WO1996012012A1 - Lipase, microorganism producing same, method for preparing said lipase and uses thereof - Google Patents

Abstract

A lipase from a Pseudomonas strain is disclosed. The lipase is active in a broad alkaline pH range. Novel microorganism strains producing the lipase, and methods for preparing said lipase, are also disclosed. In addition, the uses of the lipase and compositions containing same are disclosed.

Description

 Lipase. microorganism producing it, process for the preparation of this material and utiilliissaattiinonnss d dee c ceellllee - ccii

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 the 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 to form more water-soluble products, 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 strains of Pseudomonas, for example the lipase produced by the strain of Pseudomonas stutzeri (British patent application 1372034), the lipase produced by the strain of Pseudo¬ monas 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. 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 deter¬ gente 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 comes from an aerobic bacterium capable of producing lipase in an appropriate nutritive medium containing sources of carbon and nitrogen and mineral salts under aerobic condition. biosis.

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 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 range of temperatures. The isolated and purified lipase of the invention develops an optimal enzymatic activity, measured at a pH of 9.5, in a temperature range greater than 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 below 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 about 40 ° C and about 60 ° C. Particularly preferably, the lipase of the invention develops an optimal enzymatic activity, measured at a pH of approximately 9.5, at a temperature of approximately 55 ° C.

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, the maximum enzymatic activity being measured at a temperature of 55 ° C. and at 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., - 4 -

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 at 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 approximately 10. In a particularly preferred manner, the lipase of the invention develops an optimal enzymatic activity, measured at a temperature of about 30 ° C, in a pH range between about 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 ° C., the maximum enzymatic activity 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 solution of hardness 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 DNA mutagenesis techniques, such as exposure to ultraviolet radiation, to chemicals, such as ethyl methane sulfonate (EMS), N-methyl-N -nitro-N-nitrosoguanidine (MNNG), sodium nitrite or O-methylhydroxy lamine or by genetic engineering techniques, such as, for example, site-directed mutagenesis or random mutagenesis. These techniques are known to those skilled in the art and are described in particular in MOLECULAR CLONING - a laboratory manual - SAMBROOK, FRITSCH, MANIAΗS - second edition, 1989, chapter 15. The invention also relates to a mutated lipase obtained by modification of the nucleotide sequence of the gene that codes for lipase. The techniques for obtaining such mutated lipases are known to a person skilled in the art and are in particular described in MOLECULAR CLONING - a laboratory manual - SAMBROOK, FRITSCH, MANIAΗS - 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 NH, 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 taken 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 6 -

Pseudomonadaceae. Preferably it belongs to the genus Pseudomonas. In a particularly preferred manner, it is a strain of Pseudo¬ monas 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 MICROORGANISMS (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 - 7 -

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, FRITSCH, MANIAΗS - second edition, 1989, chapter 15. Usually, the modified sequence derived from the DNA molecule comprises at least 70% homology with the nucleotide sequence (SEQ ID NO: 2) of the gene which codes for the lipase of the invention, that is to 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 wiscon¬ sinensis 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. 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. Usually, centrifugation, filtration, ultrafiltration, evaporation, microfiltration, crystallization or a combination of one or the other of these techniques such as centrifugation followed by ultrafiltration are used. 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 stabilizers of the enzyme, 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 as glucose and sorbitol, a salt such as sodium chloride, calcium chloride, potassium sorbate and sodium benzoate or a mixture of two or many of these products. Good results have been obtained with 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 is part of detergency compositions. The present invention therefore also relates to detergent compositions containing lipase. The components of the detergent 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 a 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. The present invention is illustrated by the following examples. FIG. 1 (FIGS. 1 a and 1 b) 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). Figure 2 (Figures 2a and 2b) shows 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 Pseudomonas wisconsinensis T 92.677 / 1 strain 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 / 1 of NaCl. This suspension is diluted 10 times with demineralized water containing 9 g / 1 of NaCl.

1 ml of the diluted soil suspension is spread on an agar A nutrient medium

Medium A contains 10 g / 1 of tryptone (Difco), 5 g / 1 of yeast extract, 5 g / 1 of NaCl, 20 g / 1 of agar, 2.5 g / 1 of NaHCO ₃ , 7 , 5 g / 1 of Na ₂ CO ₃ , 10 g / 1 of olive oil, 1 g / 1 of polyvinyl alcohol (25/140) and

0.01 g / 1 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 distilled water. The agar suspension obtained is sterilized at 121 ° C for 30 minutes.

