DE3908131A1 - DNA WITH GENETIC INFORMATION FROM LIPASE - Google Patents

Abstract

DNA from e.g. chromobacterium viscosum varietas pararipoliticum having genetic information of lipase and comprising a base sequence encoding a specific amino acid sequence (I) is disclosed. Disclosed also are a vector comprising such a DNA, a transformant comprising such a DNA, and a polypeptide exhibiting the lipase activity and comprising said amino acid sequence, as well as a process for preparing such a polypeptide. The process ensures a large scale production of polypeptide having a high degree of stability and high lipase activity. The lipase is useful for the production of fatty acid from triglyceride, as a reagent for quantitative analysis of triglyceride, and as a catalyst for oil and fat reforming process using ester exchange reaction.

Description

The invention relates to a DNA with genetic information from lipase. In particular, the invention relates a DNA with genetic information from lipase and with a base sequence encoding a 319 amino acid sequence, a vector comprising this DNA, a transformant, which comprises this DNA, and a polypeptide, which shows the activity of the lipase and that mentioned Amino acid sequence includes, as well as a method for producing such a polypeptide.  

Lipase is an enzyme that catalyzes a reaction triglycerides or diglycerides to fatty acids and Glycerol be hydrolyzed, or the reverse reaction catalyzed. It will be widely used on one Variety of uses, such as as a digestive agent, as a diagnostic agent, as a catalyst for the production of various fatty acids or for Reformation of glycerides, and the like. Known sources for lipase are gastric juice of animals, pancreatic juice of Including animals, plant seeds and microorganisms Yeasts and bacteria. From the standpoint of a stable Supply of the raw materials is made of microorganisms derived lipase most widely used.

Characteristics of lipase used as a diagnostic reagent or used as a catalyst for the production of fatty acids is to be high stability against pH, Heat, Surfaktantien and the like. One has new microorganisms then investigated whether they were used to make Lipase are suitable, the such advantageous characteristics have (see for example JP-OS 29787/1971).

As a result of recent developments of genetic Recombination techniques was about a number DNAs which encode different polypeptides Have types of physiological activity. There are also some reports in the field of genetic Engineering that deals with lipase, eg. A report, for example a method for cloning a DNA Lipase-producing microorganisms of the genus Pseudomonas or Bacillus and for the production of Lipase using such a DNA (JP-OS  2 28 279/1987); another report concerns a procedure for cloning a DNA from lipase-producing microorganisms of the genus Aspergillus and for the production of lipase using such DNA (JP-OS 2 72 988/1987).

From the inventors were extensive investigations performed with the goal, using Genetic Engineering techniques to obtain a DNA for encodes such useful lipase. The have Inventor made a recombinant DNA using a DNA that is produced from a lipase Microorganism of the genus Chromobacterium separated has been. The inventors have found that this DNA a new DNA is with a base sequence that is a specific Amino acid sequence encoded, and that the two Sequences are clearly different from those of conventional polypeptides distinguish which lipase activity show.

Object of the present invention is thus the creation a DNA with genetic information from lipase and comprising a base sequence having the following amino acid sequence (I) from the first amino acid (Ala) the N-terminal side up to and including 319th amino acid (Val) coded:  

In a preferred embodiment of the present invention Invention has the base sequence which for the mentioned Amino acid sequence encodes the following formula (II):  

It is a further object of the present invention to provide a To provide a vector comprising such a DNA, a Transformants comprising such DNA, and a Polypeptide showing lipase activity and mentioned Amino acid sequence includes, as well as a method for the preparation such a polypeptide.

Other objects, features and advantages of the invention become even clearer from the following description.

Fig. 1 shows an endonuclease plan for plasmid pLIP1 and

Fig. 2 shows a similar plan for plasmid pLIP10.

