GB2216528A - DNA sequence coding lipase - Google Patents

Description

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TITLE OF THE INVENTION

DNA HAVING GENETIC INFORMATION OF LIPASE BACKGROUND OF THE INVENTION Field of the Invention:

This invention relates to a DNA having genetic information of lipase, and, more particularly, to a DNA having genetic information of lipase and comprising a base sequence encoding a 319 amino acid sequence, a vector comprising said DNA, a transformant comprising said DNA, and a polypeptide exhibiting the activity of the lipase comprising said amino acid sequence, as well as a process for preparing-such a polypeptide. Description of the Backqround:

Lipase is an enzyme catalyzing a reaction hydrolyzing triglycerides or diglycerides into fatty acids and glycerol, or catalyzing the reverse reaction. It is widely used in a variety of fields as a digestant, a diagonostic reagent, a catalyst for producing various fatty acids or for reforming glycerides, and the like. The known sources of lipase are animal gastric juice, animal pancreatic juice, plant seeds, and microorganisms, including yeasts and bacteria. From the aspect of stable supply of the raw materials, lipase derived from microorganisms are most widely used.

Characteristics demanded of lipase used as a 1 diagonostic reagent or a catalyst for the production of fatty acids are high stability against pH, heat, surfactants, and the like. Investigation into new microorganisms has been carried out for the purpose of producing lipase possessing such favorable characteristics (e.g, Japanese Patent Laid-open No. 29787/1971).

As a result of the recent development of genetic recombination techniques a number of DNAs encoding polypeptide having different types of physiological activities have been reported. Also, there are several reports in the field of.genetic engineering concerning lipase, e.g. a report relating to a process for cloning of a DNA from lipase-producing microorganisms belonging to the genus Pseudomonus or Bacillus, and f or producing lipase utilizing such a DNA (Japanese Patent Laid-open No. 228279/1987); a report concerning a process for cloning of a DNA from lipase-producing microorganisms belonging to the genus Aspergillus and for producing lipase utilizing such a DNA (Japanese Patent Laid-open No. 272988/1987).

The present inventors have undertaken extensive studies to obtain a DNA coding for such a useful lipase using genetic engineering techniques. As a result, the inventors prepared a recombinant DNA using a DNA separated from a lipase-producing microorganism belonging to the genus Chromobacterlum. The inventors 2 found that this DNA is a novel DNA having a base sequence encoding a specific amino acid sequence, and that the both sequences are quite different from those of conventional polypeptides exhibiting lipase activity.

SUMMARY OF THE INVENTION

Accordingly, an object of this invention is to provide 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 through the 319th amino acid (Val):

AlaAspThrTyrAlaAlaThrArgTyrProValIleLeuValHisGlyLeuAlaGlyThr AspLysPheAlaAsnValValAspTyrTrpTyrGlyIleGlnSerAspLeuGlnSerHis GlyAlaLysValTyrValAlaAsnLeuSerGlyPheGlnSerAspAspGlyProAsnGly ArgGlyGluGlnLeuLeuAlaTyrValLysGlnValLeuAlaAlaThrGlyAlaThrLys ValAsnLeuIleGlyHisSerGlnGlyGlyLeuThrSerArgTyrValAlaAlaValAla ProGlnLeuValAlaSerValThrThrIleGlyThrProHisArgGlySerGluPheAla AspPheValGlnAspValLeuLysThrAspProThrGlyLeuSerSerThrValIleAla AlaPheValAsnValPheGlyThrLeuValSerSerSerHisAsnThrAspGlnAspAla LeuAlaAlaLeuArgThrLeuThrThrAlaGlnThrAlaThrTyrAsnArgAsnPhePro SerAlaGlyLeuGlyAlaProGlySerCysGlnThrGlyAlaAlaThrGluThrValGly GlySerGlnHIsLeuLeuTyrSerTrpGlyGlyThrAlaIleGlnProThrSerThrVal LeuGlyValThrGlyAlaThrAspThrSerThrGlyThrLeuAspValAlaAsnValThr AspProSerThrLeuAlaLeuLeuAlaThrGlyAlaValMetIleAsnArgAlaSerGly GlnAsnAspGlyLeuValSerArgCysSerSerLeuPheGlyGlnValIleSerThrSer TyrHisTrpAsnHisLeuAspGluIleAsnGlnLeuLeuGlyValArgGlyAlaAsnAla GluAspProValAlaValIleArgThrHisValAsnArgLeuLysLeuGlnGlyVal M 3 In a preferred embodiment of this invention the base sequence encoding said amino acid sequence has the following formula (II):