1 1 of carbonate buffer (pH 9.5), containing 25 g / 1 of NaHCO _β and 75 g / 1 of Na2Cθ3, is prepared, then sterilized at 121 ° C for 30 minutes. Then, 1 ml of an aqueous solution of [0.01% (w / v)] rhoda¬ mine B (Sigma 6626) is prepared. This solution is sterilized by fύtration on a - 11

0.45 μ sterilization membrane (MILLIPORE).

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 ,pase are cultured on an agar B nutrient medium.

Medium B contains 10 g / 1 of tryptone (Difco), 5 g / 1 of yeast extract, 5 g / 1 of NaCl, 20 g / 1 of agar, 2.5 g / 1 of NaHCO ₃ , 7 , 5 g / 1 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 / 1 of NaHCO ₃ and 75 g / 1 of Na ₂ CO 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 μm x 1.5-4.0 μm. 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-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 that it develops aerobically.

It does not develop anaerobically, i.e. under an atmosphere of 84% (v / v) N ₂ , 8% (v / v) CO ₂ , 8% (v / v) H ₂ to 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 tage expressed in weight by weight.

This strain is not thermophilic. It exhibits 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 appears clearly on reading Table 1 gathering the main biochemical characteristics of these 5 strains.

Table 1

Characteristics Pseudomonas

wisconsinensis stutzeri mendo¬ alcali- pseudoalca- T 92.677 / 1 cina genes ligenes

Size μm x 0.5-0.7 0.7-0.8 0.7-0.8 0.5 0.7-0.8 μm 1.5-4.0 1.4-2.8 1 , 4-2.8 2.0-3.0 1, 2-2.5

Yellow pigment + - + d -

hydrolysis - - -

⁺ starch ^'

Arginine - - + 4- d dehydrolase

Use of + + + - - Glucose

Use of + d + - d Gluconate

Use of + - + - - Géraniol

Use of + - + d d L-histidine

Use of + - + + + L-arginine

Use of + - -1- ~ + Betaine

+ = 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 / 12012 -.- „„ -.

PCT / BE95 / 00094

- 14

The isolated bacterium therefore belongs to the genus Pseudomonas, no known species could be determined.

The strain of Pseudomonas wisconsinensis T 92.677 / 1 has been deposited in the collection named BELGIAN COORDINATED COLLECTIONS OF MICRORGANISMS (LMG culture collection) under number

LMG P-15151 October 12, 1994.

Example 2

Lipase production by the Pseudomonas wisconsinensis strain

T 92.677 / 1 The strain of Pseudomonas wisconsinensis T 92.677 / 1 is cultured on a Petri dish containing the medium B agar 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 / 1 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

0.1N NaOH, 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 K ₂ HPO ₄ 2.5 g / 1, KH ₂ PO ₄ 2.5 g / 1, MgSO _4. 7H ₂ 0

1 g / 1, (NH ₄ ) ₂ SO ₄ 2 g / 1, (NH ₂ ) 2CO 2 g / 1, CaCl ₂ 1 g / 1, soy flour

20 g / 1, yeast extract 2 g / 1, glucose 20 g / 1, Mazuôl antifoam (Mazes

Chemicals) 5 g / 1. 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 23 ° C., under a pressure of 0.15 × 10 ⁵ 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 stirring of 200 revolutions per minute, the regulation of dissolved oxygen being fixed at 10% (v / v) by regulation of the stirring speed. After 24 hours of fermentation, the enzyme activity of the culture thus obtained is measured by the following technique. - 15

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 device, which device maintains the pH constant at a set value by the addition of 0.01 N NaOH. A lipase unit (LU) is defined as the amount 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 CaCl ₂ .2H ₂ 0 and 0.61 g / 1 of Tris (2-amino-2-hydroxymethyl-1,3 -propane- diol).

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 rod, 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 0.1N NaOH. 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 0.01N NaOH. 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 μl).

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 μl).