The DNA of the present invention may be, for example are prepared using the following method from genetic engineering techniques. DNA of a microorganism, which represents a lipase gene donor and a Having lipase-producing ability, for example a lipase-producing microorganism of the genus Chromobacterium, is first separated and purified. This DNA is digested using ultrasound waves or restriction endonucleases. This digested DNA and an expression vector DNA which digests and are made linear, using a DNA ligase or similar measures on the truncated or cohesive Ends of the two DNAs combined to form a closed circle. The thus obtained, recombinant DNA vector is transformed into a reproducible host Introduced microorganism. Microorganisms which having the mentioned, recombinant DNA vector and using a screening method using a vector marker and lipase activity as indicators  were selected, are cultured. The recombinant DNA vector is then cultured out of the Cells separated and cleaned. The inventive DNA, which has the genetic information of the lipase, is then separated from the recombinant DNA vector.

Any microorganism capable of lipase production can for purposes of the present invention used as a lipase gene-donating microorganism become. Examples of lipase gene-donating microorganisms are Chromobacterium viscosum var.paralipoliticum, Chromobacterium viscosum ATCC 6918, Chrombacterium violaceum ATCC 12 472 and the like.

A transformed microorganism using from Genetic Engineering Lipase Creativity may also be lipase gene-donating Microorganism can be used.

In the following, the isolation method of DNA from the gene-donating microorganism will be explained. Any one of the above-mentioned gene-donating microorganisms is first cultured in a liquid culture medium with aeration for 1 to 3 days. The culture broth is centrifuged to collect the cells; then the cells are lysed to produce a bacteriolysate containing the lipase gene. Treatment with a cell wall lysing enzyme such as lysozyme or β- glucanase may be used for the bacteriolysate, optionally in combination with another enzyme such as protease, or a surfactant such as sodium lauryl sulfate. In addition, one can perform a physical digestion of the cell walls, z. B. by freeze-thawing or by means of a French press (French press). These measures can be used together with the treatment mentioned above.

Conventional methods of cleaning including, for example Deproteinization by phenol extraction, Protease treatment, ribonuclease treatment, alcohol precipitation and centrifuging can be applied, and Although either independently or in combination to the Separate DNA from bacteriolysate and purify.

The digestion of the microorganism DNA on this Way was separated and cleaned, can be performed be by treatment with ultrasonic waves or restriction endonucleases. To ensure that the DNA fragments and the vector DNA readily However, the use of restriction endonucleases, specifically of type II endonucleases, the act on specific nucleotide sequences, such as EcoRI, HindIII, BamHI or the like, preferred.

A particularly suitable for use vector is a Phage or a plasmid DNA with the ability to enter the Host bacterial cells grow autonomously, and those for the use as a genetic recombinant vector by artificial treatment was reconstructed.

If Escherichia coli is used as the host microorganism, for example, λ gt · λ C, λ gt · λ B or the like can be used as the phage.

The plasmid used is pBR322, pBR325, pACYC184, pUC12, pUC13, pUC18, pUC19 or the like, if used Escherichia coli is the host microorganism, while pUB110, pC194 or the like is used, if Bacillus subtilis is the host microorganism. at Use of Saccharomyces cerevisiae as host Microorganism can be YRp7, pYC1, YEp13, pJDB, YIp1 or are used. In addition, shuttle vectors which can be used in two or more microorganisms Host bacterial cells, for example, both in Escherichia coli as well as in Saccharomyces cerevisiae, can grow autonomously. These vectors are in desired Way digested using the same Restriction endonuclease like the one used for digestion the above-mentioned lipase gene-donating microorganism DNA is used.

A conventional ligation method using a DNA ligase can be used to treat the bacterial Combine DNA and the vector fragment. For example can be the cohesive end of bacterial DNA and that of the vector fragment first annealed (annealed) and then recombinant DNA from the bacterial DNA and the vector fragment under the action of a suitable DNA ligase. If necessary, the tempered, bacterial DNA vector fragment introduced into the host microorganism, to recombinant DNA with the help of a to generate in vivo DNA ligase.