20 30 40 so 60 GCGGACACCTACGCGGCGACGCGCTATCCGGTGATCCTCGTCCACGGCCTCGCGGGCACC 80 90 100 110 120 GACAAGTTCGCGAACGTGGTGGACTATTGGTACGGAATCCAGAGCGATCTGCAATCGCAT 140 150 160 170 180 GGCGCGAAGGTGTACGTCGCGAATCTCTCGGGATTCCAGAGCGACGACGGGCCGAACGGC 200 210 220 230 240 CGCGGCGAGCAGCTGCTCGCCTACGTGAAGCAGGTGCTCGCGGCCACCGGCGCGACCAAG 250 260 270 280 290 300 GTGAACCTGATCGGCCACAGCCAGGGCGGCCTGACCTCGCGCTACGTCGCGGCCGTCGCG 310 320 330 340 350 360 CCGCAACTGGTGGCCTCGGTGACGACGATCGGCACGCCGCATCGCGGCTCCGAGTTCGCC 370 380 390 400 410 420 GACTTCGTGCAGGACGTGCTGAAGACCGATCCGACCGGGCTCTCGTCGACGGTGATCGCC 430 440 450 460 470 480 GCCTTCGTCAACGTGTTCGGCACGCTCGTCAGCAGCTCGCACAACACCGACCAGGACGCG 490 500 510 520 530 540 CTCGCGGCGCTGCGCACGCTCACCACCGCGCAGACCGCCACCTACAACCGGAACTTCCCG 550 560 570 580 590 600 AGCGCGGGCCTGGGCGCGCCCGGTTCGTGCCAGACGGGCGCCGCGACCGAAACCGTCGGC 610 620 630 640 650 660 GGCAGCCAGCACCTGCTCTATTCGTGGGGCGGCACCGCGATCCAGCCCACCTCCACCGTG 670 680 690 700 710 720 CTCGGCGTGACCGGCGCGACCGACACCAGCACCGGCACGCTCGACGTCGCGAACGTGACC 730 740 750 760 770 780 GACCCGTCCACGCTCGCGCTGCTCGCCACCGGCGCGGTGATGATCAATCGCGCCTCGGGG 790 800 810 820 830 840 CAGAACGACGGGCTCGTCTCGCGCTGCAGCTCGCTGTTCGGGCAGGTGATCAGCACCAGC 850 860 870 880 890 900 4 TACCACTGGAACCATCTCGACGAGATCAACCAGCTGCTCGGCGTGCGCGGCGCCAACGCG 910 920 930 940 950 GAAGATCCGGTCGCGGTGATCCGCACGCACGTGAACCGGCTCAAGCTGCAGGGCGTG (Ii) The further object of this invention is to provide 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.

Other objects, features and advantages of the invention will hereinafter become more readily apparent from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a drawing showing an endonuclease map for plasmid pLIP1, and Fig. 2 is a similar map for plasmid pLIP10.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The DNA of the present invention can be prepared, for example, by the following method through the application of genetic engineering techniques. DNA of a microorganism which is a lipase gene donor and has lipase-producing capability, for instance, a lipaseproducing microorganism belonging to the genus Ch.romobacte.rium, is first separated and purified. This DNA is digested using ultrasonic waves or restriction endonucleases. This digested DNA and an expression I vector DNA, which is digested and rendered linear, are joined with a DNA ligase or the like at the blunt or cohesive ends of the two DNAs to form a closed circle. The recombinant DNA vector thus obtained is introduced into a reproducible host microorganism. Microorganisms having said recombinant DNA vector selected by means of screening using a vector marker and the lipase activity as indicators are cultured. Said recombinant DNA vector is then separated from the cultured cells and purified. The DNA of the present invention possessing the lipase genetic information is then separated from said recombinant DNA vector.

Any microorganisms having lipase-producing capability can be used as a lipase gene-donating microorganism for the purpose of this invention. Chromobacterium viscosum var. paralipoliticum, Chromobacte.rium viscosum ATCC 6918, Chromobacterium violaceum ATCC 12472, and the like may be given as examples of lipase gene-donating microorganisms.

A transformed microorganism which is endowed with the lipase-producing capability through the use of genetic engineering can also be used as a lipase gene donating microorganism.

The isolation method of DNA from the genedonating microorganism is now illustrated. Any one of the above-mentioned gene-donating microorganisms is le first cultured in a liquid culture medium under 6 aeration for 1 to 3 days. The culture broth is centrifuged to collect the cells, following which the cells are lysed to produce a bacteriolysate containing lipase gene. Treatment with a cell wall lysing enzyme such as lysozyme or A-glucanase can be used for the bacteriolysate, in combination, as required, with other enzyme such as protease or a surfaCe active agent such as sodium laurylsulfate. In addition. physical digestion of cell walls by means of freeze-thawing or French press, for example, may be employed together with the above-mentioned treatment.

