 ° C. The suspension is incubated for 15 minutes at 4 ° C. with moderate stirring. Then, the suspension is centrifuged for 15 minutes at 9000 revolutions per minute (BECKMAN J21, rotor JA10) at a temperature of 4 ° C. The centrifugation supernatant is kept.
- Acetone is added to the centrifugation supernatant at 4 ° C until an acetone concentration of 65% (v / v) is obtained.
- the mixture is incubated for 16 hours at 4 ° C.
- the mixture is centrifuged for 15 minutes at 9000 revolutions per minute (Beckman J21, rotor JA10) at a temperature of 4 ° C.
- the centrifugation precipitate is kept, which is suspended in 150 ml of a buffer (pH 7) containing 5 mM Brij 58 (ICI), 25 mM CaCl 2 and 20 mM Tris.
- the suspension, containing the precipitate is centrifuged for 15 minutes at 9000 rpm (Beckman J21, rotor JA10) at a temperature of 4 ° C.
- Example 3 To purify the concentrated lipase solution, as obtained in Example 3, the purification technique using hydrophobic interaction chromatography is used, followed by the purification technique using molecular sieving chromatography.
- the purification technique using hydrophobic interaction chromatography is used, followed by the purification technique using molecular sieving chromatography.
- elution buffer a 20 mM phosphate buffer at pH 7.2 containing 30% (v / v) of isopropanol.
- the flow rate is fixed at 1.5 ml per minute.
- the lipase is recovered with the fraction eluted with the phosphate buffer containing isopropanol.
- the enzymatic activity of the fraction is measured according to the method described in Example 2.
- the eluted fraction, containing the lipase, is diafiltered into an Amicon cell, equipped with a YM10 membrane, with 10 volumes of a buffer (pH 7) containing 25 mM CaCl 2 and 20 mM Tris. Then, the diafiltered fraction is concentrated to 0.5 ml by ultrafiltration using the same Amicon cell, equipped with a YM10 membrane. Then, the concentrated fraction (0.5 ml) is injected onto a molecular sieve chromatography column (Superdex 75 HR 10/30 Pharmacia column reference 17-1047-01). The separation is initiated by a flow rate of 0.5 ml per minute of a buffer (pH 7) containing 25 mM CaCl 2 and 20 mM Tris.
- Lipase corresponds to the first absorption peak at 280 nm.
- the corresponding fraction which contains the purified lipase is kept.
- Example 5 Determination of the N-terminal sequence of the lipase
- the fraction containing the purified lipase is used, as obtained in Example 4.
- N-terminal sequence (SEQ LD NO: 15) is as follows: Asn Tyr Thr Ly ⁇ Thr Lys Tyr Pro Ile Val Leu Val His Gly Val Thr 1 5 10 15
- the amino acid sequence (SEQ LD NO: 9) of the mature lipase is indirectly determined from the nucleotide sequence (SEQ LD NO.7) of the gene which codes for this lipase, the production of which is described in l Example 15. This is done using the IntelliGenetics Suite Software for Molecular Biology (Release # 5.4) computer program from IntelliGenetics, Inc. USA.
- the mature lipase contains 286 amino acids (SEQ LD NO: 9).
- the amino acid sequence (SEQ LD NO: 6) of the lipase secretion signal is identified by the same technique from the nucleotide sequence (SEQ LD NO: 4), the production of which is described in l Example 15
- the secretion signal contains 22 amino acids (SEQ LD NO: 6) which constitute the signal peptide.
- EXAMPLE 7 Amino Acid Sequence of the Lipase Modulator
- the amino acid sequence of the Pseudomonas wisconsinensis lipase modulator is indirectly determined from the nucleotide sequence (SEQ LD NO: 1) of the gene which codes for this modulator, the production of which is described. in Example 15. This is done using the IntelliGenetics Suite Software for Molecular Biology (Release # 5.4) computer program from IntelliGenetics, Inc. USA.
- FIG. 1 FIG. 1 (FIGS.
- 1a and 1b represents the amino acid sequence (SEQ LD NO: 3) of the lipase modulator of Pseudomonas wisconsinensis T 92.677 / 1 and the nucleotide sequence (SEQ LD NO: 1) coding for the modulator of Pseudomonas wisconsinensis lipase, as well as its translation into amino acids (SEQ LD NO: 2).
- the lipase modulator contains 352 amino acids (SEQ LD NO: 3).