Any microorganism which is an autonomous and stable replication of the recombinant DNA allowed and the ability to express the character of the  Foreign DNA possesses, can be used as a host bacterium become. Examples of such microorganisms include those that belong to Escherichia coli, like Escherichia coli DH1, Escherichia coli HB101, Escherichia coli W3110, Escherichia coli C600 and the like; those that cause Bacillus subtilis, such as Bacillus subtilis 207-25 [Gene, 34, 1-8 (1985)], Bacillus subtilis 207-21 [Journal of Biochemistry, 95, 87-93 (1984)], Bacillus subtilis BD170 [Nature, 293, 481-483 (1981)], Bacillus subtilis M (ATCC 6051) and the like; and those that belong to Saccharomyces cerevisiae, such as Saccharomyces cerevisiae AH-22 [Gene, 39, 117-120 (1985)], Saccharomyces cerevisiae BWG1-7A [Molecular & Cellular Biology, 6, 355-367 (1986)] and the like.

The introduction of the recombinant DNA into the host For example, microorganism can be carried out in the presence of calcium ions, when the host microorganism is a bacterium of the genus Escherichia. If a bacterium of the genus Bacillus as a host microorganism used, one can use a competent cell Method or a protoplast method. A Micro-injection method can also be used. If the host bacterium to the genus Saccharomyces heard, one can use a protoplast method or use a lithium acetate method.

The introduction of the desired DNA into the host Microorganism can be determined by means of a screening method be using a drug resistance marker of the vector and simultaneous determination of the Lipase activity. For example, you can do those Selecting bacteria that are on a selective culture medium,  based on the drug resistance marker, grow and produce lipase.

Recombinant DNA owned by the lipase gene and Once selected in this way, can from the Transformants are easily extracted for infiltration into another host bacterium. As an alternative Method can use a lipase gene DNA a restriction endonuclease or the like from a recombinant DNA having a lipase gene digested and with one end of a vector fragment which was obtained in a similar way. This can then be transformed into another host microorganism be introduced.

In the preparation of a lipase mutein, wherein it is is a mutant generated using Genetic engineering techniques from the invention Lipase gene with significant lipase activity, this can Mutant using various genetic engineering Techniques are spawned and finally with a Vector united to a recombinant DNA which then turns into a host microorganism is introduced to produce the lipase mutein.

The base sequence of the DNA of the present invention, which was prepared according to the method described above, can be determined by the dideoxy method [Science, 214, 1205-1210 (1981)]. For example, the Base sequence of a lipase gene in a plasmid using a microorganism of the genus Chromobacterium as lipase gene-donating microorganism  and Escherichia coli as a host bacterium, such as follows:

In the above base sequence, the 5'-end, which upstream with respect to the GCG codon which the first ala encoded at the N-end, any one Codon, as long as it codes for an amino acid. additionally the 5'-end may have one or more codons, which code for an amino acid. A preferred one Example of the links at the 5'-end is ATG or one Polydesoxyribonucleic acid, which is a signal peptide equivalent. The 3 'end, which is downstream with respect to GTG encoded for Val at the C end may be one Translational stop codon or any Codon coding for an amino acid. Furthermore may have one or more codons at the 3 'end, that code for an amino acid, provided that that it is desirable in this case that am 3 'end of these codons is a translation stop codon.