Conventional methods of purification, includingr for example, deproteinization by phenol extraction, protease treatment, ribonuclease treatment, alcohol precipitation, and centrifugation, can be applied either independently or in combination for separating and purifying DNA from the bacteriolysate.

Digestion of the microorganism DNA thus separated and purified can be carried out by means of treatment with ultrasonic waves or restriction endonucleases. In order to ensure ready joining of the DNA fragments and the vector DNA, however, the use of restriction endonucleases, especially type II endonucleases acting on a specific nucleotide sequences such as EcoRI, HindIII, BamHI, or the like, is preferable.

A desirable vector employed is a phage or a plasmid DNA which is capable of autonomously growing in 7 host bacterial cells and is reconstructed for use as a genetic recombinant vector through artificial treatment.

When Escherichia coli is used as the host microorganism, for example, Agt. XCI;kgt.;LB, or the like can be used as a phage.

As a plasmid, pBR322, pBR3251 pACYC184, pUC121 pUC13, pUC181 pUC19, or the like is used when Eschexichia coli is the host microorganism, while pUB110, pC194, or the like is used when Bacillus subtillis is the host microorganism. When Saccharomyces cerevisiae is used as a host microorganism, YRp7, pYC11 YEpl3j pJDBI YIpl, or the like can be used. In addition, shuttlevectors which can autonomously grow in two or more microorganism host bacterial cells, for instance, in both Escherichia coli and Saccharomyces cerevisiae, may be employed. - These vectors are desirably digested by using the same restriction endonuclease as that used for digesting the above-mentioned lipase gene-donating microorganism DNA.

A conventional ligation method using a DNA ligase can be employed to join the bacterial DNA and the vector fragment. For instance, the cohesive end of the bacterial DNA and that of the vector fragment are first annealed, and then recombinant DNA from the bacterial DNA and the vector fragment can be prepared by the action of a suitable DNA ligase. If required, the 8 annealed bacterial DNA-vector fragment is introduced into the host microorganism to produce the recombinant DNA with the aid of an in vivo DNA ligase.

Any microorganism which allows autonomic and stable replication of the recombinant DNA and is capable of expressing the character of the foreign DNA can be used as a host bacterium. Examples of such a microorganism include those belonging to Escherichia coli such as Escherichia coli DH1, Escherichia coli HB101., Escherichia coli W3110,, Escherichia coli C600, and the like; those belonging to Bacillus subtillis such as Bacillus subtillis 207-25 (Gene, 34, 1-8 (1985)], Bacillus subtillis 207-21 [journal of Biochemistxy, 95, 87-93 (1984)], Bacillus subtillis BD170 [Nature, 293, 481-483 (1981)], Bacillus subtillis M (ATCC 6051), and the like; and those belonging 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.

Introducing the recombinant DNA into the host microorganism may be performed, for example, in the presence of calcium ion when the host microorganism is a bacterium belonging to the genus Escherichia. When a bacterium belonging to the genus Bacillus is used as the host microorganism, a competent cell method or a protoplast method may be used. A micro-injection 9 method can also be used. When the host bacterium is that belonging to the genus Saccharomyces, a protoplast method or a lithium acetate method can be employed.

Introducing the desired DNA into the host microorganism can be detected by means of screening using a drug resistance marker of the vector and lipase activity at the same time. For instance, those bacteria which grow on a selective culture medium based on the drug resistance marker and which produce lipase can be selected.

Recombinant DNA possessing the lipase gene once selected in this manner may be easily extracted from the transformant for introduction into another host bacterium. Alternatively, a lipase gene DNA can be digested using a restriction endonuclease or the like from a recombinant DNA possessing a lipase gene, and is joined with a terminal of a vector fragment obtained in a similar manner. This can be then introduced into other host microorganisms.

In the production of a lipase mutein which is a mutant produced using genetic engineering techniques from the lipase gene possessing substantial lipase activity of this invention, this mutant can be proliferated using various genetic engineering techniques, and finally joined with a vector to produce recombinant DNA, which is then introduced into a host microorganism to produce the lipase mutein.

The base sequence of the DNA of this invention prepared by 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 prepared using a microorganism belonging to the genus Chromobacterium as a lipase gene-donating microorganism and Escherichia coli as a host bacterium is as follows:

20 30 40 so 60 GCGGACACCTACGCGGCGACGCGCTATCCGGTGATCCTCGTCCACGGCCTCGCGGGCACC 80 90 100 110 120 GACAAGTTCGCGAACGTGGTGGACTATTGGTACGGAA-TCCAGAGCGATCTGCAATCGCAT 140 150 160 170 180 GGCGCGAAGGTGTACGTCGCGAATCTCTCGGGATTCCAGAGCGACGACGGGCCGAACGGC 200 210 220 230 240 CGCGGCGAGCAGCTGCTCGCCTACGTGAAGCAGGTGCTCGCGGCCACCGGCGCGACCAAG 250 260 270 280 290 300 GTGAACCTGATCGGCCACAGCCAGGGCGGCCTGACCTCGCGCTACGTCGCGGCCGTCGCG 310 320 330 340 350 360 CCGCAACTGGTGGCCTCGGTGACGACGATCGGCACGCCGCATCGCGGCTCCGAGTTCGCC 370 380 390 400 410 420 GACTTCGTGCAGGACGTGCTGAAGACCGATCCGACCGGGCTCTCGTCGACGGTGATCGCC 430 440 450 460 470 480 GCCTTCGTCAACGTGTTCGGCACGCTCGTCAGCAGCTCGCACAACACCGACCAGGACGCG 490 500 510 520 530 540 CTCGCGGCGCTGCGCACGCTCACCACCGCGCAGACCGCCACCTACAACCGGAACTTCCCG 550 560 570 580 590 600 AGCGCGGGCCTGGGCGCGCCCGGTTCGTGCCAGACGGGCGCCGCGACCGAAACCGTCGGC 610 620 630 640 650 660 GGCAGCCAGCACCTGCTCTATTCGTGGGGCGGCACCGCGATCCAGCCCACCTCCACCGTG 670 680 690 700 710 720 CTCGGCGTGACCGGCGCGACCGACACCAGCACCGGCACGCTCGACGTCGCGAACGTGACC 11 730 740 750 760 770 780 GACCCGTCCACGCTCGCGCTGCTCGCCACCGGCGCGGTGATGATCAATCGCGCCTCGGGG 790 800 810 820 830 840 CAGAACGACGGGCTCGTCTCGCGCTGCAGCTCGCTGTTCGGGCAGGTGATCAGCACCAGC 850 860 870 880 890 900 TACCACTGGAACCATCTCGACGAGATCAACCAGCTGCTCGGCGTGCGCGGCGCCAACGCG 910 920 930 940 950 GAAGATCCGGTCGCGGTGATCCGCACGCACGTGAACCGGCTCAAGCTGCAGGGCGTG (II) In the above base sequence, the 51-end which is in the upstream of GCG codon encoding the first Ala at the N-terminal may be any codon so long as the same codes for an amino acid. In addition, the 51-end may have one or more codons encoding an amino acid. A preferable example of the member at the 51-end is ATG or a polydeoxyribonucleic acid corresponding to a signal peptide. The 31-end which is the downstream of GTG coding for Val at the C-terminal may have a translation termination codon or any codon encoding an amino acid. In addition, there can be one or more codons encoding an amino acid at the 31-end, provided that in this case it is desirable that a translation termination codon be present at the 31-end of these codons.

The amino acid sequence of the polypeptide produced through the expression of the DNA of this invention can be predicted from the base sequence of the DNA. The amino acid sequence of the portion constituting the N-terminal of said polypeptide can be determined through the method discussed below. A 12 lipase gene-donating microorganism capable of producing lipase is first cultured in a nutrient medium to produce and accumulate lipase in the cells. The cultured cells are collected from the broth by filtration, centrifugation, or the like means. The collected cells are then destroyed- either by mechanical means or enzymatic means using lysozyme or the like, and to the lysate EDTA and/or a suitable surface active agent are added, as required, to solubilize and separate lipase as an aqueous solution. This aqueous solution of lipase is then condensed or, without being condensed, subjected to ammonium sulfate fractionation, gel filtration, adsorption chromatography, or ion exchange chromatography to obtain highly purified lipase. The amino acid sequence of the portion constituting the N-terminal of the li pase peptide is determined for this highly purified lipase using a liquid phase protein sequencer (Beckman System 890ME, manufactured by Beckman, Inc.). In this manner, it was confirmed that the amino acid sequence at least of said portion was identical to the N-terminal amino acid sequence of lipase obtained by a genetic engineering technique. The amino acid sequence determined in this way from the base sequence (II) was equivalent to the amino acid sequence of formula (I). In the amino acid sequence of formula (I), the amino acid residue at the upstream side of N-terminal Ala may be one or more 13 amino acids. Given as preferred examples of this amino acid residue are a hydrogen atom, a Met, or a signal peptide. The C-termial Val may constitute the terminal of the peptide as is, or may have a group at its downstream such as an acid amide or one or more amino acid residues.

The transformant thus obtained, when cultured in a nutrient medium, can produce a large amount of polypeptide having lipase activity in a stable manner.