- EXAMPLE 8 Estimation of the Molecular Weight of the Lipase Modulator The molecular weight of the lipase modulator is estimated by calculation from the amino acid sequence (SEQ LD NO.3), as described in Example 7.
- the isoelectric point of the lipase modulator is estimated by calculation from the amino acid sequence (SEQ LD NO: 3), as described in example 7. This is carried out using the computer program. IntelliGenetics Suite
- the amino acid sequence of the GPW protein of Pseudomonas wisconsinensis is indirectly determined from the nucleotide sequence (SEQ LD NO: 12) of the gene which codes for this protein, the production of which is described in Example 15. This is done using the IntelliGenetics Suite Software for Molecular Biology (Release # 5.4) computer program from IntelliGenetics, Inc. USA.
- FIG. 3 represents the amino acid sequence (SEQ LD NO: 14) and the nucleotide sequence (SEQ LD NO: 12) coding for the protein GPW, as well as its translation into amino acids (SEQ LD NO: 13).
- the GPW protein contains 199 amino acids (SEQ LD NO: 14).
- Example 11 Estimation of the molecular weight of the GPW protein The molecular weight of the GPW protein is estimated, by calculation, from the amino acid sequence (SEQ LD NO: 14), as described in Example 10. This is done using the IntelliGenetics Suite Software for Molecular Biology (Release # 5.4) computer program from IntelliGenetics, Inc. USA. The estimated molecular weight of the GPW protein is approximately 22,230 Daltons.
- Example 12 Estimation of the Isoelectric Point of the GPW Protein
- the isoelectric point of the GPW protein is estimated by calculation from the amino acid sequence (SEQ LD NO: 14), as described in Example 10.
- Example 2 From the culture, as obtained in Example 2, a culture of 200 ml of the Pseudomonas wisconsinensis T 92.677 / 1 strain is carried out in liquid LB medium for 16 hours at 37 ° C.
- composition of the liquid LB medium is as follows: TRYPTONE (DLFCO) 10 g / l, yeast extract 5 g / l, NaCl 10 g / l.
- the LB agar medium also contains 20 g / l of agar (DLFCO).
- the culture obtained is centrifuged (SORVALL RC 5C Plus) at 2000 G for 15 minutes.
- the centrifugation pellet thus obtained is taken up in a solution containing 9.5 ml of TE buffer at a pH of 8.0; 500 ⁇ l of a 10% (w / v) SDS (sodium dodecyl sulfate) solution, and 50 ⁇ l of a proteinase K solution (sold by BOEHRINGER Mannheim) at 20 mg / ml (prepared immediately).
- This lysate is extracted with 15 ml of a mixture comprising 24 parts / volume of chloroform and 1 part / volume of isoamyl alcohol (3-methyl-1-butanol) under the conditions and following the procedures described.
- isoamyl alcohol (3-methyl-1-butanol)
- the DNA contained in the viscous suspension is precipitated according to the technique described in SAMBROOK et al., 1989, p 9.18.
- the precipitated DNA is wound around a Pasteur pipette, then is washed three times with 70% (v / v) of ethanol.
- the washed DNA is dried for 5 minutes in air at room temperature.
- the dried DNA is suspended in 2.5 ml of TE buffer at pH 8.0.
- the chromosomal DNA (15 ⁇ g) of Pseudomonas wisconsinensis T 92.677 / 1 is partially cleaved by the restriction enzyme Sau3AI.
- the method is implemented by successive dilution of the restriction enzyme and the restriction conditions are those described in SAMBROOK et al., 1989, pages 5.28-5.32.
- Cleavage is partially inhibited by the addition of i ⁇ l of 0.5 M d ⁇ DTA at pH 8.0 in the presence of ice. Determined on agarose gel (0.8% w / v), as described by
- the ligation thus obtained is used to transfect E. coli HB101 cells (PROMEGA) using the kit sold under the name of GIGAPACK II PACKAGING EXTRACT KIT (STRATAGENE) and following the manufacturer's recommendations for use.
- the transfected cells ⁇ . coli HB 101 are cultured on a Petri dish containing LB agar medium, 25 ⁇ g / ml of streptomycin and 50 ⁇ g / ml of spectinomycin, for approximately 24 hours at 37 ° C. This gives a collection of transfected strains which are called E. coli HB 101 (pRG930 :: WI).
- the initial N-terminal sequence of the lipase from Pseudomonas wisconsinensis T 92.677 / 1, as obtained in Example 5, is taken as the initial reference base.