The amino acid sequence of the expression of the invention DNA-generated polypeptide can be derived from the Base sequence of the DNA can be predicted. The amino acid sequence of the section which is the N-end of the Can be determined by the the method explained below. A lipase gene donating Microorganism with the ability to produce Lipase is first cultured in a nutrient medium to Lipase in the cells to produce and enrich. The cultured cells are removed from the broth by filtration, Centrifugation or similar measures collected. The collected cells are then either by mechanical Measures or by enzymatic measures Use of lysozyme or the like destroyed. To that  Lysate becomes EDTA and / or a suitable surfactant Means given, if necessary, to lipase too solubilize and separate as an aqueous solution. These aqueous solution of lipase is then concentrated or, without to be concentrated, the ammonium sulfate fractionation, Gel filtration, adsorption chromatography or ion exchange chromatography subjected to highly purified To get lipase. The amino acid sequence of Section building the N-terminus of the lipase peptide, is determined in this highly pure lipase under Use of a device for the determination of protein sequences in the liquid phase (Beckman System 890ME, manufactured by Beckman, Inc.). This way it is confirmed that the amino acid sequence of at least this section is identical to the N-terminal amino acid sequence of lipase produced by a genetic engineering Technique was obtained. The amino acid sequence. the up determined from the base sequence (II), is equivalent to the amino acid sequence of the formula (I). The amino acid sequence of formula (I) may be at the amino acid residue at the upstream Side of the N-terminal Ala to one or more amino acids act. Preferred examples of this amino acid residue are a hydrogen atom, a Met or a signal peptide. As such, the C-terminal Val can be the Build up the end of the peptide or it can be at its downstream a group, like an acid amide or one or more amino acid residues.

The transformant obtained in this way can, if he is cultured in a nutrient medium, large quantities of the polypeptide having lipase activity in a stable manner produce.  

The cultivation of the host microorganism, the one Transformat is done under conditions which are fixed be taking into account the nutrient physiological Characteristics of the host microorganism. In most cases, will be a liquid cultivation applied. For a production on an industrial scale However, it is a cultivation under deep aerobic stirring conditions more advantageous. A great variety of nutrients, as is conventional for bacterial cultures can be used to cultivate the host Microorganism can be used. Specifically, any, usable carbon compounds as carbon sources be used. These include, for example Glucose, sucrose, lactose, maltose, fructose, molasses and the like. As nitrogen sources, any, available nitrogen compounds are used, including peptones, meat extracts, yeast extracts, Casein hydrolysates and the like. Other ingredients, including Salts, such as phosphates, carbonates and sulfates, and salts of magnesium, calcium, potassium, iron, manganese, Zinc and the like, as well as certain types of amino acids or vitamins, may be used, provided that that is appropriate.

The cultivation temperature can be varied within a range in which the bacteria grow and produce lipase can. A preferred temperature range is 20 to 42 ° C for Escherichia coli; 30 to 37 ° C for Bacillus subtilis; and 25 to 35 ° C for Saccharomyces cerevisiae. The cultivation period can to a certain extent depending on the cultivation conditions be varied. Basically, the cultivation becomes  terminated when the yield of lipase Maximum reached. In the ordinary practice are to about 12 to 48 hours if the host Microorganism Escherichia coli; 18 to 42 h, though the host microorganism is Bacillus subtilis, and 24 to 48 hours if the host microorganism Saccharomyces cerevisiae is. You can check the pH of the culture media change in the area where the bacteria grow and can produce lipase. The most preferred area the pH is about 6 to 8 when the host Microorganism Escherichia coli; about 7 in the case of Bacillus subtilis and about 5 to 7 in the case of Saccharomyces cerevisiae.

Lipase may be for further use in the form of a culture broth be offered with a content of the bacteria. However, lipase contained in the culture broth generally becomes used after being removed from the cells by filtration, Centrifuging or similar measures removed has been. If lipase is present in the cells the cells first by filtration or centrifugation separated and the collected cells are then either by mechanical action or enzymatic Measures using lysozyme or the like. Destroyed. To the suspension is added a chelating agent, such as EDTA and / or a suitable surfactant, added, if necessary, to solubilize the lipase and in this way the collection of lipase as aqueous To enable solution.

The solutions thus obtained containing lipase are then by evaporation in vacuo or using of a filter and a salting out treatment  with ammonium sulfate, sodium sulfate or the like. Subjected or a fractionated precipitate using a hydrophilic organic solvent such as Methanol, ethanol, acetone or the like. The precipitate is dissolved in water and the solution is passed through a semipermeable membrane dialysed to low molecular weight Eliminate impurities. As an alternative Method can be the precipitate by means of gel filtration, Adsorption chromatography, ion exchange chromatography or the like. Purified lipase is added from the lipase containing solution using of these various measures was obtained by Evaporation in vacuo, freeze-drying or the like. manufactured.