Culturing of the host microorganism which is a transformant is carried out under the conditions determined taking the nutrient-physiological characteristics of the host microorganism into consideration. In most of the cases liquid culturing is employed. For industrial scale production, however, culturing under deep aerobic stirring conditions is more advantageous. A wide variety of nutrients conventionally used for bacterial culture can be used for culturing the host microorganism. Specifically, any carbon compounds which are utilizable can be used as carbon sources. These include, for example, glucose, sucrose, lactose, maltose, fructose, molasses, and the like. As nitrogen sources, any available nitrogen compounds can be employed, including peptones, meat extracts, yeast extracts, casein hydrolyzates, and the like. Other ingredients, including salts such as phosphates, carbonates, and sulfates, as well as salts 14 of magnesium, calcium, potassium, iron, manganese, zinc, and the like. and certain types of amino acids or vitamins, may be used as appropriate.

The culturing temperature can be varied in a range in which the bacteria can grow and produce lipase. A preferable temperature range is 20 to 420C for Escherichia coli, 30 to 3711C for Bacillus subtillis, and 25 to 350C for Saccharomyces cerevislae. The culturing time may be varied to some degree depending on the culturing conditions. Basically, the culturing is terminated at the time when the yield of lipase reaches maximum. In usual practice, this takes about 12 to 48-hours when the host microorganism is Escherichia coli, 18 to 42 hours when the host microorganism is Bacillus subtillis, and 24 to 48 hours when the host microorganism is Saccharomyces cerevislae. It is possible to change the pH of the culture media within the range in which the bacteria can grow and produce lipase. The especially preferable pH range is about 6 to 8 when the host microorganism is Esche.richia col!, about 7 when the host microorganism is Bacillus subtillis, and about 5 to 7 when the host microorganism is Saccharomyces cerevislae.

Lipase may be served for use in the form of a culture broth as it contains bacteria. Lipase contained in the culture broth, however, is generally used after being separated from the cells by filtration, centrifugation, or the like means. When lipase is contained within the cells, the cells are first separated by means of filtration or centrifugation. The collected cells are then destroyed either by mechanical means or enzymatic means using lysozyme or the like, and to the suapension a chelating agent such as EDTA and/or a suitable surfactant is added, as required, to solubilize lipase, thus allowing the collection of lipase as an aqueous solution.

The solutions containing lipase thus obtained are then condensed by evaporation in vacuo or by using a filter, and subjected to salting-out treatment with ammonium sulfate, sodium sulfate, or the like, or to fractional precipitation using a hydrophilic organic solvent such as methanol, ethanol, acetone, or the like. The precipitate is dissolved into water, and the solution is dialyzed through a semipermeable membrane to eliminate low molecular weight impurities. Alternatively, the precipitate is refined by means of gel filtration, adsorption chromatography, ion-exchange chromatography, or the like. Purified lipase is produced from the lipase-containing solution obtained by using these various means through vacuum evaporation,, Iyophilization, or the like.

The activity of the lipase thus prepared is measured according to the following method:

16 <Reaction mixture> 0.2 M Tris-hydrochloride buffer (pH 7.5) 0.2 (ml) 27.5 mM Dilinoleoyl glycerol 0.05 (15% Triton X-100) mM CaC12 0.01 mM ATP 0.005 mM CoASH 0.005 Acyl CoA Synthetase (50 Ulnzl) 0.01 Water Balance Total 0.5 (ml) Add 0.5 ml of the reaction mixture having the above composition to a-test tube and preincubate the temperature at 37C for 2-3 minutes. Add 50 gl of an enzyme solution (10 mM PIPES-NaOH buffer, containing 0.1% bovine serum albumin; pH 7.3) and incubate at 370C for 10 minutes. Add 0.5 mI of 10 mM N-ethylmaleimide and 0.5 ml of a reagent (R-2) having the composition presented below, and incubate at 371C for a further 5,minutes. Stop the reaction by adding 1.5 ml of 0.5% sodium dodecyl sulfate and measure the absorbance at 550 nm. The activity of the substance to produce 1 g mol of linoleic acid per minute was taken as 1 unit (U) - <Reagent (R-2) Composition> 0.2 M PIPES-NaOH buffer (pH 7.3) 0.05 (M1) 0.3% 4-aminoantipyrine 0.05 0.3% TOOS 1 0.05 Peroxidase (45 Ulml) 0.05 Acyl CoA oxidase (500 Ulml) 0.02 0.2 M ATP 0.01 Water 0.27 1 N-ethyl-N-(2-hydroxy-3-sulfopropyl-m-toluidine) In the description of this specification, amino acids, peptides, nucleic acids, and nucleic acidrelated compounds are abbreviated according to the prevailing standards. Some examples of the abbreviation are listed below. All designations of amino acids denote the L-isomers.

