BE1008998A3 - Lipase, microorganism producing the preparation process for the lipase and uses thereof. - Google Patents

Abstract

The invention relates to a lipase from a Pseudomonas strain. This lipase is active in a wide range of alkaline pH. The invention also relates to new strains of microorganisms producing this lipase and methods of preparing this lipase. The invention also relates to uses thereof and the compositions containing it.

Description

         

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  Lipase. microorganism producing it. process for the preparation of this lipase and uses thereof
The invention relates to a novel lipase. The invention also relates to a new strain of microorganism producing this lipase.



   The invention also relates to the methods of preparation of this lipase, the uses thereof and the compositions comprising it. It is known to include lipases in detergent compositions in order to remove fatty deposits from tissues (Enzyme Microb. Technol., 1993 (3), pages 634-645). These fatty deposits contain triglycerides supplied, for example, by sebum, various food products (oil, sauce, butter, fat), cosmetic products. Lipases hydrolyze triglycerides, forming products more soluble in water, mono and di-glycerides, glycerol and free fatty acids.



   Lipases which can be used in detergent compositions are known, such as in particular lipases originating from Pseudomonas strains, for example the lipase produced by the strain of Pseudomonas stutzeri (British patent application 1372034), the lipase produced by the strain of Pseudomonas mendocina ( European patent application 0 571 982), the lipase produced by the strain of Pseudomonas alcaligenes (US patent 5,063,160), and the lipase produced by the strain of Pseudomonas pseudoalcaligenes (European patent application 0 218 272). However, despite the properties of these lipases, the detergent compositions containing these enzymes appear to be ineffective.



   Consequently, there is currently a need for a lipase which can be used in the detergency field, which is very stable and also very active in a wide range of pH and temperature. In addition, there is also a need for a lipase which is particularly effective on oily spots, and this at a low dose of enzyme. In addition, there is also a need for a lipase which is particularly effective on oily stains from the first washing cycles.



   The present invention aims to provide a new lipase, active in a wide range of temperature, active in a wide range of alkaline pH, and effective from the first washing cycle.

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   The present invention also aims to identify, isolate and provide a strain, in particular a strain of Pseudomonas, which naturally produces said lipase.



   The present invention also aims to prepare and provide a composition containing this lipase, and in particular a detergent composition.



   To this end, the present invention relates to an isolated and purified lipase originating from a strain of Pseudomonas wisconsinensis. The present invention also relates to an isolated and purified lipase originating from a derivative or mutant of a strain of Pseudomonas wisconsinensis capable of producing this lipase. Preferably the lipase of the invention comes from the strain of Pseudomonas wisconsinensis T 92.677 / 1 or a derivative or mutant of this strain capable of producing this lipase. The lipase of the invention is derived from the strain of Pseudomonas wisconsinensis T 92.677 / 1. Lipase is classified in the international system under number E. C. 3.1. 1.3. ; it is a glycerol ester hydrolase.



   Preferably, the isolated and purified lipase has a molecular weight of about 30 kDa. It is essentially extracellular.



   The N-terminal amino acid sequence (SEQ ID NO: 1) of the lipase of the invention is as follows: Asn Tyr Thr Lys Thr Lys Tyr Pro Ile Val Leu Val His Gly Val Thr Gly 1 5 10 15 Phe Asn Thr Ile Gly Gly Leu
20
The invention relates to a lipase which originates from an aerobic bacterium capable of producing the lipase in an appropriate nutritive medium containing sources of carbon and nitrogen and mineral salts under aerobic conditions.



   The invention relates to an isolated and purified lipase comprising the amino acid sequence of 1 to 286 amino acids (SEQ ID NO: 4) or a modified sequence derived therefrom. The amino acid sequence and the nucleotide sequence (SEQ ID NO: 2) coding for mature lipase, as well as its translation into amino acids (SEQ ID NO: 3), is given in FIG. 1 (FIGS. 1a and 1b ).



   The lipase of the invention is synthesized in the form of a precursor. The precursor contains 308 amino acids (SEQ ID NO: 7). We identify the

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 nucleotide sequence (SEQ ID NO: 5) coding for the lipase precursor, as well as its translation into amino acids (SEQ ID NO: 6). FIG. 2 (FIGS. 2a and 2b) represents the nucleotide sequence (SEQ ID NO: 5) of the coding part of the lipase as well as its translation into amino acids (SEQ ID NO: 6).



   The precursor contains the 286 amino acid sequence (SEQ ID NO: 4) of the mature lipase and the 22 amino acid sequence (SEQ ID NO: 10) of the presequence.



   The mature lipase sequence is preceded by a presequence. It is an additional sequence of 22 amino acids (SEQ ID NO: 10).



  The corresponding nucleotide sequence is identified (SEQ ID NO: 8), as well as its translation into amino acids (SEQ ID NO: 9). This presequence codes for the signal peptide for the lipase of the invention.



   Preferably, said lipase has an estimated isoelectric point of between approximately 9.8 and approximately 10.1. In a particularly preferred manner, said lipase has an estimated isoelectric point equal to approximately 9.95.



   The lipase of the invention is active over a wide temperature range. The isolated and purified lipase of the invention develops an optimal enzymatic activity, measured at a pH of 9.5, in a temperature range above about 40 C. The isolated and purified lipase of the invention develops an optimal enzymatic activity, measured at a pH of 9.5, in a temperature range of less than about 60 C. More particularly, the lipase of the invention develops an optimal enzymatic activity, measured at a pH of 9.5, in a temperature range between approximately 40 ° C. and approximately 60 ° C. In a particularly preferred manner, the lipase of the invention develops an optimal enzymatic activity, measured at a pH of approximately 9.5,

   at a temperature of about 55 OC.



   The isolated and purified lipase of the invention develops an enzymatic activity of more than 50% of the maximum enzymatic activity in a temperature range between approximately 40 ° C. and approximately 60 ° C. for a pH of approximately 9.5, maximum enzyme activity being measured at a
 EMI3.1
 temperature of 55 C and a pH of 9.5.



  The lipase of the invention is active over a wide range of alkaline pH.



  Usually, the isolated and purified lipase of the invention develops an optimal enzymatic activity, measured at a temperature of approximately 30 ° C.,

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 in a range of pH greater than or equal to approximately 8. Usually, the isolated and purified lipase of the invention develops an optimal enzymatic activity, measured at a temperature of approximately 30 ° C., in a range of pH less than or equal to approximately 10. More particularly, the lipase of the invention develops an optimal enzymatic activity, measured at a temperature of approximately 30 ° C., in a pH range of between approximately
8 and about 10.

   In a particularly preferred manner, the lipase of the invention develops an optimal enzymatic activity, measured at a temperature of approximately 30 C, in a pH range between approximately
8 and about 9.5.



   The isolated and purified lipase of the invention develops an enzymatic activity of more than 85% of the maximum enzymatic activity in a pH range of between approximately 8 and approximately 10 for a temperature of approximately 30 oC, the activity maximum enzymatic being measured at a temperature of 30 C and a pH of 9.5.



   The lipase of the invention is thermostable at an alkaline pH. Indeed, the isolated and purified lipase of the invention shows a relative enzymatic activity of at least 55% measured after an incubation of 160 minutes at a temperature of 55 C and at a pH of 10 in a buffered hardness solution 15. It shows a relative enzymatic activity of at least 70% measured after an incubation of 80 minutes under these same conditions.



   By relative enzymatic activity is meant the ratio between the enzymatic activity, measured during a test carried out under given conditions of pH, temperature, substrate and duration, and the maximum enzymatic activity measured during this same test, the enzymatic activity being measured from the hydrolysis of the triolein and the maximum enzymatic activity being arbitrarily fixed at the value of 100.



   The invention also relates to a modified lipase, that is to say an enzyme whose amino acid sequence differs from that of the wild-type enzyme by at least one amino acid. These modifications can be obtained by conventional techniques of DNA mutagenesis, such as exposure to ultraviolet radiation, to chemicals, such as
 EMI4.1
 as ethyl methane sulfonate (EMS), te N-methyl-N-nitro-N-mtrosoguanidine (MNNG), sodium nitrite or O-methylhydroxylamine or by genetic engineering techniques, such as, for example, site-directed mutagenesis

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 or random mutagenesis.

   These techniques are known to those skilled in the art and are in particular described in MOLECULAR CLONING-a laboratory manual-SAMBROOK, FRITSCH, MANIATIS-second edition,
1989, chapter 15.



   The invention also relates to a mutated lipase obtained by modification of the nucleotide sequence of the gene which codes for lipase. The techniques for obtaining such mutated lipases are known to those skilled in the art and are in particular described in MOLECULAR CLONING-a laboratory manual-SAMBROOK, FRITSCH, MANIATIS-second edition, 1989, chapter 15.



   The invention also relates to a lipase having immunochemical properties identical or partially identical to the lipase obtained from the strain of Pseudomonas wisconsinensis T 92.677 / 1. The immunochemical properties can be determined immunologically by identity tests, in particular by using specific, polyclonal or monoclonal antibodies. Identity tests are known to those skilled in the art, such as in particular the Ouchterlony immunodiffusion method, or the immunoelectrophoresis method. Examples of such methods are described by Axelsen N.

   H., Handbook of Immunoprecipitation Gel Techniques, Blackwell Scientific Publications, 1983, chapters 5 and 14, the terms "antigenic identity" and "partial antigenic identity" are described in this document in chapters 5.19 and 20. A serum containing the specific antibody is prepared according to the method described by immunizing animals (for example mice, rabbits or goats) with a preparation of purified lipase. This preparation can be mixed with an additive, such as Freund's adjuvant, and the mixture obtained is injected into animals. The polyclonal antibody is obtained after one or more immunizations. An example consists in injecting, subcutaneously two weeks apart, four fractions each containing 150 micrograms of purified lipase, the immunization then lasts 8 weeks.

   The serum is collected after the immunization period and the immunoglobulin can be isolated according to the method described by Axelsen N. H. (1983).



   The present invention also relates to the isolation, identification and supply of a novel lipase producing bacteria. This aerobic bacteria has been isolated and purified. Generally it belongs to the family of

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Pseudomonadaceae. Preferably it belongs to the genus Pseudomonas.



   In a particularly preferred manner, it is a strain of Pseudomonas wisconsinensis. Good results have been obtained with the Pseudomonas wisconsinensis T 92.677 / 1 strain or a derivative or mutant of this strain.



   By derivative of this strain is meant any naturally modified bacteria, that is to say by natural selection. By mutant of this strain is meant any artificially modified bacteria. Mutants of this strain can be obtained by known modification techniques, such as ultraviolet radiation, X-rays, mutagens or genetic engineering.