The activity of the lipase produced in this way is determined by the following method:

(Reaction mixture) 0.2 M Tris hydrochloride buffer (pH 7.5) 0.2 (ml) 27.5 mM dilinoleoylglycerol (15% Triton X-100) 0.05 50 mM CaCl₂ 0.01 200 mM ATP 0.005 100 mM CoASH 0.005 Acyl-CoA synthetase (50 U / ml) 0.01 water rest a total of 0.5 (ml)

Add 0.5 ml of the reaction mixture having the above composition into a test tube and preincub 2 to 3 min at 37 ° C. Add 50 μl of an enzyme solution (10 mM PIPES-NaOH buffer containing 0.1% bovine serum albumin; pH 7.3) and incubate at 37 ° C for 10 min. Give 0.5 ml  10 mM N-ethylmaleimide and 0.5 ml of a reagent (R-2) with the composition below and incubate another 5 min at 37 ° C. Finish the reaction Add 1.5 ml of 0.5% sodium dodecyl sulfate and Measures absorption at 550 nm. Activity of substance to produce 1 micromole of linoleic acid / min is called 1 unit (E) taken.

[Reagent (R-2) composition] 0.2 M PIPES-NaOH buffer (pH 7.3) 0.05 (ml) 0.3% 4-aminoantipyrine 0.05 0.3% TOOS [N-ethyl-N- (2-hydroxy-3-sulfopropyl-m-toluidine)] 0.05 Peroxidase (45 e / ml) 0.05 Acyl-CoA oxidase (500 U / ml) 0.02 0.2 M ATP 0.01 water 0.27

In the present specification, amino acids, Peptides, nucleic acids and nucleic acid-related compounds abbreviated according to the usual standards. Some Examples of the abbreviations used are below specified. All names for amino acids denote the L-isomers.

DNA = Deoxyribonucleic acid RNA = Ribonucleic acid A = Adenine T = Thymine G = Guanine C = Cytosine Ala = Alanine bad = Arginine Asn = Asparagine Asp = Aspartate Cys = Cysteine Gln = glutamine Glu = Glutamate Gly = Glycine His = Histidine Ile = Isoleucine Leu = Leucine Lys = Lysine mead = Methionine Phe = Phenylalanine Per = Proline Ser = Serine Thr = Threonine Trp = Tryptophan Tyr = Tyrosine

With reference to the following exemplary embodiments, which serve to illustrate the invention and no limitation will be more features the invention even clearer.

Example 1 Production of chromosomal DNA

A chromosomal DNA is made from the strain Chromobacterium viscosum varietas pararipoliticum (JP OS 29 787/1971), according to the following method. The strain is shaken overnight at 37 ° C in 150 ml of a broth medium containing 0.5% sodium thiosulfate, cultured. The cultured broth will Centrifuged at 3000 rpm for 10 minutes to allow the cells to collect. The cells are suspended in 5 ml of a solution, containing 10% sucrose, 50 mM tris-hydrochloric acid  (pH 8.0) and 50 mM EDTA. To the suspension 1 ml of a lysozyme solution (10 mg / ml) is added. The mixture is incubated for 15 min at 37 ° C and then Add 1 ml of 10% SDS (sodium dodecyl sulfate). To the resulting suspension is added the same Volume of a mixed solvent of chloroform and phenol (1: 1). The mixture is stirred and added for 3 min Centrifuged at 10,000 rpm to remove the water and solvent layers to separate. To the separated water layer slowly add a double volume of ethanol. The mixture is slowly stirred with a glass rod to to cause the DNA to wrap around the rod. The DNA separated in this way is dissolved in 10 ml of a Dissolved solution containing 10 mM tris-hydrochloric acid (pH 8.0) and 1 mM EDTA (such a solution hereinafter referred to as "TE"). This solution is mixed with an equal volume of chloroform-phenol Treated solvent mixture and centrifuged again, to separate the water layer. To this water layer Add 2 times the volume of ethanol and separate the DNA again in the manner described above from the mixture from. This DNA is then dissolved in 2 ml of TE.