- sequence of this synthetic oligonucleotide is the following SEQ LD NO: 16
- This synthetic oligonucleotide is labeled at its termination by means of ⁇ 32P ATP with a T4 polynucleotide kinase enzyme using the technique described in the SEQUITHERM CYCLE SEQUENCING KIT (BYOZYME). Screening is carried out on the genomic library by the so-called “colony” technique blot "(AMERSHAM) using as probe the synthetic oligonucleotide as prepared above and following the technique indicated by the manufacturer.
- the colonies of the genomic bank (E. coli HB101 (pRG930 :: WI)) are cultured for 18 hours at 37 ° C. on membranes called "hybond-N +" (AMERSHAM) according to the technique indicated by the manufacturer.
- the membranes (400 crn ⁇ ) are placed in plastic bags containing 45 ml of prehybridization solution.
- the prehybridization solution contains 15 ml of 20X SSC (3 M NaCl and 0.3 M sodium citrate, pH 7.0), 5 ml of Denhardt's solution and 500 ⁇ g of denatured salmon sperm DNA and fragmented (AMERSHAM).
- Denhardt's solution contains 1 g of FICOLL type 400 (PHARMACIA), 1 g of polyvinylpyrrolidone and 1 g of bovine serum albumin.
- the membranes placed in plastic bags are incubated at 68 ° C. in a water bath with shaking (100 revolutions per minute, amplitude of about 2.54 cm) for 4 hours.
- the membranes are then incubated with a hybridization solution at 68 ° C. in a water bath with stirring (100 revolutions per minute, amplitude of approximately 2.54 cm) for 18 hours.
- the hybridization solution is prepared by mixing 5 ml of the prehybridization solution preheated to 68 ° C and the labeled synthetic oligonucleotide, and having been incubated in a water bath for 5 minutes, the final concentration of the synthetic oligonucleotide is 0.3 picomoles.
- the membranes are then recovered.
- the membranes are then washed with a solution containing 100 ml of 2X SSC (3 M NaCl and 0.03 M sodium citrate, pH 7.0) and 0.1% (w / v) of SDS for 5 minutes at ambient temperature.
- 2X SSC 3 M NaCl and 0.03 M sodium citrate, pH 7.0
- 0.1% (w / v) of SDS for 5 minutes at ambient temperature.
- the washed membranes are dried between two absorbent papers.
- the dried membranes are covered with a transparent food-grade plastic sheet. They are then subjected to X-ray autoradiography using the technique described by the manufacturer (AMERSHAM).
- the screening of the genomic library highlights 80 colonies giving a signal. This shows that these colonies contain a DNA fragment carrying the nucleotide sequence which codes for the lipase of Pseudomonas wisconsinensis.
- the Pseudomonas wisconsinensis T 92.677 / 1 strain is used as a positive control and the d ⁇ strains. coli HB 101 and d ⁇ . coli HB 101 (pRG930) are used as a negative control. There is a clear and strong signal for the wild strain of Pseudomonas wisconsinensis T 92.677 / 1 and no signal for the d ⁇ strains. coli HB 101 and d ⁇ . coli HB 101 (pRG930).
- the strain of. coli HB 101 (pRG930) was obtained by the transformation technique described in Molecular Cloning, a laboratory Manual -
- a new hybridization test is carried out, according to the technique described above. This test confirms the results obtained.
- a colony of the genomic bank presenting a strong signal is isolated and cultured on a Petri dish containing the LB agar medium, 25 ⁇ g / ml of streptomycin and 50 ⁇ g / ml of spectinomycin, for approximately 24 hours at 37 ° C. This colony is called E. coli HB101 (pRG930 :: WI12).
- Example 14 Analysis of the plasmid pRG930 :: WI12 present in the strain d ⁇ . coli HBlOl (pRG930:: WI12) 1. Analysis by the so-called Southern Blot technique
- the DNA is isolated from the d ⁇ strain. coli HB101 (pRG930 :: WI12), obtained in Example 13, according to the technique described by SAMBROOK et al., 1989, pages 1.25-1.28, and a restriction analysis is carried out using the enzyme EcoRI restriction.
- the DNA fragments obtained are separated by agarose gel electrophoresis (0.8% w / v) according to the technique described in SAMBROOK et al., 1989, pages 6.01-6.19.
- the DNA is transferred to a membrane called "hybond-N +" (AMERSHAM) and hybridization is carried out according to the technique indicated by the manufacturer as illustrated in example 13.