A lipase unit (LU) is determined as follows: 1 LU / ml = (V1-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 - 16

Example 2, at a pH of 8 with 10N concentrated caustic soda. Then, 1% (v / v) of Triton X-114 (SERVA 37214) is added to this culture. The mixture is gently stirred 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 gently stirred 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 stored. The centrifuge 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. Then, the mixture is centrifuged for 15 minutes at 9000 rpm

(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. Then the suspension, containing the precipitate, is centrifuged for 15 minutes at

9000 revolutions per minute (Beckman J21, rotor JA10) 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. . By following the directions for use prescribed by the supplier

(Pharmacia) about the hydro- interaction chromatography column - 17

phobe, a Pharmacia 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 diafiltered in 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 column

Pharmacia reference 17-1047-01). The separation is initiated by a flow of

0.5 ml per minute of a buffer (pH 7) containing 25 mM CaCl and 20 mM Tris.

Three absorption peaks at 280 nm are separated. Lipase corresponds to the first peak of absorption at 280 nm. The corresponding fraction which contains the purified lipase is kept. Example 5 Determination of the N-terminus 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 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 # 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. It 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 1b) represents 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:) of the lipase (Example 6), is summarized in Table 2. ΓABLE 2

Symbol Amino acids Number% mole (in molecular weight)

A alanine 28 9,790

B aspartic acid 0 0

C cysteine 2 0.699

D aspartic acid 10 3,497

E glutamic acid 7 2,448

F phenylalanine 7 2,448

G glycine 35 12,238

H histidine 10 3,497

I isoleucine 14 4,895

K lysine 9 3 147

L leucine 22 7i 692

M methionine 1 0.350

N asparagine 25 8.741

P proline 11 3,846

Q glutamine 6 2,098

R arginine 13 4,545

S serine 24 8.392

T threonine 18 6,294

V valine 29 10,140 w tryptophan 5 1,748

Unknown X 0 0

Y tyrosine 10 3,497 z glutamine 0 0 glutamic acid

Example 8

Molecular weight estimate

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

Pharmacia, user dossier no. 200) of the gel reveals a polypepptide 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.

An isoelectric point is deduced from 9.95 for the mature form and from 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. Table 3

Temperature Relative activity ° C%

18 19 24 26

30 41 34 45

40 56 45 52

49 91 55 100

60 54 76 43

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 a relative enzymatic activity of 100%. 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 approximately 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 Example 4.

The results are collated in Table 4.

Table 4

1 PH Relative activity

%

7.0 17

8.0 100

9.0 100

9.5 100

10.0 91

10.5 71

11.072

12.0 47

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 a relative enzymatic activity of 100%. The lipase of the invention develops an enzymatic activity of more than approximately 90% of the maximum enzymatic activity in a pH range of 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 between approximately 8 and approximately 11 for a temperature of approximately 30 ° C. Example 13

Stability of the lipase with respect to temperature 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. (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. The results are collated in Table 5.

Table 5

Incubation time Relative activity minutes%

0 100

10 84

20 81

30 79

40 93

50 86

60 80

80 72

100 59

120 59

130 60

140 61

160 61

260 39

1140 25

The deactivation constant (k) over the 0-260 minute interval is 0.000333 min-1. [We obtain the deactivation constant k according to the definition In (AJ A) = -kt, t being the incubation time, AQ the activity relating to the incubation time 0 and A _j the activity relating to the time incubation t.] During this test, the maximum enzymatic activity was measured for the sample at time 0. By definition, this sample was therefore assigned a relative enzymatic activity of 100%.

From this example it is concluded that the 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 solution of hardness 15 It shows a relative enzymatic activity of at least 70% measured after an incubation of 80 minutes 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. No. F 1000) and bacon fat (Laru Gmbh) is prepared by adding 0.1% (w / w) of Sudan red with bacon fat. The mixture is heated to 90 ° C. until the Sudan red is completely dissolved. A roll of fabric is immersed in the solution of Sudan red and bacon fat kept 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 by 10 cm pieces. Then the pieces of impregnated fabric are stored at -18 ° C in the dark.