example 2 Preparation of pACYC184 Plasmid DNA

Escherichia coli pM191 carrying pACYC184, [J. Bacteriol, 134, 1141 (1981); ATCC 37033] is under Shake in 1 L of BHI medium (manufactured by Difco Co.) cultured. After the turbidity of the broth has a value of OD₆₆₀ = 1.0, spectinomycin is up to added to a final concentration of 300 ug / ml. The Shake the broth at 37 ° C for at least 16 h.  set. After completion of shaking cultivation is the broth is centrifuged at 3000 rpm for 10 min To collect cells. The plasmid DNA collected from the Cells are analyzed according to the lysozyme SDS method and the Cesium chloride-ethidium bromide method produced [Maniatis et al., Molecular Cloning, 86-94, Cold Spring Harbor (1982)].

example 3 Construction of plasmid pLIP1 with lipase gene

• (1) 2 μl (about 0.5 μg) Chromobacterium viscosum var. pararipolitic chromosomal DNA prepared in Example 1.1 μl of a 10-fold concentration of H buffer [Maniatis et al., Molecular Cloning, 104, Cold Spring Harbor (1982)], 1 μl BglII (10 U / μl, manufactured by Takara Shuzo Co., Ltd.) and 6 μl of water are mixed and incubated for 1 h at 37 ° C for digestion. plasmid pACYC184 DNA (about 0.3 μg), prepared separately is made using BamHI according to the same Method digested. Add 0.6 E alkaline Phosphatase (manufactured by Takara Shuzo Co., Ltd .; hereinafter also referred to as "BAP") and incubated the mixture for 1 h at 65 ° C. The produced in this way Both digested DNA solutions will be together mixed. One gives to this mixed DNA solution 0.1 vol. 3M sodium acetate. Then the solution becomes with an equal volume of chloroform-phenol Treated solvent mixture and to separate the Water layer centrifuged. To the water layer there a 2-fold volume of ethanol. The precipitated DNA is collected by centrifugation and dried in vacuo. The dried DNA is dissolved in 89 μl of water.  To do this, 10 μl of a 10-fold concentration Ligation buffer [0.5 M tris-hydrochloric acid (pH 7.6), 0.1 M MgCl₂, 0.1 M dithiothreitol, 10 mM spermidine, 10 mM ATP] and 1 μl T4 DNA ligase (350 E; by Takara Shuzo Co., Ltd.) and mixes the whole thing. you allow the mixture to stand overnight at 4 ° C. This DNA Solution is treated with a chloroform-phenol mixture and the DNA is precipitated with ethanol, in vacuo dried and dissolved in 10 ul TE.
• (2) Escherichia coli DH1 (strain No. ME7778, ATCC 27325; supplied by National Genetic Research Institute) is prepared in 100 ml of BHI medium (brain-heart infusion; from Difco Co.), while the logarithmic Growth phase collected by centrifugation (10,000 rpm, 2 minutes) and in 40 ml of an ice-cold solution (pH 5.8) containing 30 mM potassium acetate, 100 mM RbCl, 10 mM CaCl₂, 50 mM MnCl₂ and 15% glycerol contains. The suspension is allowed to stand at 0 ° C. for 5 min. Subsequently, the suspension is centrifuged to the Remove supernatant. The cells are in 4 ml of a Suspended solution containing 10 mM MOPS buffer (prepared from Dotite Co.), 75 mM CaCl₂, 10 mM RbCl and 15% glycerol (pH 6.5), and the suspension is left for 15 min allowed to stand at 0 ° C to obtain competitor cells.
• (3) To 200 μl of the Escherichia coli cell suspension 10 μl of the product prepared in the above step (1) DNA solution. After the mixture stood at 0 ° C for 30 min Add 1 ml of BHI medium and store the mixture at 37 ° C for 90 min. An aliquot of the mixture (100 ml) is loaded on a BHI agar plate containing  25 μg / ml chloramphenicol, spread and over Cultured at 37 ° C overnight to produce transformants. These transformants are grown on a Crosslee agar medium (manufactured by Eiken Chemical Co., Ltd.) and further cultivated overnight at 37 ° C.