DNA: Deoxyribonucleic acid RNA: Ribonucleic acid A: Adenine T: Thymine G: Guanine C: Cytosine Ala: Alanine Arg: Arginine Asn: Asparagine Asp: Aspartate Cys: Cysteine G1n: Glutamine 18 Glu: Glutamate Gly: Glycine His: Histidine Ile: Isoleucine Leu: Leucine Lys: Lysine Met: Methionine Phe: Phenylalanine Pro: Proline Ser: Serine Thr: Threonine Trp: Tryptophan Tyr: Tyrosine Other features of the invention will become apparent in the course of the following description of the exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof.

Example 1 [Preparation of Chromosomal DNA] A chromosomal DNA was prepared from strain Chromobacterium viscosum varietas pararipoliticum (Japanese Patent Laid-open No. 29787/1971) according to the following method. The strain was cultured with shaking overnight at 370C in 150 ml of a bouillon medium containing 0.5% sodium thiosulfate. The cultured broth was centrifuged at 3,000 rpm for 10 19 minutes to collect the cells. The cells were suspended into 5 ml of a solution containing 10% saccharose, 50 mM Tris hydrochloric acid (pH 8.0), and 50 mM EDTA. To the suspension, 1 ml of a lysozyme solution (10 mglml) was added, and the mixture was incubated at 370C for 15 minutes, followed by the addition of 1 ml of 10% SDS (sodium dodecylsulfate). To the suspension thus obtained an equal volume of a mixed solvent of chloroform and phenol (1:1) was added, and the mixture was stirred and centrifuged at 10,000 rpm for 3 minutes to separate the water and solvent layers. To the separated water layer, a 2-fold volume of ethanol was added slowly, and the mixture was stirred slowly with a glass rod so as to cause the DNA to wind around the rod. The DNA separated in this manner was dissolved into 10 ml of a solution containing 10 mM Tris hydrochloric acid (pH 8.0) and 1 mM EDTA (such a solution is hereinafter referred to as 'ITE"). This solution was treated with an equal volume of a chloroform- phenol mixed solvent, and was again centrifuged to separate the water layer. To this water layer, a 2-fold volume of ethanol was further added, and the DNA was again separated from the mixture in the same manner as described above. This DNA was then dissolved into 2 ml of TE. Example 2 [Preparation of pACYC184 Plasmid DNA] Esche.richia coli pM191 carrying pACYC184 therein (J. Bacteriol,, 134, 1141 (1981); ATCC 37033) was cultured with shaking in 1 1 of BHI medium (produced by Difco Co.). When the turbidity of the broth reached OD660 1.0, spectinomycin was added at a final concentration of 300 gglml. Shaking of the broth at 37C was continued for at least 16 hours. Upon termination of the shake culturing the broth was centrifuged at 3,000 rpm for 10 minutes to collect the cells. The plasmid DNA from the collected cells was prepared according to the lysozyme-SDS method and the cesium chloride-ethidium bromide method [Maniatis et al, Molecular Cloning, 86-94, Cold Spring Harbor (1982)]. Example 3 [Construction of plasmid pLIP1 having a lipase gene] (1) Two (2) gl (about 0.5 gg) of Chromoba cterium viscosum varietas pararipoliticum chromosomal DNA prepared in Example 1, 1 gl of a 10-fold concentration of H-buffer [Maniatis et al, Molecular Cloning, 104, Cold Spring Harbor'(1982)], 1 gl of BglII (10 unit/gl; produced by Takara Shuzo Co., Ltd.). and 6 gl of water were mixed and incubated at 370C for 1 hour for digestion. Plasmid pACYC184 DNA (about 0.3 gg) which was separately prepared was digested using BamHI according to the same method. To this was added 0.6 unit of alkaline phosphatase (produced by Takara Shuzo Co., Ltd.; hereinafter referred to from time to time as 21 "BAP") and the mixture was incubated at 651C for 1 Z:

hour. The two digested DNA solutions thus prepared were mixed together, and to this mixed DNA solution, 0.1 volume of 3M sodium acetate was added. Subsequently, the solution was treated with an equal volume of a chloroform-phenol mixed solvent and centrifuged to separate the water layer. To the water layer a 2-fold amount of ethanol was added, and the precipitated DNA was collected by means of centrifugation and dried in vacuo. The dried DNA was dissolved into 89 gl of water, and to this 10 gl of a 10-fold concentration ligation buffer [0.5 M Tris hydrochloric acid (pH 7.6), 0,1 M MgCl2r 0-1 M dithiothreitol, 10 mM spermidine, 10 mM ATP) and I gl of T4 DNA ligase (350 unit; produced by Takara Shuzo Co., Ltd.) were added and mixed, and the mixtur e was allowed to stand at 40C overnight. This DNA solution was treated with a chloroform-phenol mixture, and the DNA was precipitated with ethanol, dried in vacuo, and dissolved into 10 gl of TE.