A liquid detergent composition is prepared containing 4 g / l of a compact washing powder (Eurocompact powder from UNILEVER) and various lipase concentrations equivalent to 500, 1000 and 2000 LU / 1, 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. 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 rinsing cycle on the same tissue sample 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 which does not contain 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 - 25

iance (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 a 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 92.677 / 1 Lipase Gene 1. Extraction of the chromosomal AFN from the Pseudomonas wisconsinensis T 92.677 / 1 strain

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 Pseudomonas wisconsinensis T 92.677 / 1 strain is carried out in LB growth medium for 16 hours at 37 ° C. The composition of the LB growth medium is as follows: TRYPTONE (DIFCO) 10 g / 1, yeast extract 5 g / 1, NaCl 10 g / 1.

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 .mu.l ^of proteinase K solution (sold by

BOEHRINGER Mannheim) at 20 mg / ml (temporarily repaired).

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. 26

Then, 1.8 ml of a 5 M NaCl solution and 15 ml of a 10% (w / v) CTAB / NaCl solution (CTAB = cetyltrimethyl ammonium bromide, NaCl at 0) are added to this suspension. , 7 M). The suspension thus obtained is incubated for 20 minutes at 65 ° C. 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, MANIAΗS - 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) 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.

2. Construction of a genomic library of Pseudomonas wisconsinensis T 92.677 / 1

From the suspension obtained above, the chromosomal DNA (15 μg) 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 iμl 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 BamHI site as described by

SAMBROOK et al. , 1989, pages 1.60-1.61. We get a collection of 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 cells of E. coli HB101 (PROMEGA) using the kit sold under the name of

GIGAPACK π PACKAGING EXTRACT KIT (STRATAGENE) and following the manufacturer's recommendations for use.

The transfected cells of E. coli HB101 are cultured on a Petri dish containing LB agar medium, 25 μg / ml of streptomycin and 50 μg / ml 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 HB101 (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 μg / ml of streptomycin and 50 μg / ml of spectinomycin, for approximately 24 hours at 37 ° C. These 1500 colonies of E. coli HB101 (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 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 / 3-cyanoethyl phosphoramidites in a BIOSEARCH CYCLONE SYNTHESIZER apparatus. The sequence of this synthetic oligonucleotide is as follows: SEQ ID NO: 11 '- AACTACACCAAGACCAAATACCCCATCGTGCTGGTCCA - - CGGCGTGACCGGCTTCAACACTATCGGCGGGCTC -

This synthetic oligonucleotide is labeled at its termination by means of T - ^ ² P ATP with a T4 polynucleotide kinase enzyme according to the technique described in the SEQUITHERM CYCLE SEQUENCING KIT (BYOZYME). Screening is carried out on the genomic bank by the so-called "colony blot" technique (AMERSHAM) using as probe the synthetic oligonucleotide as prepared above.

The 1,500 colonies of the genomic bank (E. coli HB101 (pRG930:: WI)) are cultured for 18 hours at 37 ° C. on membranes called "hybond-N ^" (AMERSHAM) using the technique indicated by the maker.

The membranes (400 cmr) are placed in plastic bags containing 45 ml of prehybridization solution.

The solution of

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 and fragmented salmon sperm DNA (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 - 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 (1U0 revolutions per minute, amplitude of about 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 Manual - MANIAΗS 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 E. strains. 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 LB agar medium, 25 μg / ml of streptomycin and 50 μg / 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. 30

Example 16

Analysis of the plasmid pRG930 :: WI12 present in the strain of E. coli HB101 (pRG930 :: WI12) - Analysis by the so-called Southern Blot technique 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 so-called "hybond-N ⁺ " membrane (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

Restriction analysis of the plasmid pRG930 :: WI12 is carried out using the restriction enzyme EcoRI. The DNA fragments obtained are separated by electrophoresis on an agarose gel.

An analysis is carried out according to the Southern 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: 11), the other 4 fragments give no signal.

The 8.5 kbp fragment attached to the vector pRG930 is then ligated 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 DH5α (GIBCO) as described in SAMBROOK et al., 1989, page 1.82-1.84.

We get a colony of E. coli DH5α (pRG930 :: WI13) which is isolated and cultured on a Petri dish containing the LB agar medium, 25 μg / ml of streptomycin and 50 μg / ml of spectinomycin, for approximately 24 hours at 37 ° C. A partial restriction map of the plasmid pRG930 :: WI13 is established using the technique described in Molecular Cloning, a laboratory Manual - MANIAΗS et al., 1982, Cold Spring Harbor Laboratory, pages 374-379, and using the enzymes restriction restriction Clal, EcoRI, Ncol, Sali, Hindiπ, Bgiπ, Xhol, BamHI, Smal, Sacl, SacII, Kpnl, SphI, Xbal, 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 Pst1. The DNA fragments obtained are separated by electrophoresis on an agarose gel. An analysis is carried out according to the Southern Blot technique, as described in Example 16.