About 50,000 colonies of transformants that are on this Are generated are examined. A blue colored Strain is obtained. This strain is called as Escherichia coli DHI pLIP1 designated.

Example 4 Mapping of pLIP1 and determination of the base sequence of the lipase gene

Plasmid pLIP1 DNA is made from Escherichia coli DH1 pLIP1 in the same way as in the preparation of pACYC 184 receive.

A cleavage map of the pLIP1 DNA thus obtained is prepared using the restriction endonucleases BamHI, ClaI, EcoRV, SalI, MluI, PstI and XhoI (all manufactured by Takara Shuzo Co., Ltd.). The results are shown in FIG .

example 5

1 μl of a 10-fold concentration of M buffer [Maniatis et al., Molecular Cloning, 104, Cold Spring Harbor (1982)] and 1 μl ClaI (10 U / μl, manufactured by Takara Shuzo Co., Ltd.) are added to approximately 1.0 μg (8 μl) of plasmid pLIP1 DNA is added from the strain Escherichia coli DHI pLIP1 according to the method described in Example 2 was prepared to the DNA for 2 h at 37 ° C to  digest. About 3.2 kb of the DNA fragments are passed through Electrophoresis separated. The DNA fragments become treated with phenol and precipitated in ethanol. This Precipitate is dissolved in 20 μl TE [containing 10 mM Tris-hydrochloric acid and 1 mM EDTA (pH 8)]. To about 0.2 μg (1 μl) of pBR322 plamid DNA prepared separately and purified according to the method of Example 2 was added, 1 ul of a 10-fold M buffer, 7 μl of water and 1 μl of ClaI (10 U / μl, prepared by Takara Shuzo Co., Ltd.). After 2 hours digesting the DNA at 37 ° C is added 5 ul 1M tris-hydrochloric acid Buffer (pH 8.0), 80 μl of water and 5 μl of alkaline Phosphatase (0.5 U / μl, manufactured by Takara Shuzo Co., Ltd.) and reacts at 65 ° C for 2 hours. To the reaction is the resulting product with three times an equal volume of a chloroform-phenol solvent mixture treated and the DNA is replaced by ethanol Precipitation isolated. The DNA thus obtained is dissolved in 20 μl of TE dissolved. 5 μl each of two types of DNA solutions, which were made in this way will be mixed. To this mixture is added 2 .mu.l of a 10-fold concentration of ligation buffer, 7 ul of water and 1 μl of T4 DNA ligase (175 U / μl, manufactured by Takara Shuzo Co., Ltd.) and allowed to mix overnight 4 ° C stand. This DNA solution is washed with a chloroform Phenol mixture is treated and the DNA is mixed with ethanol like. The precipitate is dissolved in 10 μl of TE and Escherichia coli DH1 competitor cells, which are based on the same as in Example 3 (2) were prepared, are transformed. The transformants are on an agar medium containing 50 μg / ml Ampicillin, and cultured overnight at 37 ° C. The generated Colonies become on a Crosslee agar medium  same as in Example 3 (3) replicated to a To obtain lipase-producing Escherichia coli. This Strain is called Escherichia coli DH1 pLIP10 and was at Fermentation Research Institute, Agency deposited by Industrial Science and Technology; deposit No. 2266, FERM BP-2266.