  These techniques are known to those skilled in the art and are described in particular in SAMBROOK et al., 1989, chapter 15. Examples of mutagenic agents are described in particular by R. Scriban, Biotechnologie, (Technique et Documentation Lavoisier), 1982, p. 365-368.



   The strain of Pseudomonas wisconsinensis T 92.677 / 1 was deposited in the collection named BELGIAN COORDINATED COLLECTIONS OF MICROORGANISM (LMG culture collection, University of Ghent, Laboratory of Microbiology-KL Ledeganckstraat 35, B-9000 Ghent, Belgium) in accordance with the Budapest Treaty under the number LMG P-15151 October 12, 1994. The invention relates to an isolated and purified culture of the strain of Pseudomonas wisconsinensis and a culture derived or mutated thereof. More particularly, the invention relates to an isolated and purified culture of the strain of Pseudomonas wisconsinensis T 92.677 / 1 and a culture derived or mutated therefrom.



   The strain of the present invention has been identified by its biochemical characteristics. It is an aerobic Gram negative bacterium. It does not develop anaerobically. No spores are formed. The oxidase test is positive in the presence of 1% (w / v) of tetramethyl-1,4-phenylene-diammoniumdichloride. This bacteria is not thermophilic. It does not produce gas from glucose.



   The invention also relates to the isolation and supply of a DNA molecule comprising the nucleotide sequence (SEQ ID NO: 2) which codes for the mature lipase of Pseudomonas wisconsinensis T 92.677 / 1 or a modified sequence derived from that -this.



   By modified sequence derived from the DNA molecule is meant any DNA molecule obtained by modification of one or more nucleotides

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 of the gene which codes for the lipase of the invention. The techniques for obtaining such sequences are known to those skilled in the art and are in particular described in MOLECULAR CLONING-a laboratory manual-
SAMBROOK, FRISCH, MANIATIS - second edition, 1989, chapter 15.



  Usually, the modified sequence derived from the DNA molecule comprises at least 70% of homology with the nucleotide sequence (SEQ ID NO: 2) of the gene which codes for the lipase of the invention, that is to say say at least 70% of identical nucleotides and having the same position in the sequence. Preferably, the modified sequence derived from the DNA molecule comprises at least 80% of homology with the nucleotide sequence (SEQ ID NO: 2) of the gene which codes for the lipase of the invention.



  In a particularly preferred manner, the modified sequence derived from the DNA molecule comprises at least 90% of homology with the nucleotide sequence (SEQ ID NO: 2) of the gene which codes for the lipase of the invention.



   Usually, the DNA molecule according to the invention comprises at least the nucleotide sequence (SEQ ID NO: 5) which codes for the lipase precursor or a modified sequence derived therefrom. This nucleotide sequence (SEQ ID NO: 5) comprises the nucleotide sequence (SEQ ID NO: 2) coding for the mature lipase of Pseudomonas wisconsinensis T 92.677 / 1 and its signal sequence (pre-sequence) (SEQ ID NO: 8). Preferably, this DNA molecule comprises the entire lipase gene from Pseudomonas wisconsinensis T 92. 677/1.



   The present invention also relates to a process for the production of a lipase. This process comprises the cultivation of an aerobic bacterium capable of producing lipase in an appropriate nutritive medium containing carbon and nitrogen sources and mineral salts under aerobic conditions and the harvesting of the lipase thus obtained. This culture medium can be solid or liquid. Preferably the culture medium is liquid. Usually, the aerobic bacterium is a strain of Pseudomonas or a derivative or mutant of this strain capable of producing lipase. More particularly, the aerobic bacterium is a strain of Pseudomonas wisconsinensis or a derivative or mutant of this strain capable of producing lipase.

   Preferably, the aerobic bacterium is a strain of Pseudomonas wisconsinensis T 92.677 / 1 or a derivative or mutant of this strain capable of producing lipase.

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   The culture conditions of these bacteria, which make it possible to obtain the lipase 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. Examples of culture conditions are described in particular in European patent application 0 571 982.



   Lipase harvesting techniques are well known to those skilled in the art and are chosen according to the intended uses of the lipase. Typically, centrifugation, filtration, ultrafiltration, evaporation, microfiltration, crystallization, or a combination of any of these techniques such as centrifugation followed by ultrafiltration are employed. Examples of such techniques are notably described by R. Scriban, Biotechnologie, (Technique et Documentation Lavoisier), 1982, p. 267-276.



   The lipase can then be purified, if necessary and according to the intended uses. Enzyme purification techniques are well known to those skilled in the art, such as precipitation using a salt, such as ammonium sulfate, or a solvent, such as acetone or an alcohol. . Examples of such techniques are notably described by R. Scriban, Biotechnologie, (Technique et Documentation Lavoisier), 1982, p. 267-276.



  The lipase can also be dried by atomization or lyophilization. Examples of such techniques are notably described by R. Scriban, Biotechnologie, (Technique et Documentation Lavoisier), 1982, p. 267-276.



  The present invention also relates to enzymatic compositions comprising the lipase according to the invention and at least one additive. According to the envisaged uses, the enzymatic compositions comprising the lipase of the present invention can be in solid or liquid form.



   The additives, included in the composition according to the invention, are known to those skilled in the art and are chosen according to the intended use of the composition. They must be compatible with lipase and not affect the enzymatic activity of lipase. Usually these additives are enzyme stabilizers, preservatives and formulants. Examples of additives are described in particular in European patent application 0 218 272. Examples of additives that may be mentioned include ethylene glycol, glycerol, 1,2-propanediol, starch, a sugar such as glucose and sorbitol, a salt such as sodium chloride, calcium chloride, potassium sorbate and sodium benzoate or a mixture of two or

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 many of these products. Good results have been obtained with the
1,2-propanediol.

   Good results have also been obtained with sorbitol.



   The lipase according to the invention has multiple outlets in various industries, such as, for example, the food industries, the pharmaceutical industries or the chemical industries.



   Lipase can in particular be used in detergency. The present invention also relates to the use of lipase, as defined above, in detergency. An example of such a use is described in particular in British patent application 1372034 and in European patent application 0 218 272. In this context, it forms part of detergency compositions. The present invention therefore also relates to detergent compositions containing lipase. The components of the detergency compositions are known to those skilled in the art and are adapted according to the intended use of the composition.

   Such components are in particular enzymes, such as for example proteases, amylases and / or cellulases; bulking agents, such as sodium tripolyphosphate; bleaching agents, such as perborate; formulation additives; surfactants. The detergency compositions of the invention can be used, depending on their formulation, as washing powder, granule or liquid for washing clothes; as a stain remover for detaching or degreasing objects or detaching laundry prior to cleaning; and as a powder, granule or liquid for washing dishes.



   Lipase can in particular be used during the treatment of old paper in order to remove the oil-based inks. An example of such a use is described in particular in the summary Chemical Abstract 113/154607.



   Lipase can in particular be used during the processing of paper pulp to avoid sticky deposits known under the name of "pitch". An example of such use is described in particular in the document Enzyme Microb. Technol., 1993 (3), pages 634-645.



   Lipase can in particular be used in the food industry to develop the flavor of certain food products, such as cheeses; when making special margarines. An example of such a use is described in particular in the document Enzyme Microb.



  Technol., 1993 (3), pages 634-645.

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   The present invention is illustrated by the following examples.



   FIG. 1 (FIGS. 1a and 1b) represents the amino acid sequence and the nucleotide sequence (SEQ ID NO: 2) coding for mature lipase, as well as its translation into amino acids (SEQ ID NO: 3).



   FIG. 2 (FIGS. 2a and 2b) represents the nucleotide sequence (SEQ ID NO: 5) of the coding part of the lipase as well as its translation into amino acids (SEQ ID NO: 6).



  Example 1 Isolation and Characterization of the Pseudomonas wisconsinensis T 92.677 / 1 Strain
The strain Pseudomonas wisconsinensis T 92. 677/1 was isolated from a soil sample taken from 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.



   1 ml of the diluted soil suspension is spread on an agar nutrient medium A.
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  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 7.5 g / l Na2COg, 10 g / 1 olive oil, 1 g / l polyvinyl alcohol (25/140) and 0.01 g / l 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).
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  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 distilled water. The agar suspension obtained is sterilized at 121 C for 30 minutes.
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  1 1 of carbonate buffer (pH 9.5), containing 25 g / l of NaHCO3 and 75 g / l of NaCOg, is prepared, then sterilized at 121 C for 30 minutes.
Then, 1 ml of an aqueous solution of [0.01% (w / v)] rhodamine B (Sigma 6626) is prepared. This solution is sterilized by filtration on a

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 0.45 u sterilization membrane (MILLIPORE).



   The sterilized olive oil emulsion and the sterilized agar are cooled to 60 ° C., then mixed under sterile conditions. 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 rpm (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 extract of
 EMI11.1
 yeast, 5 g / l of NaCl, 20 g / l of agar, 2.5 g / l of NaHCO3, 7.5 g / l of NaCOo. Tryptone, yeast extract, NaCl, agar, which make up medium B, are mixed with 900 ml of distilled water, then sterilized at 121 OC for 30 minutes. The pH is adjusted to 9.5 by the addition of 100 ml of pre-sterilized carbonate buffer (containing 25 g / l of NaHCO3 and 75 g / l of Na2CO3) '
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 p. m x 1.5-4.0 pm. 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) detetramethyl-1,4-phenylene-diammoniumdichloride.



  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 anaerobically, i.e. under an atmosphere of 84% (v / v) N2, 8% (v / v) CO2, 8% (v / v) H2 at 37 C.



   The abbreviation% (v / v) represents a percentage expressed in volume by volume. 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

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 EMI12.1
 tage expressed in weight by weight.



  C>
This strain is not thermophilic. It exhibits normal development after incubation on medium B agar 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 d 'a strain of Pseudomonas stutzeri. This is clear from a reading of Table 1 gathering the main biochemical characteristics of these 5 strains.