- AMERSHAM a membrane called "hybond-N +"
- the plasmid pRG930 is used as negative control. A single band is observed, giving a hybridization signal with the synthetic oligonucleotide (SEQ LD NO: 16) on the electrophoresis gel. The fragment carried by this band has a size of approximately 24.5 kbp, it is linked to the vector pRG930.
- the strain of. coli HB101 (pRG930 :: WI12) is cultured in a liquid LB medium containing, in addition, 25 ⁇ g / ml of streptomycin and 50 ⁇ g / ml of spectinomycin, for approximately 18 hours at 37 ° C. From the culture, a new culture is prepared at 30 ° C. which is stopped halfway through the exponential growth phase. This culture is called culture A.
- the strain of. coli DH5ct (pRK2013) is cultured in a liquid LB medium containing, in addition, 100 ⁇ g / ml of kanamycin, for approximately 18 hours at 37 ° C. From the culture, a new culture is prepared at 30 ° C. which is stopped halfway through the exponential growth phase. This culture is called culture B.
- the strain of. coli DH5 ⁇ was obtained by the transformation technique described in SAMBROOK et al., 1989, using the d ⁇ strain. coli DH5 ⁇ (GIBCO) and the plasmid pRK2013 (Proc. Natl. Acad. Sci. USA, 1979, vol. 76, N ° 4, pages 1648-1652).
- the Pseudomonas wisconsinensis T 92.677 / 1 strain is cultured in liquid LB medium for approximately 18 hours at 37 ° C. From the culture, a new culture is prepared at 30 ° C. which is stopped halfway through the exponential growth phase. This culture is called culture C. 1 mi of cultures A, B and C is centrifuged with a centrifuge
- each suspension 50 ⁇ l of each suspension is then mixed.
- the mixture is poured into a petri dish containing LB agar medium, then is incubated for 18 hours at 30 ° C. After incubation, the mixture is recovered by washing the agar medium contained in the Petri dish with 3 ml of liquid LB medium.
- the Pseudomonas wisconsinensis RC12 strain is cultured in 7 A medium for 18 hours at 30 ° C.
- the fragment inserted into the plasmid pRG930 :: WI12 is formed, on the one hand, of 4 fragments which together have a size of approximately 18.5 kbp and, on the other hand, of an attached fragment to the vector pRG930. This fragment has a size of approximately 8.5 kbp.
- the 8.5 kbp fragment attached to the vector pRG930 is then ligated onto itself according to the method described by SAMBROOK et al., 1989, pages 1.68-1.69.
- the plasmid pRG930 :: WI13 is obtained.
- the ligation thus obtained is used to transform cells of E. coli DH5 ⁇ (GLBCO) as described in SAMBROOK et al., 1989, page 1.82-1.84.
- a strain called Pseudomonas wisconsinensis RC13 is obtained, which is cultured using the technique described in Example 2.
- the enzyme activity (lipase) of the culture is measured using the technique described in Example 2.
- the 2.5 kbp fragment gives a hybridization signal with the synthetic oligonucleotide (SEQ LD NO.16), the other 5 fragments give no signal
- the 2.5 kbp fragment is ligated with the vector pRG930 according to the method described by SAMBROOK et al., 1989, pages 1.68-1.69.
- the plasmid pRG930 :: WI 14 is obtained.
- the 2.5 kbp fragment is inserted into the plasmid pBLUESCRIPT by the technique described in SAMBROOK et al., 1989, page 1.85.
- the plasmid pBLUESCRLPT :: WI14 is obtained.
- the plasmid pBLUESCRIPT can be obtained from the company STRATAGENE.
- the 2.5 kbp fragment sequence inserted into the plasmid pBLUE S CRIPT:: WI 14 is obtained by using the SEQUITHERM CYCLE SEQUENCLNG KIT (BIOZYM) kit and by following the technique recommended by the manufacturer
- the nucleotide sequence (SEQ LD NO: 7) coding for the mature lipase of Pseudomonas wisconsinensis T 92.677 / 1 is identified according to the method described above.
- amino acid sequence (SEQ LD NO: 9) of the mature lipase of Pseudomonas wisconsinensis T 92.677 / 1 is obtained by translation (SEQ LD NO: 8) of the nucleotide sequence (SEQ LD NO.7). This translation is carried out according to the method described in Example 6.
- the amino acid sequence (SEQ LD NO: 6) of the Pseudomonas wisconsinensis T 92.677 / 1 lipase secretion signal is obtained by translation (SEQ LD NO: 5) of the nucleotide sequence (SEQ LD NO: 4) .