This demonstrates that the fragment inserted into the plasmid pRG930 :: WT13 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 JD 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 32

pBLUESCRIPT:: WI14 is obtained by using the SEQUΓΓHERM CYCLE SEQUENCING KIT kit (BIOZYM) and by 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, Kpnl or SacII successively in place of the restriction enzyme Pst1.

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. LIST OF SEQUENCES (1) GENERAL INFORMATION:

(i) DEPOSITOR:

(A) NAME: SOLVAY (Société -anonyme)

(B) STREET: rue du Prince Albert, 33

(C) CITY: Brussels

(E) COUNTRY: Belgi-que

(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-DETACHABLE FORM:

(A) TYPE OF SUPPORT: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentln Release # 1.0, Version # 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) ORGANIZATION: 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 (C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: DNA (genomics) (v) TYPE OF FRAGMENT: internal

(vi) ORIGIN:

(A) ORGANIZATION: 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 GTTCAûTACC 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 TTCGTGGCTG GACCGAACCG GTGTCGCTGT ATCGCCAGCA CGCCAACCGC 840

CTGAAGAACA AGGGCGTCTG A 861

(2) INFORMATION FOR SEQ ID NO: 3:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 861 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: DNA (genomics) (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 Aia 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 Gin Arg Gly Ala Glu Leu Ala Arg Gin 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 Gin. 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 A C GGT ATC AAC GCG CTA GGC ACC 480 Leu Ser Gly Ser Ser Asn Pro Gin 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 -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 Gin 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 Gin 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 "rg Val His Val Ala S-. * - Val Ala 35 40 45

Ala Phe Asn Asp Ser Glu Gin Arg Gly Ala Glu Leu Ala Arg Gin 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 Gin Gly Ser Pro Thr Ser Arg Val Ala Ala Ser Leu Arg Pro Asp 85 90 95 - 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 Gin 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 Gin 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 Gin 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: single

(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 GACCGGGGGGACGCTTCATACATCT -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) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 927 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: Single

(D) CONFIGURATION: linear

(n) TYPE OF MOLECULE: DNA (genomics)

(ix) CHARACTERISTIC:

(A) NAME / KEY: sigjpeptide

(B) LOCATION: 1..66

(ix) CHARACTERISTIC:

(A) NAME / KEY: mat_peptιde

(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 39 -

GCC GTC GAG GCC CAG GCC AAC TAC ACC AAG ACC AAG TAT CCG ATC GTG 96 Ala Val Glu Ala Gin 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

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 Gin 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 Gin 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 Gin 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 Gin 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 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 Gin 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 Gin 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 Gin 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 Va]. 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 Gin Arg Gly Ala 45 50 55

Glu Leu Ala Arg Gin Ile Val Pro Trp Ala Ala Gly Gly Gly Gly Lys 60 65 70 41

Val Asn Leu Ile Gly His Ser Gin 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 Gin 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 Gin 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 Gin 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: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: DNA (genomics)

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

(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 Gin 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 Gin Ala 20

(2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 72 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: Other nucleic acid

(A) DESCRIPTION: / desc = "synthetic oligonucleotide"

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11:

AACTACACCA AGACCAAATA CCCCATCGTG CTGGTCCACG GCGTGACCGG CTTCAACACT 60

ATCGGCGGGC TC 72

Claims

/ 12012 -., -_ -.

PCT / BE95 / 00094

44

R E V E N D I C A T I O N S

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 from 1 to 286 amino acids (SEQ ID NO: 4) or a modified sequence derived therefrom.

5 - 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.

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 aerobic conditions.

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. 10 - Lipase, characterized in that it develops an optimal enzymatic activity, measured at a pH of 9.5, in a temperature range 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 about 40 ° C and about 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 , 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.

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 of approximately 30 ° C, the activity maximum enzymatic 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 d <s a buffered solution of - 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 from this.

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 - Process 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 - Process according to claim 23, characterized in that the aerobic bacterium is a strain of Pseudomonas.

25 - Process 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.