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  Table 1
 EMI13.1
 
 <tb>
 <tb> Features <SEP> Pseudomonas
 <tb> wisconsinensis <SEP> stutzeri <SEP> mendo- <SEP> alkali <SEP> - <SEP> pseudoalcaT <SEP> 92.677 / 1 <SEP> cina <SEP> genes <SEP> ligenes
 <tb> Size <SEP> #m <SEP> x <SEP> 0.5-0, <SEP> 7 <SEP> 0, <SEP> 7-0, <SEP> 8 <SEP> 0.7-0, <SEP> 8 <SEP> 0.5 <SEP> 0, <SEP> 7-0, <SEP> 8
 <tb> / you <SEP> 1, <SEP> 5-4.0 <SEP> 1, <SEP> 4-2, <SEP> 8 <SEP> 1.4-2, <SEP> 8 <SEP> 2.0-3, <SEP> 0 <SEP> 1,2-2,

    <SEP> 5
 <tb> Pigment <SEP> yellow <SEP> + - + <SEP> hydrolysis <SEP> +
 <tb> starch
 <tb> Arginin - + <SEP> + <SEP> d
 <tb> dehydrolase
 <tb> Usage <SEP> from <SEP> + <SEP> + <SEP> +
 <tb> Glucose
 <tb> Usage <SEP> from <SEP> + <SEP> d <SEP> + <SEP> d
 <tb> Gluconate
 <tb> Usage <SEP> from <SEP> + - + - Géraniol
 <tb> Usage <SEP> from <SEP> + <SEP> - <SEP> + <SEP> d <SEP> d
 <tb> L-histidine
 <tb> Usage <SEP> from <SEP> + <SEP> - <SEP> + <SEP> + <SEP> +
 <tb> L-arginine
 <tb> Usage <SEP> from <SEP> + <SEP> - <SEP> + <SEP> - <SEP> +
 <tb> Betaine
 <tb>
 + = positive test for 90% or more of the strains - = negative test for 90% or more of the strains d = positive test for more than 10%, but less than 90% of the strains

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The isolated bacterium therefore belongs to the genus Pseudomonas,

   no known species could be determined.



   The strain of Pseudomonas wisconsinensis T 92. 677/1 was deposited in the collection named BELGIAN COORDINATED COLLECTIONS OF MICRORGANISMS (LMG culture collection) under the number LMG P-15151 on October 12, 1994.



  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, starting from this culture, a culture is carried out in 25 ml of a liquid medium C. Medium C contains 10 gll of tryptone (Difco), 5 g / 1 of yeast extract, 10 g / 1 of NaCl, the pH of the medium is adjusted to 7.0 with NaOH O, 1N, 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.



   After 16 hours of incubation, this culture is introduced into a 20 liter fermenter containing 13 liters of sterilized liquid medium D.



   Medium D contains K2HPO4 2.5 gll, KH2PO4 2.5 gll, MgSO4. 7H20 1 gel, (NH4) 2SO4 2 g / 1, (NH2) 2CO 2 g / l, CaC12 1 g / l, soy flour 20 g / l, yeast extract 2 g / l, glucose 20 gll, Mazuôl antifoam (Mazes Chemicals) 5 gel. 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). 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 culture in the fermenter is carried out at a temperature of 3 ° C., under a pressure of 0.15. 10 "Pa (Pa = Pascal) (0.15 bar), with an aeration of 0.3 VVM (volume of air per volume of culture medium per minute), with axial agitation of 200 revolutions per minute, regulation dissolved oxygen being fixed at 10% (v / v) by regulating the stirring speed.



   After 24 hours of fermentation, the enzyme activity of the culture thus obtained is measured by the following technique.

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   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 (LU) 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 (Fluka 51200) are mixed in 100 ml of distilled water. This mixture is emulsified using an Ultra-Turrax mixer at 13,500 rpm (axial stirring) for 3 times 5 minutes, keeping the mixture under nitrogen and in an ice bath.



   A dilution buffer is prepared containing 2.34 g / l of NaCl, 2.94 g / l of CaCl2. 2H20 and 0.61 g / l Tris (2-amino-2-hydroxymethyl-1, 3-propanediol).



   An automatic titration apparatus is used, equipped with a burette containing 0.01N NaOH, a temperature probe and a pH probe and equipped with a thermostatically controlled reactor.



   Into the reactor thermostatically controlled at 30 ° C., a magnetic stirring bar, 10 ml of triolein emulsion and 20 ml of dilution buffer are introduced. The pH of the solution thus obtained is adjusted to 9.5 with NaOH O, IN. Then, 0.5 ml of the test sample containing the lipase is introduced, the sample is optionally diluted so that it contains only a maximum of 5 LU. The pH is checked for the first two minutes with NaOH O, ISO. Then, the soda consumption is recorded between 2 and 4 minutes, while keeping the pH constant (volume of soda consumed between 2 and 4 minutes = VI in 1).



   Then, the same test is carried out by replacing the sample containing the lipase with 0.5 ml of dilution buffer (volume of soda consumed between 2 and 4 minutes = V2 in 1).



   A lipase unit (LU) is determined as follows:
1 LU / ml = (VI-V2) x possible dilution of the sample x 10
By this method, lipase activity is detected in the culture.



  EXAMPLE 3 Preparation of a Concentrated Lipase Solution
The pH of the culture is adjusted, as obtained at the end of fermentation to

  <Desc / Clms Page number 16>

 Example 2, at a pH of 8 with concentrated 10N caustic soda



   Then, 1% (v / v) of Triton X-114 (SERVA) is added to this culture.
37214). The mixture is stirred gently for 2 hours at 15 C.



   Next, 1% (v / v) of Optifloc FC205 (SOLVAY) is added to the mixture in the form of a 10% (v / v) solution. The mixture is stirred gently for 1 hour at 15 C.



   The mixture is centrifuged for 15 minutes at 9000 revolutions per minute (Beckman J21, rotor JA10) at a temperature of 4 C. The centrifuge supernatant is kept.



   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 at 4 ° C. are added to the lower phase. The suspension is incubated for 15 minutes at 4 ° C. with moderate agitation.



   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.



   Then, 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 (HERE), 25 mM CaC12 and 20 mM Tris.



   Then, the suspension, containing the precipitate, is centrifuged for 15 minutes at 9000 revolutions per minute (Beckman J21, rotor JAI0) at a temperature of 4 C. The centrifugation supernatant is stored, which forms a concentrated solution of lipase.



  EXAMPLE 4 Purification of the Lipase
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.



   Following the directions for use prescribed by the supplier (Pharmacia) for the hydro- interaction chromatography column

  <Desc / Clms Page number 17>

 phobe, a Phannacia Hiload 16/10 Phenyl-Sepharose column (ref 17-1085-01) is loaded with 140 ml of the concentrated lipase solution, as obtained in Example 3.



   As a balance buffer, a 20 mM phosphate buffer at pH 7.2 is used; as 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 devised in a cell
 EMI17.1
 Amicon, provided with a Yam10 membrane, with 10 volumes of a buffer (pH 7) containing 25 mM CaC12 and 20 mM Tris.



   Then, the diaflltrated fraction is concentrated to 0.5 ml by ultrafiltration using the same Amicon cell, equipped with a Yam10 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 CaCl2 and 20 mM Tris.



   Three absorption peaks at 280 nm are separated. Lipase corresponds to
 EMI17.2
 first absorption peak at 280 nm. The corresponding fraction which contains the purified lipase is kept.



  Example 5 Determination of the N-terminal sequence
The method described by Vandekerkhove J. et al., Eur. J.



  Biochemistry, 152.9 (1985), to determine the N-terminal sequence of the lipase.



   The fraction containing the purified lipase is used, as obtained in Example 4.



   The N-terminal sequence (SEQ ID NO: 1) is as follows: Asn Tyr Thr Lys Thr Lys Tyr Pro Ile Val Leu Val His Gly Val Thr Gly 1 5 10 15
Phe Asn Thr Ile Gly Gly Leu
20
This sequence differs from the N-terminal sequences of other lipases

  <Desc / Clms Page number 18>

 secreted by other strains of Pseudomonas, sequences notably published in Enzyme Microb. Technol., 1993 (3), pages 634-645.



   Example 6
Amino acid sequence
The amino acid sequence of the lipase of the present invention is indirectly determined from the nucleotide sequence (SEQ ID NO: 5) of the gene which codes for this lipase, the production of which is described in Example 17. This is done using the IntelliGenetics Suite Software for Molecular Biology (Release &num; 5. 4) computer program from IntelliGenetics, Inc. USA.



   FIG. 2 (FIGS. 2a and 2b) represents the nucleotide sequence (SEQ ID NO: 5) of the coding part of the lipase as well as its translation into amino acids (SEQ ID NO: 6).



   Lipase is synthesized in the form of a precursor. The lipase precursor contains 308 amino acids (SEQ ID NO: 7). The nucleotide sequence (SEQ ID NO: 5) coding for the lipase precursor is identified as well as its translation into amino acids (SEQ ID NO: 6).



   The presequence of the lipase synthesized in the form of a precursor is identified. n is a sequence of 22 amino acids (SEQ ID NO: 10) which constitutes the signal peptide. The corresponding nucleotide sequence is identified (SEQ ID NO: 8).



   Next, the amino acid sequence of the mature lipase is identified.



  The mature lipase contains 286 amino acids (SEQ ID NO: 4).



   Figure 1 (Figure 1a and Figure Ib) shows the nucleotide sequence (SEQ ID NO: 2) coding for mature lipase as well as its translation into amino acids (SEQ ID NO: 3).



  EXAMPLE 7 Distribution in Amino Acids
The amino acid distribution of the mature lipase, determined from the amino acid sequence (SEQ ID NO: 4) of the lipase (Example 6), is summarized in Table 2.

  <Desc / Clms Page number 19>

 



  TABLE 2
 EMI19.1
 
 <tb>
 <tb> Symbol <SEP> Acids <SEP> amines <SEP> Number <SEP>% <SEP> mole
 <tb> (in <SEP> weight <SEP> molecular)
 <tb> A <SEP> alanine <SEP> 28 <SEP> 9,790
 <tb> B <SEP> acid <SEP> aspartic <SEP> 0 <SEP> 0
 <tb> C <SEP> cysteine <SEP> 2 <SEP> 0, <SEP> 699
 <tb> D <SEP> acid <SEP> aspartic <SEP> 10 <SEP> 3, <SEP> 497
 <tb> E <SEP> acid Glutamic <SEP> <SEP> 7 <SEP> 2, <SEP> 448
 <tb> F <SEP> phenylalanine <SEP> 7 <SEP> 2,448
 <tb> G <SEP> wisteria <SEP> 35 <SEP> 12,238
 <tb> H <SEP> histidine <SEP> 10 <SEP> 3,497
 <tb> 1 <SEP> isoleucine <SEP> 14 <SEP> 4,895
 <tb> K <SEP> lysine <SEP> 9 <SEP> 3.147
 <tb> L <SEP> leucine <SEP> 22 <SEP> 7,692
 <tb> M <SEP> methionine <SEP> 1 <SEP> 0.350
 <tb> N <SEP> asparagine <SEP> 25 <SEP> 8.741
 <tb> P <SEP> proline <SEP> 11 <SEP> 3,846
 <tb> Q <SEP> glutamine <SEP> 6 <SEP> 2,098
 <tb> R <SEP> arginine <SEP> 13 <SEP> 4,

  545
 <tb> S <SEP> serine <SEP> 24 <SEP> 8.392
 <tb> T <SEP> threonine <SEP> 18 <SEP> 6,294
 <tb> V <SEP> valine <SEP> 29 <SEP> 10, <SEP> 140
 <tb> W <SEP> tryptophan <SEP> 5 <SEP> 1,748
 <tb> X <SEP> unknown <SEP> 0 <SEP> 0
 <tb> y <SEP> tyrosine <SEP> 10 <SEP> 3,497
 <tb> Z <SEP> glutamine <SEP> 0 <SEP> 0
 <tb> acid Glutamic <SEP>
 <tb>
 
 EMI19.2
 Example 8 Estimation of the molecular weight
The molecular weight of the lipase is estimated by calculation from the amino acid sequence of the mature form of lipase and from the amino acid sequence of lipase including the signal peptide, as described in example 6.