- the translation is carried out according to the technique described in Example 6.
- the nucleotide sequence (SEQ LD NO: 1) which codes for the pseudomonas wisconsinensis T 92.677 / 1 lipase modulator is identified according to the method described above.
- This nucleotide sequence (SEQ LD NO: 1) comprises 1056 nucleotides.
- the amino acid sequence (SEQ LD NO 3) of the Pseudomonas wisconsinensis T 92.677 / 1 lipase modulator is obtained by translation (SEQ LD NO.2) of the nucleotide sequence (SEQ LD NO 1) The translation is carried out according to the technique described in example 7.
- the nucleotide sequence (SEQ LD NO: 12) which codes for the protein GPW isolated from the lipase operon of Pseudomonas wisconsinensis T is identified according to the method described above. 92 677/1.
- the amino acid sequence (SEQ LD NO: 14) of the GPW protein is obtained by translation (SEQ LD NO: 13) of the nucleotide sequence (SEQ LD NO: 12). The translation is carried out according to the technique described in Example 10.
- GGC CTT ACC CTG TAC TGG CGC TGG CCG GCA GCA GTG CCT GAA GCG CAG 96 Gly Leu Thr Leu Tyr Trp Arg Trp Pro Ala Ala Val Pro Glu Ala Gin 20 25 30
- GGCCACAGTC AGGGCTCGCC GACCTCGCGC GTGGCGGCTT CGTTGCGGCC GGATCTGGTG 360
- CTCTGTCAGT ACGCGGGCAA GCCGCTGGTG GTGGTCAATA CCGCCAGCTT TTGCGGCTTC 120
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Abstract
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AU72080/96A AU7208096A (en) | 1995-10-12 | 1996-10-14 | Expression system, vector and cell transformed thereby |
EP96933282A EP0800576A1 (fr) | 1995-10-12 | 1996-10-14 | Systeme d'expression, vecteur et cellule transformee par ce vecteur |
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BE9500851A BE1009650A5 (fr) | 1995-10-12 | 1995-10-12 | Systeme d'expression, vecteur et cellule transformee par ce vecteur. |
BE9500851 | 1995-10-12 |
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Cited By (1)
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WO1998006836A2 (fr) * | 1996-08-16 | 1998-02-19 | Genencor International, Inc. | Systeme d'expression pour niveaux d'expression modifies |
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WO1992004444A1 (fr) * | 1990-09-12 | 1992-03-19 | Zymogenetics, Inc. | Procedes de production d'hydrolase d'acyloxyacyle |
WO1993020214A1 (fr) * | 1992-03-30 | 1993-10-14 | Genencor International, Inc. | Expression genique heterologue dans le bacillum subtilis: approche par fusion |
WO1994002617A2 (fr) * | 1992-07-23 | 1994-02-03 | Gist-Brocades N.V. | Clonage et expression d'un gene modulateur de lipase a partir de genes pseudoalcalins de pseudomonas |
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1995
- 1995-10-12 BE BE9500851A patent/BE1009650A5/fr not_active IP Right Cessation
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1996
- 1996-10-14 CA CA 2207598 patent/CA2207598A1/fr not_active Abandoned
- 1996-10-14 WO PCT/BE1996/000109 patent/WO1997013847A1/fr not_active Application Discontinuation
- 1996-10-14 EP EP96933282A patent/EP0800576A1/fr not_active Withdrawn
- 1996-10-14 AU AU72080/96A patent/AU7208096A/en not_active Abandoned
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WO1998006836A2 (fr) * | 1996-08-16 | 1998-02-19 | Genencor International, Inc. | Systeme d'expression pour niveaux d'expression modifies |
WO1998006836A3 (fr) * | 1996-08-16 | 1998-04-30 | Genencor Int | Systeme d'expression pour niveaux d'expression modifies |
US6048710A (en) * | 1996-08-16 | 2000-04-11 | Genecor International, Inc. | Expression system for altered expression levels |
US6313283B1 (en) | 1996-08-16 | 2001-11-06 | Genencor International, Inc. | Expression system for altered expression levels |
Also Published As
Publication number | Publication date |
---|---|
CA2207598A1 (fr) | 1997-04-17 |
EP0800576A1 (fr) | 1997-10-15 |
BE1009650A5 (fr) | 1997-06-03 |
AU7208096A (en) | 1997-04-30 |
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