This strain turns into BHI medium overnight after purification cultured at 37 ° C. The lipase-producing ability of the strain is determined on the cultured cells under Use of the aforementioned lipase assay method. It was found that the strain has an activity of about 0.01 U / ml. The one held by this tribe Plasmid is separated according to Example 2. The plasmid contains a lipase gene and a plasmid pBR322 gene and is called pLIP10.

example 6 Mapping of pLIP10 and determination of the base sequence of the lipase gene pLIP10 plasmid DNA is extracted from Escherichia coli DH1 pLIP10 in the same way as in the preparation of pACYC 184 received described.

A cleavage map of the pLIP10 DNA thus obtained is prepared using the restriction endonucleases BamHI, SalI, XhoI and PstI (all from Takara Shuzo Co., Ltd.). The results are shown in FIG . The base sequence of the DNA encoding the lipase gene is determined by the dideoxy method [Science, 214, 1205-1210 (1981)] using the M13 phage. The base sequence of the lipase gene and the amino acid sequence are given as formulas (I) and (II).

example 7 Production of lipase

Escherichia coli DH1 pLIP10 is maintained at 37 ° C for 18 h in 20 L of BHI medium (Difco Co.) using a 30 l bottle fermenter cultivated. The cultivated Cells are centrifuged at 5000 rpm during Collected for 10 minutes. The cells become physiological with 2 l Saline and washed in 2 l of 0.1 mM Phosphatalbuminpuffer (Containing 13.61 g KH₂PO₄, 3.92 g NaOH, 1 g bovine fetal serum albumin, 1 g NaN₃ and 1 l Water; pH 8.0). To the thus produced Suspension are lysozyme, EDTA-2Na and Triton X-100 in a concentration of 1 mg / ml or 2mM or 0.1%. After incubation at 37 ° C for 30 minutes, the mixture becomes Centrifuged at 5000 rpm for 10 minutes to give the resulting To collect supernatant. 1.9 l of this supernatant are subjected to acetone (50-80%) precipitation. The precipitate is centrifuged for 30 minutes collected at 5000 rpm, in 100 ml of phosphate albumin buffer and then the DEAE-Sephallose CL-6B- Subjected to ion exchange chromatography, wherein the active fraction is collected. This faction will desalted and freeze-dried, taking a powdery Product receives. In this enzyme sample you put a lipase activity of 350 E, if you take the Lipase activity measurement according to the method mentioned above performs.  

The present invention ensures production on a large scale of high-level polypeptide Degree of stability and with high lipase activity. The Lipase is useful in the production of fatty acid from triglyceride, as a reagent for quantitative analysis triglyceride and as a catalyst for oil and grease Reforming method using the ester exchange reaction.

Claims (9)

A DNA having genetic information of lipase and comprising a base sequence encoding the following amino acid sequence (I) from the first amino acid (Ala) on the N-terminal side up to and including the 319th amino acid (Val): 2. DNA according to claim 1, characterized in that said base sequence 957 comprises continuous bases which, starting from the 5 'end, are represented by the following base sequence (II): 3. A vector comprising the defined in claim 1 DNA.   4. Vector according to claim 3, characterized in that that the vector is plasmid pLIP1 or pLIP10 acts. 5. A transformant with DNA that is related to the Host microorganism is foreign and as in claim 1 is defined. 6. Transformant according to claim 5, characterized that the host microorganism is a microorganism from the strain Escherichia coli. 7. A method of producing a polypeptide comprising
Cultivating a transformant having DNA foreign to the host microorganism and defined in claim 1 to cause the transformant to express genetic information of said DNA, and
Collection of the polypeptide with lipase activity from the culture broth. 8. The method according to claim 7, characterized that the host microorganism is a microorganism from the strain Escherichia coli. A polypeptide having the activity of lipase having the following amino acid sequence (I) from the first amino acid (Ala) on the N-terminal side up to and including the 319th amino acid (Val):