   A molecular weight of 30093 Daltons for the mature form and a molecular weight of 32365 Daltons for the form comprising the signal peptide are deduced by calculation.



  Example 9 Determination of the molecular weight of the lipase by SDS-PAGE analysis
Is carried out on the fraction, containing the purified lipase, as obtained in Example 4, an electrophoresis in polyacrylamide gel under denaturing conditions (SDS-PAGE). The gel system used is the PHASTSYSTEM system from PHARMACIA LKB BIOTECHNOLOGY (file

  <Desc / Clms Page number 20>

 of use NO 110), with gels containing a polyacrylamide gradient of 10-15% (v / v). The electrophoresis conditions are those prescribed by the supplier. Molecular weight markers are used as a control
PHARMACIA LMW (Low Molecular Weight), reference 17-0446-01.

   The markers used are phosphorylase b (94 kD), albumin (67 kD), ovalbumin (43 kD), carboanhydrase (30 kD), trypsin inhibitor (20.1 kD) and alpha-lactalbumin (14.4 kD).



   The gel thus obtained shows that the fraction containing the purified lipase, as obtained in Example 4, is pure.



   A Coomassie blue staining (Fast Coomassie staining, Phannacia, use file no 200) of the gel reveals a polypeptide with a molecular weight of approximately 30 (+/- 0.5) kD.



    Example 10 Estimation of the isoelectric point
The isoelectric point of the lipase is estimated from the amino acid sequence of the mature form of the lipase and from the amino acid sequence of the lipase including the signal peptide, as described in Example 6.



   We deduce an isoelectric point of 9.95 for the mature form and
 EMI20.1
 10, 12 for the form comprising the signal peptide.



  Example 11 Determination of the optimum pH of the lipase
The enzymatic activity of the lipase is measured at different pHs according to the method described in Example 2. The hydrolysis of the substrate (triolein emulsion) is therefore determined following the action of the lipase at different pHs, all the others conditions being identical to standard conditions, as described in Example 2, that is to say at a temperature of 30 ° C. and a duration of two minutes.



   The fraction containing the purified lipase is used, as obtained in Example 4.



   The results are collated in Table 3.

  <Desc / Clms Page number 21>

 
 EMI21.1
 Table 3
 EMI21.2
 
 <tb>
 <tb> Temperature <SEP> Activity <SEP> relative
 <tb> oc <SEP>%
 <tb> 18 <SEP> 19
 <tb> 24 <SEP> 26
 <tb> 30 <SEP> 41
 <tb> 34 <SEP> 45
 <tb> 40 <SEP> 56
 <tb> 45 <SEP> 52
 <tb> 49 <SEP> 91
 <tb> 55 <SEP> 100
 <tb> 60 <SEP> 54
 <tb> 76 <SEP> 43
 <tb>
 
During the test, the maximum enzymatic activity was measured for the sample placed at a pH of approximately 9.5 and at a temperature of approximately 55 C. By definition, this sample was therefore assigned an activity 100% relative enzyme.



   This example shows that the lipase of the invention has an optimal enzymatic activity measured at a pH of 9.5 in a temperature range between about 40 and about 60 C.



   This example also shows that the lipase of the invention develops an optimal enzymatic activity, measured at a pH of 9.5, at a temperature of around 55 C.



   The lipase of the invention develops an enzymatic activity of more than 50% of the maximum enzymatic activity in a temperature range between approximately 40 and 60 C for a pH of approximately 9.5.



  Example 12 Determination of the Lipase pH Optimum
The enzymatic activity of the lipase is measured at different pHs according to the method described in Example 2.



   The hydrolysis of the substrate (triolein emulsion) is therefore determined following the action of the lipase at different pH, all the other conditions being identical to the standard conditions, as described in Example 2, that is to say say at a temperature of 30 C and a duration of two minutes.



   The fraction containing the purified lipase is used, as obtained at

  <Desc / Clms Page number 22>

 Example 4.



   The results are collated in Table 4.



   Table 4
 EMI22.1
 
 <tb>
 <tb> pH <SEP> Activity <SEP> relative
 <tb>%
 <tb> 7.0 <SEP> 17
 <tb> 8, <SEP> 0 <SEP> 100
 <tb> 9.0 <SEP> 100
 <tb> 9, <SEP> 5 <SEP> 100
 <tb> 10.0 <SEP> 91
 <tb> 10.5 <SEP> 71
 <tb> 11.0 <SEP> 72
 <tb> 12.0 <SEP> 47
 <tb>
 
 EMI22.2
 This example shows that the lipase of the invention develops an optimal enzymatic activity, measured at a temperature of approximately 30 ° C., in a pH range between approximately 8 and 10.



  During the test, the maximum enzymatic activity was measured for the sample placed at a pH of approximately 9.5 and at a temperature of approximately 30 C. By definition, this sample was therefore assigned an activity 100% relative enzyme.



  The lipase of the invention develops an enzymatic activity of more than approximately 90% of the maximum enzymatic activity in a pH range between approximately 8 and approximately 10 for a temperature of approximately 30 C.



  The lipase of the invention develops an enzymatic activity of more than approximately 70% of the maximum enzymatic activity in a pH range of between approximately 8 and approximately 11 for a temperature of approximately 30 C.



  EXAMPLE 13 Temperature Stability of the Lipase The part of the fraction containing the purified lipase, as obtained in Example 4, is incubated at pH 10 and at 55 ° C. in an aqueous hardness buffer. 15 (buffer containing 1.98 mM calcium chloride, 0.69 mM magnesium chloride and 2.5 mM sodium bicarbonate).



  At regular intervals, as specified in Table 4 (incubation time in minutes), a sample is taken from which the enzymatic activity is measured according to the method described in Example 2.

  <Desc / Clms Page number 23>

 The results are collated in Table 5.



   Table 5
 EMI23.1
 
 <tb>
 <tb> Time <SEP> incubation <SEP> Activity <SEP> relative
 <tb> minutes <SEP>%
 <tb> 0 <SEP> 100
 <tb> 10 <SEP> 84
 <tb> 20 <SEP> 81
 <tb> 30 <SEP> 79
 <tb> 40 <SEP> 93
 <tb> 50 <SEP> 86
 <tb> 60 <SEP> 80
 <tb> 80 <SEP> 72
 <tb> 100 <SEP> 59
 <tb> 120 <SEP> 59
 <tb> 130 <SEP> 60
 <tb> 140 <SEP> 61
 <tb> 160 <SEP> 61
 <tb> 260 <SEP> 39
 <tb> 1140 <SEP> 25
 <tb>
 
The deactivation constant (k) over the 0-260 minute interval is 0.000333 min-l. [We obtain the deactivation constant k according to the definition In (AAo) = -k. t, t being the incubation time, Ao the activity relating to the incubation time 0 and At the activity relating to the incubation time t.]
During this test, the maximum enzymatic activity was measured for the sample at time 0.

   By definition, therefore, a relative enzyme activity of 100% was assigned to this sample.



   From this example it is concluded that the lipase of the invention shows a
 EMI23.2
 relative enzymatic activity of at least 55% measured after an incubation of 160 minutes at a temperature of 55 C and a pH of 10 in a buffered solution of hardness 15. It shows a relative enzymatic activity of at least 70% measured after an 80-minute incubation under these same conditions.



  Example 14 Use of a detergent composition containing lipase
A piece of white fabric (R. Hoppe Gmbh) is prepared, based on cotton (65%) and polyester (35%), 10 cm by 10 cm, impregnated with bacon fat and Sudan red. The impregnation of the fabric is carried out as follows.



   A homogeneous solution of Sudan red 7B (SIGMA cat.



    N F 1000) and bacon fat (Laru Gmbh) by adding 0.1% (w / w) of

  <Desc / Clms Page number 24>

 Sudan red with bacon fat. The mixture is heated to 90 ° C. until the Sudan red is completely dissolved. A roll of cloth is immersed in the solution of Sudan red and bacon fat maintained at 90 ° C. and continuously moved into this solution at the speed of 0.5 m / minute. Then, the fabric is spun under constant linear pressure between two rollers.



  The fabric thus impregnated contains 31.5% (w / w) of fat. The impregnated fabric is placed at -18 ° C. for 22 hours until the fat has completely crystallized. The fabric is cut into 10 cm pieces on
10 cm. Then, the pieces of impregnated fabric are stored at −18 ° C. in the dark.



   A liquid detergent composition containing 4 g / l of a compact detergent powder (Eurocompact powder from UNILEVER) and various lipase concentrations equivalent to 500.1000 and 2000 LU / 1 is prepared in water of hardness 15 (water containing 1.98 mM calcium chloride, 0.69 mM magnesium chloride and 2.5 mM sodium bicarbonate). The initial pH of the liquid detergent composition is approximately 10. The lipase as obtained in Example 4 is used, which is diluted with water of hardness 15 in order to obtain the concentrations desired.



   Then, the fabric impregnated with bacon fat and Sudan red is washed with 200 ml of the liquid detergent composition in a 250 ml reactor. The washing takes place at 35 ° C. for 45 minutes with stirring at 40 RPM (back and forth stirring for 20 seconds). After washing, the fabric is rinsed three times with 200 ml of distilled water, then dried at 22 ° C. between two papers for 24 hours.



   Then, the reflectance (RL) at 460 nm of the dried fabric is determined using a colorimeter (Tricolor LFM 3).