(2) Escherichia coli DHl (Stock No. ME 7778, ATCC 27325; provided by National Genetic Research Institute] was cultured in 100 ml of BHI medium [Brain Heart Infusion, produced by Difco Co.], collected during logarithmic growth phase by centrifugation (10,000 rpm, 2 minutes), and suspended into 40 ml of an ice-cold solution (pH 5.8) containing 30 mM potassium acetate, 22 mM RbCl,, 10 mM CaC12, 50 mM MnC12, and 15% glycerine. After having been allowed to stand at OC for 5 minutes, the suspension was centrifuged to remove the supernatant. The cells were suspended into 4 ml of a solution containing 10 mM MOPS buffer (produced by Dotite Co.), 75 mM CaCl2,, 10 mM RbCl,, and 15% glycerine (pH 6.5), andthe suspension was left at OOC for 15 minutes to obtain competent cells.

(3) To 200 gl of the Escherichia coli cell suspension 10 gl of the DNA solution prepared in (1) above was added. After the mixture was allowed to stand at OC for 30 minutes, 1 ml of BHI medium was added to it, and the mixture was kept at 370C for 90 minutes. An aliquot of the mixture (100 ml) was spread on a BHI agar plate containing 25 gglml of chloramphenicol, and cultured overnight at 37C to produce transformants. These transformants were replicated on a Crosslee agar medium (produced by Eiken Chemical Co., Ltd.), and was further cultured overnight at 370C.

About 50,000 colonies of transformants thus produced were investigated, and 1 blue colored strain was obtained. This strain was named Escherichia coli DHI pLIP1.

Example 4 [Mapping of pLIP1 and determination of the base sequence of the lipase gene] 23 Plasmid pLIP1 DNA was prepared from the Esche.richia coli DH1 pLIP1 in the same manner as that employed for the preparation of pACYC 184.

A cleavage map of the pLIP1 DNA thus obtained was prepared using the restriction endonucleases BamHI, ClaI, EcoRV, SalI. MluI, PstI, and.XhoI (all produced by Takara Shuzo Co., Ltd.). The results are shown in Figure 1. Example 5 One (1) gl of a 10-fold concentration M buffer [Maniatis et al, Molecular Cloning, 104,, Cold Spring Harbor (1982)] and 1 gl of Cla 1 (10 units/gl, produced by Takara Shuzo Co., Ltd.) were added to about 1.0 gg (8 gl) of plasmid pLIP1 DNA which was prepared from the strain Escherichia coli DHI pLIP1 according to the same method as described in Example 2 to digest the DNA at 37C for 2 hours. About 3.2 kb of DNA fragments were separated by electrophoresis. The DNA fragments were treated by phenol and precipitated in ethanol. This precipitate was dissolved into 20 gl of TE [containing 10 mM Tris hydrochloric acid and 1 mM EDTA (pH 8)]. To about 0.2 gg (1 gl) of pBR322 plasmid DNA which was separately prepared and purified according to the same manner as in Example 2 were added 1 gl of a 10-fold X buffer, 7 gl of water, and 1 gl of ClaI (10 units/gl, produced by Takara Shuzo Co., Ltd.). After digesting the DNA at 370C for 2 hours, 5 gl of 1 M Tris 24 hydrochloric acid buffer (pH 8.0), 80 gl of water, and 5 gl of alkaline phosphatase (0.5 u/gl; produced by Takara Shuzo Co., Ltd.) were added and reacted for 2 hours at 65C. After the reaction, the resulting product was treated three times with an equal volume of a chloroform-phenol mixed solvent and the DNA was recovered by ethanol precipitation. The DNA thus recovered was dissolved into 20 gl of TE. Each 5 gl of 2 kinds of DNA solutions thus prepared were mixed, and to this mixture were added 2 gl of a 10-fold concentration ligation buffer, 7 gl of water, and 1 gl of T4 DNA ligase (175 unit/gl, produced by Takara Shuzo Co., Ltd.), and the mixture was allowed to stand at 4C overnight. This DNA solution was treated with a chloroform-phenol mixture, and the DNA was precipitated with ethanol. The precipitate was dissolved into 10 gl of TE, and Escherichia coli DHl competent cells prepared according to the same method as in example 3 (2) were transformed. The transformants were spread on an agar medium containing 50 gglml of ampicillin, and cultured overnight at 370C. Colonies produced were replicated on a Crosslee agar medium in the same manner as in Example 3 (3) to obtain a lipase- producing Esche.richia coli. This strain was named Escherichia coli DH1 pLIP10, and deposited with Fermentation Research Institute, Agency of Industrial Science and Technology; deposition No. 2266, FERM BP-2266.

This strain, after purification, was cultured in BHI medium at 370C overnight. The lipase-producing ability of the strain was determined on the cultured cells using the aforementioned lipase assay method to find that the strain has about 0.01 Ulml activity.