   This test is repeated three times, that is to say then the washing cycle is carried out three times with the liquid detergent composition containing the lipase and the su rinse cycle, the same sample of tissue without impregnating it with Sudan red. and bacon fat between each cycle. At each end of the cycle, the reflectance at 460 nm of the dried fabric is determined.



   A strictly identical test is carried out with a detergent composition not containing lipase. At each end of the cycle, the reflectance (RO) at 460 nm of the dried fabric is determined.



   The washing performance in% is defined as the difference between the reflectance (RL) obtained with the washing in the presence of lipase and the reflect

  <Desc / Clms Page number 25>

 tance (RO) obtained with washing in the absence of lipase, that is to say RL-
RO expressed in%.



   The results of this example show that the lipase of the invention is effective at low dose of enzyme in the detergent composition.



   The results of this example also show that the lipase of the invention is particularly effective from the second washing cycle and that it retains increased efficiency after three and four washing cycles.



   In particular, lipase is particularly effective at the concentration of 500 LU / 1 after three washing cycles.



   In addition, lipase is particularly effective at the concentration of 1000 LU / 1 from the first washing cycle. Lipase is particularly effective at the concentration of 1000 LU / 1 during all the washing cycles.



    Example 15 Cloning of the Pseudomonas wisconsinensis T 92 lipase gene. 677/1 1. Extraction of the chromosomal DNA from the strain of Pseudomonas wisconsinensis T 92.677 / 1
Genomic DNA is prepared using the technique described by WILSON, 1990, Current Protocols in Molecular Biology, vol. 1 (Unit 2.4), with the modifications described below.



   From the culture, as obtained in Example 2, a culture of 200 ml of the strain of Pseudomonas wisconsinensis T 92.677 / 1 is carried out in LB growth medium for 16 hours at 37 C. The composition of the medium LB growth is as follows: TRYPTOSE (DIFCO) 10 g / l, yeast extract 5 g / l, NaCl 10 g / l.



   The culture obtained is centrifuged (SORVALL RC 5C Plus centrifuge, SS-34 rotor) 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 µl of a 10% (w / v) SDS (sodium dodecyl sulfate) solution; and 50 μl of a proteinase J solution: (sold by BOEHRINGER Mannheim) at 20 mg / ml (prepared immediately).



   The TE buffer (pH 8.0) consists of 10 mM TRIS-HC1 (TRIS-HC1 = Tris (hydroxymethyl) aminomethane) -HCl) and ImM EDTA (ethylenediaminetetraacetic acid).



   The suspension thus obtained, containing the centrifugation pellet, is incubated for 90 minutes at 65 C.

  <Desc / Clms Page number 26>

 
Then, 1.8 ml of a 5 M NaCl solution and 15 ml of a 10% (w / v) CTAB / NaCl solution (CTAB = bromide) are added to this suspension.
 EMI26.1
 cetyltrimethyl ammonium, 0.7 M NaCl). The suspension thus obtained is incubated for 20 minutes at 65 oC. A lysate is obtained.



   This lysate is extracted with 15 ml of a chloroform / isoamyl alcohol mixture (3-methyl-1-butanol) 24/1 under the conditions and following the procedures described in Molecular Cloning-a laboratory manual - SAMBROOK , FRITSCH, MANIA TIS - second edition,
1989, on page E. 3, until a clean interface is obtained, as described there.



   To the aqueous phase recovered, 0.6 volumes (v / v) of isopropanol is added, a viscous suspension is obtained.



   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.
 EMI26.2
 



  2. Construction of a genomic library of Pseudomonas wisconsinensis T 92. 677/1 From the suspension obtained above, the chromosomal DNA (15 gg) 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 11 of 0.5 M EDTA at pH 8.0 in the presence of ice.



   The fractions, obtained after cleavage, which contain fragments having a size between 20 and 40 kbp, are determined on an agarose gel.



   All the fragments thus obtained are then subjected to precipitation according to the technique described in SAMBROOK et al., 1989, p.



  9.18.



   The DNA fragments are then ligated according to the method described by SAMBROOK et al., 1989, pages 1.68-1. 69, with the plasmid pRG930 (750 ng), previously cut at the BamID site as described by SAMBROOK et al., 1989, pages 1.60-1. 61. We obtain a collection of

  <Desc / Clms Page number 27>

 plasmids which are called pRG930:: WI.



   The process for obtaining the plasmid pRG930 is described in Molecular
Plant-Microbe Interactions, 1992, vol. 5 (3), pages 228-234.



   The ligation thus obtained is used to transfect E. coli HBIOI cells (PROMEGA) using the kit sold under the name of GIGAPACK II PACKAGING EXTRACT KIT (STRATAGEM and following the manufacturer's recommendations for use.



   The transfected cells of E. coli HB101 are cultured on a petri dish containing the agar LB medium, 25 g / ml of streptomycin and 50 μg of spectinomycin, for approximately 24 hours at 37 ° C. This gives a collection of transfected strains which are called E. coli HB101 (pRG930:: WI).



   From 12 colonies of E. coli HBI01 (pRG930:: WI) chosen at random, DNA fragments are isolated according to the technique described in SAMBROOK et al., 1989, page 1.85.



   Restriction analysis of these DNA fragments is carried out (SAMBROOK et al., 1989, page 1.85) after cleavage with the restriction enzymes EcoRI and Pstl.



   This analysis indicates that the size of the inserted fragments present in the plasmids pRG930:: WI is between approximately 20 and 30 kbp (kbp = 1000 base pairs) and that these fragments are different from each other.



  This indicates that the genomic bank which has been constituted is representative.



   1500 colonies of E. coli HB101 (pRG930:: WI) are then 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. These 1500 colonies of E. coli HBIOI (pRG930:: WI) constitute the genomic library.



  3. Obtaining a chromosomal fragment containing the lipase gene from Pseudomonas wisconsinensis T 92.677 / 1
Finally, to establish the nucleotide sequence of a probe capable of screening the genomic library, the 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. .



   To remove the maximum ambiguity in the translation of amino acids to nucleotides, we take into account on the one hand the sequences of

  <Desc / Clms Page number 28>

 nucleotides of several lipases produced by different strains of
Pseudomonas and published in Gilbert J. et al., Enzyme Microbiological
Technology, 1993,15, p. 634-645, and, on the other hand, the property known as the "codon usage" of the strain of Pseudomonas aeruginosa and published in West S. et al., Nucleic Acid research, 1988,16, p. 9323-9335.



   On the basis of these elements, the sequence of a synthetic oligonucleotide of 72 bp is established (bp = base pair). This synthetic oligonucleotide is prepared according to the technique described in BEAUCAGE et al.



   (1981), Tetrahedron Letters, 22, pages 1859-1882 and using ss-cyanoethyl phosphoramidites in a BIOSEARCH apparatus
CYCLONE SYNTHESIZER.



   The sequence of this synthetic oligonucleotide is as follows: SEQIDNO:! ! '-AACTACACCAAGACCAAATACCCCATCGTGCTGGTCCA- - CGGCGTGACCGGCTTCAACACTATCGGCGGGCTC-3'
This synthetic oligonucleotide is labeled at its termination by means of T P 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 bank by the technique known as "colony Mot" (AMERSHAM) using as probe synthetic oligonucleotide as prepared above.



   The 1,500 colonies of the genomic bank (E. coli HBIOI (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 cm2) 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 μg of denatured salmon sperm DNA and fragmented (AMERSHAM).



   500 ml of Denhardt's solution contains 5 g of FICOLL type 400 (PHARMACIA), 5 g of polyvinylpyrrolidone and 5 g of bovine serum albumin.



   The membranes placed in plastic bags are incubated at 68 C

  <Desc / Clms Page number 29>

 in a water bath with stirring (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 pMol.



   The membranes are then recovered. The membranes are then washed with a solution containing 100 ml of 2X SSC (0.3 M NaCl and 0.03 M sodium citrate, pH 7.0) and 0.1% (w / v) of SDS for 5 minutes at room temperature.



   Then, 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 an X-ray autoradiography (AMERSHAM).



   The strain of Pseudomonas wisconsinensis T 92. 677/1 is used as a positive control and the strains of E. coli HB101 and E. coli HB101 (pRG930) are used as a negative control.



   The strain of E. coli HB101 (pRG930) was obtained by the transformation technique described in Molecular Cloning, a laboratory ManualMANIAIS et al., 1982, Cold Spring Harbor Laboratory, pages 150-151, using the strain of E. coli HB101 and the plasmid pRG930.



   There is a clear and strong signal for the wild strain of Pseudomonas wisconsinensis T 92. 677/1 and no signal for the strains of E. coli HB101 and E. coli HB101 (pRG930). The screening of the genomic library highlights 80 colonies giving a signal.



   This is confirmed by a new hybridization test.



   A colony of the genomic bank presenting a strong signal is isolated and cultured on a Petri dish containing the agar LB medium, 25 ILg / ml of streptomycin and 50 ug / ml of spectinomycin, for approximately 24 hours at 37 C. This colony is called E. coli HB101 (pRG930:: WI12).



   A hybridization test is again carried out with the colony of E. coli HB101 (pRG930:: WI12) in order to confirm the results obtained.

  <Desc / Clms Page number 30>

 



   Example 16 Analysis of the plasmid pRG930:: WI12 present in the strain of B. coli
HB101 (pRG930:: WI12) - Analysis using the so-called Southem Blot technique
The DNA is isolated from the strain of E. coli HB101 (pRG930:: WI12), obtained in Example 15, according to the technique described by
SAMBROOK et al., 1989, pages 1.25-1. 28, and restriction analysis is carried out using the restriction enzyme EcoRI. The DNA fragments obtained are separated by agarose gel electrophoresis according to the technique described in SAMBROOK et al., 1989, pages 6.01-6. 19.



   This analysis shows that the DNA fragment inserted into the plasmid pRG930:: WI12 has a size of approximately 27 kbp.



   Then, 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 15. As negative control, the plasmid pRG930 is used.



   A single band is observed giving a hybridization signal with the synthetic oligonucleotide (SEQ ID NO: 11) 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.



  Example 17 Nucleotide sequence of the complete gene which codes for the lipase of Pseudomonas wisconsinensis T 92.677 / 1 1. Obtaining the strain of Pseudomonas wisconsinensis RC13
A restriction analysis of the plasmid pRG930:: WI12 is carried out using the restriction enzyme EcoRI. The DNA fragments obtained are separated by agarose gel electrophoresis.



   An analysis is carried out according to the Southem Blot technique, as described in Example 16.



   This demonstrates that 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, gives a hybridization signal with the synthetic oligonucleotide (SEQ ID NO: the other 4 fragments give no signal.



   The 8.5 kbp fragment attached to the vector pRG930 is then ligated

  <Desc / Clms Page number 31>

 on 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 DH5a (GIBCO) as described in SAMBROOK et al., 1989, page
1.82-1. 84.



   We get a colony of E. coli DH5a (pRG930:: WI13) which is isolated and cultured on a Petri dish containing the agar LB medium, 25 g / ml of streptomycin and 50%, g / ml of spectinomycin, for approximately 24 hours at 37 "VS.



   A partial restriction map of the plasmid pRG930:: WI13 is established using the technique described in Molecular Cloning, a laboratory Manual-MANIATIS et al., 1982, Cold Spring Harbor Laboratory, pages 374-379, and using the enzymes restriction ClaI, EcoRI, NcoI, Sali, HindIn, Bgin, XhoI, BamHI, Smal, Sacl, Sacn, KpnI, SphI, XbaI, EcoRV and Spel.



  2. Identification of the nucleotide sequence of the lipase
A restriction analysis of the plasmid pPRG930:: WI13 is carried out, using the restriction enzyme Pst. The DNA fragments obtained are separated by agarose gel electrophoresis.



   An analysis is carried out according to the Southem Blot technique, as described in Example 16.



   This demonstrates that the fragment inserted into the plasmid pRG930:: WI13 is formed, on the one hand, of 5 fragments and, on the other hand, of a small fragment. This small fragment is approximately 2.5 kbp in size.



   The 2.5 kbp fragment gives a hybridization signal with the synthetic oligonucleotide (SEQ ID NO: 11), the other 5 fragments give no signal.



   The 2.5 kbp fragment is ligated with the vector pPRG930 according to the method described by SAMBROOK et al., 1989, pages 1.68-1. 69. The plasmid pRG930:: WI14 is obtained.



   From the plasmid pRG930:: WI14, the 2.5 kbp fragment is inserted into the plasmid pBLUESCRIPT.



   The plasmid pBLUESCRIPT can be obtained from the company STRATAGENE.



   The plasmid pBLUESCRIPT:: WI14 is obtained.



   The 2.5 kbp fragment sequence inserted into the plasmid

  <Desc / Clms Page number 32>

   pBLUESCRIPT:: WI14 is obtained by implementing the kit
SEQUITHERM CYCLE SEQUENCING KIT (BIOZYM) and following the technique recommended by the manufacturer.



   To complete and verify the nucleotide sequence obtained, the example above is repeated with the following modification. The restriction analysis of the plasmid pPRG930:: WI14 is carried out, using the restriction enzymes SalI, KpnI or SacII successively in place of the restriction enzyme PstI.



   The nucleotide sequence of the Pseudomonas wisconsinensis T 92.677 / 1 lipase is identified. The amino acid sequence and the nucleotide sequence (SEQ ID NO: 2) coding for mature lipase, as well as its translation into amino acids (SEQ ID NO: 3), is given in FIG. 1 (FIGS. 1a and 1b ).



   It is verified that the first amino acids of the lipase sequence, thus identified, correspond to the N-terminal sequence determined in Example 5.



  * Lipase is synthesized in the form of a precursor. The precursor contains 308 amino acids (SEQ ID NO: 7). The nucleotide sequence (SEQ ID NO: 5) coding for the lipase precursor is identified, as well as its translation into amino acids (SEQ ID NO: 6).



   The precursor contains the 286 amino acid sequence (SEQ ID NO: 4) of the mature lipase and the 22 amino acid sequence (SEQ ID NO: 10) of the presequence.



   The mature lipase sequence is preceded by a presequence. It is an additional sequence of 22 amino acids (SEQ ID NO: 10).



  The corresponding nucleotide sequence is identified (SEQ ID NO: 8), as well as its translation into amino acids (SEQ ID NO: 9). This presequence codes for the signal peptide of the lipase of Pseudomonas wisconsinensis T 92.677 / 1.

  <Desc / Clms Page number 33>

 



   SEQUENCE LIST (1) GENERAL INFORMATION: (i) DEPOSITOR: (A) NAME: SOLVAY (Public limited company) (B) STREET: rue du Prince Albert, 33 (C) CITY: Brussels (E) COUNTRY: Belgium (F) POSTAL CODE: 1050 (ii) TITLE OF THE INVENTION: Lipase, microorganism producing it, process for preparing this lipase and uses thereof (iii) NUMBER OF SEQUENCES: 11 (iv) COMPUTER-DEPENDABLE FORM: (A) MEDIA TYPE: Floppy disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patenten Release &num; 1. 0, Version &num; 1. 30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) NUMBER OF STRANDS: (D) CONFIGURATION : linear (ii) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: N-terminal (vi) ORIGIN: (A) ORGANISM:

   Pseudomonas (B) STRAIN: Pseudomonas wisconsinensis (C) INDIVIDUAL / ISOLATED: T 92.677 / 1 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:
Asn Tyr Thr Lys Thr Lys Tyr Pro Ile Val Leu Val His Gly Val Thr
1 5 10 15
Gly Phe Asn Thr Ile Gly Gly Leu
20 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 861 base pairs (B) TYPE: nucleotide

  <Desc / Clms Page number 34>

 (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomics) (v) TYPE OF FRAGMENT: internal (vi) ORIGIN: (A) ORGANISM: Pseudomonas (B) STRAIN: Pseudomonas wisconsinensis (C) INDIVIDUAL / ISOLATED: T 92.677 / 1 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:

   AACTACACCA AGACCAAGTA TCCGATCGTG CTGGTACACG GCGTGACCGG GTTCAATACC 60 ATCGGCGGGC TGGTCAATTA CTTCCATACC ATTCCCTGGA ACCTAGAGCG CGATGGCGCC 120 CGGGTGCACG TCGCCAGTGT CGCTGCCTTC AATGACAGCG AGCAGCGCGG CGCCGAGCTG 180 GCCCGGCAGA TCGTGCCCTG GGCCGCAGGC GGAGGCGGCA AGGTCAACCT GATCGGCCAC 240 AGTCAGGGCT CGCCGACCTC GCGCGTGGCG GCTTCGTTGC GGCCGGATCT GGTGGCATCG 300 GTGACCTCGA TCAACGGCGT CAACAAGGGC TCCAAGGTCG CCGATGTGGT GCGCGGCGTG 360 CTGCCACCGG GTAGCGGTAT CGAAGGCGGC GCCAATGCCA TCGCCAACGC CCTCGGTGCG 420 GTGATCAATC TGCTGTCTGG CTCAAGCAAC CCGCAAAACG GTATCAACGC GCTAGGCACC 480 CTGACCACCG CGGGCACCAG TGCGCTGAAC AGTCGCCACC CGTGGGGCGT CAACACCAGC 540 AGCTACTGCG CCAAGTCCAC CGAAGTGCAC AATGTGCGCG GTCACAGCAT CCGCTACTAC 600 TCCTGGACCG GTAATGCCGC CTATACCAAC GTGCTCGATG CGGCCGATCC CTTCCTGGCC 660 TTCACCGGCC TGGTGTTCGG CAGCGAGAAG AACGACGGTC TGGTGGGCGT ATGTTCCACC 720 TATCTGGGGC AGGTGATCGA CGACAGCTAC AACATGAACC ACGTCGATGC

  GATCAACCAC 780 CTGTTCGGCA INCL. OF STRANDS: simple (D) CONFIGURATION: linear (il) TYPE OF MOLECULE: DNA (genomics)

  <Desc / Clms Page number 35>

 (ix) CHARACTERISTIC: (A) NAME / KEY: CDS (B) LOCATION: 1 .. 858 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:

   AAC TAC ACC AAG ACC AAG TAT CCG ATC GTG CTG GTA CAC GGC GTG ACC 48 Asn Tyr Thr Lys Thr Lys Tyr Pro Ile Val Leu Val His Gly Val Thr
1 5 10 15 GGG TTC AAT ACC ATC GGC GGG CTG GTC AAT TAC TTC CAT ACC ATT CCC 96 Gly Phe Asn Thr Ile Gly Gly Leu Val Asn Tyr Phe His Thr Ile Pro
20 25 30 TGG AAC CTA GAG CGC GAT GGC GCC CGG GTG CAC GTC GCC AGT GTC GCT 144 Trp Asn Leu Glu Arg Asp Gly Ala Arg Val His Val Ala Ser Val Ala
35 40 45 GCC TTC AAT GAC AGC GAG CAG CGC GGC GCC GAG CTG GCC CGG CAG ATC 192 Ala Phe Asn Asp Ser Glu Gln Arg Gly Ala Glu Leu Ala Arg Gln Ile
50 55 60 GTG CCC TGG GCC GCA GGC GGA GGC GGC AAG GTC AAC CTG ATC GGC CAC 240 Val Pro Trp Ala Ala Gly Gly Gly Gly Lys Val Asn Leu Ile Gly His
65 70 75 80 AGT CAG GGC TCG CCG ACC TCG CGC GTG GCG GCT TCG TTG CGG CCG GAT 288 Ser Gln Gly Ser Pro Thr Ser Arg Val Ala Ala Ser Leu Arg Pro Asp
85 90 95 CTG GTG GCA TCG GTG ACC TCG ATC AAC GGC GTC

  AAC AAG GGC TCC AAG 336 Leu Val Ala Ser Val Thr Ser Ile Asn Gly Val Asn Lys Gly Ser Lys
100 105 110 GTC GCC GAT GTG GTG CGC GGC GTG CTG CCA CCG GGT AGC GGT ATC GAA 384 Val Ala Asp Val Val Arg Gly Val Leu Pro Pro Gly Ser Gly Ile Glu
115 120 125 GGC GGC GCC AAT GCC ATC GCC AAC GCC CTC GGT GCG GTG ATC AAT CTG 432 Gly Gly Ala Asn Ala Ile Ala Asn Ala Leu Gly Ala Val Ile Asn Leu
130 135 140 CTG TCT GGC TCA AGC AAC CCG CAA AAC GGT ATC AAC GCG CTA GGC ACC 480 Leu Ser Gly Ser Ser Asn Pro Gln Asn Gly Ile Asn Ala Leu Gly Thr 145 150 155 160 CTG ACC ACC GCG GGC ACC AGT GCG CTG AAC AGT CGC CAC CCG TGG GGC 528 Leu Thr Thr Ala Gly Thr Ser Ala Leu Asn Ser Arg His Pro Trp Gly
165 170 175 GTC AAC ACC AGC AGC TAC TGC GCC AAG TCC ACC GAA GTG CAC AAT GTG 576 Val Asn Thr Ser Ser Tyr Cys Ala Lys Ser Thr Glu Val His Asn Val
180 185 190

  <Desc / Clms Page number 36>

 
CGC GGT CAC AGC ATC CGC TAC TAC TCC

  TGG ACC GGT AAT GCC GCC TAT 624 Arg Gly His Ser Ile Arg Tyr Tyr Ser Trp Thr Gly Asn Ala Ala Tyr
195 200 205 ACC AAC GTG CTC GAT GCG GCC GAT CCC TTC CTG GCC TTC ACC GGC CTG 672 Thr Asn Val Leu Asp Ala Ala Asp Pro Phe Leu Ala Phe Thr Gly Leu
210 215 220 GTG TTC GGC AGC GAG AAG AAC GAC GGT CTG GTG GGC GTA TGT TCC ACC 720 Val Phe Gly Ser Glu Lys Asn Asp Gly Leu Val Gly Val Cys Ser Thr 225 230 235 240 TAT CTG GGG CAG GTG ATC GAC GAC AGC TAC AAC ATG AAC CAC GTC GAT 768 Tyr Leu Gly Gln Val Ile Asp Asp Ser Tyr Asn Met Asn His Val Asp
245 250 255 GCG ATC AAC CAC CTG TTC GGC ATT CGT GGC TGG ACC GAA CCG GTG TCG 816 Ala Ile Asn His Leu Phe Gly Ile Arg Gly Trp Thr Glu Pro Val Ser
260 265 270 CTG TAT CGC CAG CAC GCC AAC CGC CTG AAG AAC AAG GGC GTC TGA 861 Leu Tyr Arg Gln His Ala Asn Arg Leu Lys Asn Lys Gly Val
275 280 285 (2) INFORMATION FOR SEQ ID NO: 4:

   (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 286 amino acids (B) TYPE: amino acid (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4 : Asn Tyr Thr Lys Thr Lys Tyr Pro Ile Val Leu Val His Gly Val Thr
1 5 10 15 Gly Phe Asn Thr Ile Gly Gly Leu Val Asn Tyr Phe His Thr Ile Pro
20 25 30 Trp Asn Leu Glu Arg Asp Gly Ala Arg Val His Val Ala Ser Val Ala
35 40 45 Ala Phe Asn Asp Ser Glu Gln Arg Gly Ala Glu Leu Ala Arg Gln Ile
50 55 60 Val Pro Trp Ala Ala Gly Gly Gly Gly Lys Val Asn Leu Ile Gly His
65 70 75 80 Ser Gln Gly Ser Pro Thr Ser Arg Val Ala Ala Ser Leu Arg Pro Asp
85 90 95

  <Desc / Clms Page number 37>

 
Leu Val Ala Ser Val Thr Ser Ile Asn Gly Val Asn Lys Gly Ser Lys
100 105 110
Val Ala Asp Val Val Arg Gly Val Leu Pro Pro Gly Ser Gly Ile Glu
115 120 125
Gly Gly Ala Asn Ala Ile Ala Asn Ala Leu Gly Ala Val Ile

  Asn leu
130 135 140
Leu Ser Gly Ser Ser Asn Pro Gln Asn Gly Ile Asn Ala Leu Gly Thr
145 150 155 160
Leu Thr Thr Ala Gly Thr Ser Ala Leu Asn Ser Arg His Pro Trp Gly
165 170 175 Val Asn Thr Ser Ser Tyr Cys Ala Lys Ser Thr Glu Val His Asn Val
180 185 190 Arg Gly His Ser Ile Arg Tyr Tyr Ser Trp Thr Gly Asn Ala Ala Tyr
195 200 205 Thr Asn Val Leu Asp Ala Ala Asp Pro Phe Leu Ala Phe Thr Gly Leu
210 215 220 Val Phe Gly Ser Glu Lys Asn Asp Gly Leu Val Gly Val Cys Ser Thr 225 230 235 240 Tyr Leu Gly Gln Val Ile Asp Asp Ser Tyr Asn Met Asn His Val Asp
245 250 255 Ala Ile Asn His Leu Phe Gly Ile Arg Gly Trp Thr Glu Pro Val Ser
260 265 270 Leu Tyr Arg Gln His Ala Asn Arg Leu Lys Asn Lys Gly Val
275 280 285 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE:

   (A) LENGTH: 927 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomics) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: ATGCGTCGCG TCTATACCGC TGCCCTGGCA ACACTCGCTC TGCTTGGCGC CGTCGAGGCC 60 CAGGCCAACT ACACCAAGAC CAAGTATCCG ATCGTGCTGG TACACGGCGT GACCGGGTTC 120 AATACCATCG GCGGGCTGGAT CATCACTTC

  <Desc / Clms Page number 38>

 
GGCGCCCGGG TGCACGTCGC CAGTGTCGCT GCCTTCAATG ACAGCGAGCA GCGCGGCGCC 240
GAGCTGGCCC GGCAGATCGT GCCCTGGGCC GCAGGCGGAG GCGGCAAGGT CAACCTGATC 300
GGCCACAGTC AGGGCTCGCC GACCTCGCGC GTGGCGGCTT CGTTGCGGCC GGATCTGGTG 360
GCATCGGTGA CCTCGATCAA CGGCGTCAAC AAGGGCTCCA AGGTCGCCGA TGTGGTGCGC 420
GGCGTGCTGC CACCGGGTAG CGGTATCGAA GGCGGCGCCA ATGCCATCGC CAACGCCCTC 480
GGTGCGGTGA TCAATCTGCT GTCTGGCTCA AGCAACCCGC

  AAAACGGTAT CAACGCGCTA 540
GGCACCCTGA CCACCGCGGG CACCAGTGCG CTGAACAGTC GCCACCCGTG GGGCGTCAAC 600
ACCAGCAGCT ACTGCGCCAA GTCCACCGAA GTGCACAATG TGCGCGGTCA CAGCATCCGC 660
TACTACTCCT GGACCGGTAA TGCCGCCTAT ACCAACGTGC TCGATGCGGC CGATCCCTTC 720
CTGGCCTTCA CCGGCCTGGT GTTCGGCAGC GAGAAGAACG ACGGTCTGGT GGGCGTATGT 780 TCCACCTATC TGGGGCAGGT GATCGACGAC AGCTACAACA TGAACCACGT CGATGCGATC 840 AACCACCTGT TCGGCATTCG TGGCTGGACC GAACCGGTGT CGCTGTATCG CCAGCACGCC 900 AACCGCCTGA AGAACAAGGG CGTCTGA 927 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 927 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomics) (ix) CHARACTERISTICS: (A) NAME / KEY: sig-peptide (B) LOCATION: 1 .. 66 (ix) CHARACTERISTIC:

   (A) NAME / KEY: mat-peptide (B) LOCATION: 67 .. 924 (ix) CHARACTERISTIC: (A) NAME / KEY: CDS (B) LOCATION: 1 .. 924 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: ATG CGT CGC GTC TAT ACC GCT GCC CTG GCA ACA CTC GCT CTG CTT GGC 48 Met Arg Arg Val Tyr Thr Ala Ala Leu Ala Thr Leu Ala Leu Leu Gly - 22-20-15-10

  <Desc / Clms Page number 39>

 GCC GTC GAG GCC CAG GCC AAC TAC ACC AAG ACC AAG TAT CCG ATC GTG 96 Ala Val Glu Ala Gln Ala Asn Tyr Thr Lys Thr Lys Tyr Pro Ile Val - 5 1 5 10 CTG GTA CAC GGC GTG ACC GGG TTC AAT ACC ATC GGC GGG CTG GTC AAT 144 Leu Val His Gly Val Thr Gly Phe Asn Thr Ile Gly Gly Leu Val Asn
15 20 25
 EMI39.1
 TAC TTC CAT ACC ATT CCC TGG AAC CTA GAG CGC GAT GGC GCC CGG GTG 192 Tyr Phe His Thr Ile Pro Trp Asn Leu Glu Arg Asp Gly Ala Arg Val
30 35 40 CAC GTC GCC AGT GTC GCT GCC TTC AAT GAC AGC GAG CAG CGC GGC GCC

  240 His Val Ala Ser Val Ala Ala Phe Asn Asp Ser Glu Gln Arg Gly Ala
45 50 55 GAG CTG GCC CGG CAG ATC GTG CCC TGG GCC GCA GGC GGA GGC GGC AAG 288 Glu Leu Ala Arg Gln Ile Val Pro Trp Ala Ala Gly Gly Gly Gly Lys
60 65 70 GTC AAC CTG ATC GGC CAC AGT CAG GGC TCG CCG ACC TCG CGC GTG GCG 336 Val Asn Leu Ile Gly His Ser Gln Gly Ser Pro Thr Ser Arg Val Ala
75 80 85 90 GCT TCG TTG CGG CCG GAT CTG GTG GCA TCG GTG ACC TCG ATC AAC GGC 384 Ala Ser Leu Arg Pro Asp Leu Val Ala Ser Val Thr Ser Ile Asn Gly
95 100 105 GTC AAC AAG GGC TCC AAG GTC GCC GAT GTG GTG CGC GGC GTG CTG CCA 432 Val Asn Lys Gly Ser Lys Val Ala Asp Val Val Arg Gly Val Leu Pro
110 115 120 CCG GGT AGC GGT ATC GAA GGC GGC GCC AAT GCC ATC GCC AAC GCC CTC 480 Pro Gly Ser Gly Ile Glu Gly Gly Ala Asn Ala Ile Ala Asn Ala Leu
125 130 135 GGT GCG GTG ATC AAT CTG CTG TCT GGC TCA AGC AAC CCG CAA AAC GGT 528 Gly Ala Val

  Ile Asn Leu Leu Ser Gly Ser Ser Asn Pro Gln Asn Gly
140 145 150 ATC AAC GCG CTA GGC ACC CTG ACC ACC GCG GGC ACC AGT GCG CTG AAC 576 Ile Asn Ala Leu Gly Thr Leu Thr Thr Ala Gly Thr Ser Ala Leu Asn 155 160 165 170 AGT CGC CAC CCG TGG GGC GTC AAC ACC AGC AGC TAC TGC GCC AAG TCC 624 Ser Arg His Pro Trp Gly Val Asn Thr Ser Ser Tyr Cys Ala Lys Ser
175 180 185 ACC GAA GTG CAC AAT GTG CGC GGT CAC AGC ATC CGC TAC TAC TCC TGG 672 Thr Glu Val His Asn Val Arg Gly His Ser Ile Arg Tyr Tyr Ser Trp
190 195 200

  <Desc / Clms Page number 40>

 
ACC GGT AAT GCC GCC TAT ACC AAC GTG CTC GAT GCG GCC GAT CCC TTC 720
Thr Gly Asn Ala Ala Tyr Thr Asn Val Leu Asp Ala Ala Asp Pro Phe
205 210 215
CTG GCC TTC ACC GGC CTG GTG TTC GGC AGC GAG AAG AAC GAC GGT CTG 768
Leu Ala Phe Thr Gly Leu Val Phe Gly Ser Glu Lys Asn Asp Gly Leu
220 225 230
GTG GGC GTA TGT TCC ACC TAT CTG GGG CAG GTG ATC GAC GAC AGC TAC 816
Val

  Gly Val Cys Ser Thr Tyr Leu Gly Gln Val Ile Asp Asp Ser Tyr
235 240 245 250 AAC ATG AAC CAC GTC GAT GCG ATC AAC CAC CTG TTC GGC ATT CGT GGC 864 Asn Met Asn His Val Asp Ala Ile Asn His Leu Phe Gly Ile Arg Gly
255 260 265 TGG ACC GAA CCG GTG TCG CTG TAT CGC CAG CAC GCC AAC CGC CTG AAG 912 Trp Thr Glu Pro Val Ser Leu Tyr Arg Gln His Ala Asn Arg Leu Lys
270 275 280 AAC AAG GGC GTC TGA 927 Asn Lys Gly Val
285 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 308 amino acids (B) TYPE: amino acid (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:

   Met Arg Arg Val Tyr Thr Ala Ala Leu Ala Thr Leu Ala Leu Leu Gly - 22-20-15-10 Ala Val Glu Ala Gln Ala Asn Tyr Thr Lys Thr Lys Tyr Pro Ile Val - 5 1 5 10 Leu Val His Gly Val Thr Gly Phe Asn Thr Ile Gly Gly Leu Val Asn
15 20 25 Tyr Phe His Thr Ile Pro Trp Asn Leu Glu Arg Asp Gly Ala Arg Val
30 35 40 His Val Ala Ser Val Ala Ala Phe Asn Asp Ser Glu Gln Arg Gly Ala
45 50 55 Glu Leu Ala Arg Gln Ile Val Pro Trp Ala Ala Gly Gly Gly Gly Lys
60 65 70

  <Desc / Clms Page number 41>

 
Val Asn Leu Ile Gly His Ser Gln Gly Ser Pro Thr Ser Arg Val Ala
75 80 85 90 Ala Ser Leu Arg Pro Asp Leu Val Ala Ser Val Thr Ser Ile Asn Gly
95 100 105 Val Asn Lys Gly Ser Lys Val Ala Asp Val Val Arg Gly Val Leu Pro
110 115 120
Pro Gly Ser Gly Ile Glu Gly Gly Ala Asn Ala Ile Ala Asn Ala Leu
125 130 135 Gly Ala Val Ile Asn Leu Leu Ser Gly Ser Ser Asn Pro Gln Asn Gly
140

  145 150 Ile Asn Ala Leu Gly Thr Leu Thr Thr Ala Gly Thr Ser Ala Leu Asn 155 160 165 170 Ser Arg His Pro Trp Gly Val Asn Thr Ser Ser Tyr Cys Ala Lys Ser
175 180 185 Thr Glu Val His Asn Val Arg Gly His Ser Ile Arg Tyr Tyr Ser Trp
190 195 200 Thr Gly Asn Ala Ala Tyr Thr Asn Val Leu Asp Ala Ala Asp Pro Phe
205 210 215 Leu Ala Phe Thr Gly Leu Val Phe Gly Ser Glu Lys Asn Asp Gly Leu
220 225 230 Val Gly Val Cys Ser Thr Tyr Leu Gly Gln Val Ile Asp Asp Ser Tyr 235 240 245 250 Asn Met Asn His Val Asp Ala Ile Asn His Leu Phe Gly Ile Arg Gly
255 260 265 Trp Thr Glu Pro Val Ser Leu Tyr Arg Gln His Ala Asn Arg Leu Lys
270 275 280 Asn Lys Gly Val
285 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 66 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE:

   DNA (genomics) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:

  <Desc / Clms Page number 42>

 
ATGCGTCGCG TCTATACCGC TGCCCTGGCA ACACTCGCTC TGCTTGGCGC CGTCGAGGCC 60
CAGGCC 66 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 66 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomics) (ix) CHARACTERISTIC: (A) NAME / KEY: CDS (B) LOCATION: 1 .. 66 (ix) CHARACTERISTIC: (A) NAME / KEY: sig-peptide ( B) LOCATION: 1 .. 66 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9:

   ATG CGT CGC GTC TAT ACC GCT GCC CTG GCA ACA CTC GCT CTG CTT GGC 48 Met Arg Arg Val Tyr Thr Ala Ala Leu Ala Thr Leu Ala Leu Leu Gly
1 5 10 15 GCC GTC GAG GCC CAG GCC 66 Ala Val Glu Ala Gln Ala
20 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: Met Arg Arg Val Tyr Thr Ala Ala Leu Ala Thr Leu Ala Leu Leu Gly
1 5 10 15 Ala Val Glu Ala Gln Ala
20 (2) INFORMATION FOR SEQ ID NO: 11:

  <Desc / Clms Page number 43>

 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 72 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE:

   Other nucleic acid (A) DESCRIPTION: / desc = "synthetic oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: AACTACACCA AGACCAAATA CCCCATCGTG CTGGTCCACG GCGTGACCGG CTTCAACACT 60 ATCGGCGGGC TC 72


      

Claims (1)

 CLAIMS 1-Lipase, characterized in that it comes from a strain of Pseudomonas wisconsinensis or a derivative or mutant of this strain capable of producing this lipase.    2 - Lipase, characterized in that it comes from the strain of Pseudomonas wisconsinensis T 92.677 / 1 (LMG P-15151) or a derivative or mutant of this strain capable of producing this lipase.    3-Lipase, characterized in that its N-terminal amino acid sequence (SEQ ID NO: 1) is as follows: Asn Tyr Thr Lys Thr Lys Tyr Pro Ile Val Leu Val His 1 5 10 Gly Val Thr Gly Phe Asn Thr Ile Gly Gly Leu 15 20 4-Lipase, isolated and purified, characterized in that it comprises the amino acid sequence of 1 to 286 amino acids (SEQ ID NO: 4) or a modified sequence derived therefrom.  S-Lipase, isolated and purified, characterized in that the sequence of mature lipase is preceded by a pre-sequence of 22 amino acids (SEQ ID NO: 10) which codes for the signal peptide for lipase.    6-Lipase, isolated and purified, characterized in that it has a molecular weight of approximately 30 kDa.  EMI44.1   7-Lipase, isolated and purified, characterized in that it has an estimated isoelectric point of between approximately 9.8 and approximately 10.1.    8-Lipase, characterized in that it comes from an aerobic bacterium capable of producing lipase in an appropriate nutritive medium containing sources of carbon and nitrogen and mineral salts under the condition of aerobiosis.    9-Lipase, characterized in that it develops an optimal enzymatic activity, measured at a pH of 9.5, in a temperature range above about 40 C.  <Desc / Clms Page number 45>      10-Lipase, characterized in that it develops an optimal enzymatic activity, measured at a pH of 9.5, in a range of  EMI45.1  temperature below about 60 C. 11-Lipase, characterized in that it develops an optimal enzymatic activity, measured at a pH of 9.5, in a temperature range between approximately 40 C and approximately 60 C. 12-Lipase, characterized in that it develops an optimal enzymatic activity, measured at a pH of 9.5, at a temperature of around 55 C.    13-Lipase, characterized in that it develops an enzymatic activity of more than 50% of the maximum enzymatic activity in a temperature range between approximately 40 C and approximately 60 C for a pH of approximately 9.5, l the maximum enzymatic activity being measured at a temperature of 55 ° C. and at a pH of 9.5.    14-Lipase, characterized in that it develops an optimal enzymatic activity, measured at a temperature of approximately 30 C, in a range of pH greater than or equal to approximately 8.  EMI45.2   15-Lipase, characterized in that it develops an optimal enzymatic activity, measured at a temperature of approximately 30 C, in a pH range less than or equal to approximately 10. 16-Lipase, characterized in that it develops an optimal enzymatic activity, measured at a temperature of approximately 30 C, in a pH range between approximately 8 and approximately 10.    17-Lipase, characterized in that it develops an enzymatic activity of more than 85% of the maximum enzymatic activity in a range of pH between approximately 8 and approximately 10 for a temperature  EMI45.3  about 30 oC, the maximum enzymatic activity being measured at a temperature of 30 C and a pH of 9.5. 18-Lipase, characterized in that it shows a relative enzymatic activity of at least 55% measured after an incubation of 160 minutes at a temperature of 55 C and at a pH of 10 in a buffered solution of  <Desc / Clms Page number 46>  hardness 15.    19-An isolated and purified culture of Pseudomonas wisconsinensis and culture derived or mutated therefrom.    20-An isolated and purified culture of Pseudomonas wisconsinensis T 92.677 / 1 (LMG P-15151) and culture derived or mutated therefrom.  21-DNA molecule comprising the nucleotide sequence (SEQ ID NO: 2) which codes for the mature lipase of Pseudomonas wisconsinensis T 92.677 / 1 (LMG P-15151) or a modified sequence derived therefrom.  22-DNA molecule according to claim 21, characterized in that it comprises the nucleotide sequence (SEQ ID NO: 5) which codes for the lipase precursor of Pseudomonas wisconsinensis T 92.677 / 1 or a modified sequence derived from this one.    23-A method for the production of a lipase according to any one of claims 1 to 18, characterized in that it comprises the culture of a bacterium capable of producing the lipase in an appropriate nutritive medium containing carbon sources and nitrogen and mineral salts and the harvest of the lipase thus obtained.    24-A method according to claim 23, characterized in that the aerobic bacteria is a strain of Pseudomonas.  25-A method according to claim 24, characterized in that the aerobic bacterium is the strain of Pseudomonas wisconsinensis T 92.677 / 1 (LMG P-15151) and a derivative or mutant of this strain capable of producing lipase.    26-An enzymatic composition containing the lipase according to any one of claims 1 to 18 and at least one additive.  27-The enzymatic composition according to claim 26, characterized in that it is a detergency composition.  28- Use of the lipase according to any one of claims 1 to 18 in detergency.