The plasmid held by this strain was separated in the same manner as in Example 2. The plasmid containing a lipase gene and a plasmid pBR322 gene was named pLIP10.

Example 6 [Mapping of pLIP10 and determination of the base sequence of the lipase gene] pLIP10 plasmid DNA was prepared from the Escherichia coli DH1 pLIP10 in the same manner as that employed for the preparation of pACYC 184.

A cleavage map of the pLIP10 DNA thus obtained was prepared using the restriction endonucleases BamHI, SalI, XhoI, and PstI, (all produced by Takara Shuzo Co., Ltd.). The results are shown in Figure 2. The base sequence of the DNA encoding lipase gene was determined by means of the dideoxy method [Sclence, 214, 1205-1210 (1981)] using M13 phage. The base sequence of the lipase gene and the amino acid sequence were those shown as formulae (I) and (II).

Example 7 [preparation of lipase] Escherichia col! DH1 pLIP10 was cultured in 20 1 of BHI medium (produced by Difco Co.) at 370C for 18 26 hours using a 30-liter jar fermenter. The cultured cells were collected by centrifugation at 5,000 rpm for 10 minutes. The cells were washed with 2 1 of physiological saline and suspended into 2 1 of 0.1 mM phosphate albumin buffer (containing 13.61 g of KH2PO41 3.92 g of NaOH, 1 g of bovine fetus serum albumin, 1 g of NaN3, and 1 1 of water; pH 8.0). To the suspension thus prepared lysozyme, EDTA-2Na, and Triton X-100 were added at a concentration of 1 mglml, 2 mM, and 0.1%, respectively. After incubation at 370C for 30 minutes, the mixture was centrifuged at 5,000 rpm for 10 minutes to collect the resulting supernatant. 1.9 1 of this supernatant was subjected to acetone (50-80%) precipitation. The precipitate was collected by centrifugation at 5,000 rpm for 30 minutes, dissolved into 100 ml of phosphate albumin buffer, and was then subjected to DEAE-Sephallose CL-6B ion exchange chromatography to collect the active fraction. This fraction was desalted and freeze dried to obtain a powdery product. This enzyme sample was found to have a lipase activity of 350 U as a result of the lipase activity measurement according to the above-mentioned method.

The present invention 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 27 triglyceride, as a reagent for quantitative analysis of triglyceride, and as a catalyst for oil and fat reforming process using ester exchange reaction.

Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

i t, 28 4

Claims (9)

1. 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 through the 319th amino acid (Val):

AlaAspThrTyrAlaAlaThr-krgTyrProVal1leLeuValHisGlyLeuAlaGlyThr AsDLysPheAlaAsnValValAspTyrTrDTy--GlyIleGlnSerAsDLeuGlnSerHis GlyAlaLysValTyrValAlaAsnLeuSerGlyPheGlnSerAspAspGlyProAsnGly ArgGlyGluGlnLeuLeuAlaTyrValLysGlnValLeuAlaAlaThrGlyAlaThrLys ValAsnLeuIleGlyHisSerGlnGlyGlyLeuThrSerArgTyrValAlaAlaValAla ProGlnLeuValAlaSerValThrThrIleGlyThrProHisAraGlySerGluPheAla AspPheValGlnAspValLeuLysThrAspProThrGlyLeuSerSerThrValIleAla A!aPheValAsnValPheGl,,rThrLeuValSerSerSerHisAsnThrAspGlrlAspAla LeuAlaAlaLeuA.,--gThrLeuThrThr.A-laGlnThrAlaThrTyrAsnArgAsnPhePro SerAlaGlyLeuGlyAlaProGlySerCysG!nThrGlyAlaAlaThrGluThrValGly GlySerGlnHIsLeuLeuTyrSerTrpGlyGlyThrA!aIleGlnProThrSerThrVal LeuGlyValThrGlyAlaThrAspThrSerThrGlyThrLeuAspValAlaAsnValThr AsDProSerThrLeuAlaLeuLeuAlaThrGlyAlaValMetIleAsnArgAlaSerGly GlnAsnAspGlyLeuValSerA--gCysSerSerLauPheGlyGlnValIleSerThrSer Tyrl, lisTrpAsnHisLeuAspGluIleAsnGlnLeuLeuGlyValArgGlyAlaAsnAla G!uAspProValAlaValIleArgThrHisValAsnA.rgLeuLysLeuGlnGlyVa1 (1)

2. The DNA according to Claim 11 wherein said base sequence is comprised of consecutive 957 bases starting from 51-end represented by the following base sequence (II):

29 20 30 40 50 60 GCGGACACCTACGCGGCGACGCGCTATCCGGTGATCCTCGTCCACGGCCTCGCGGGCACC 80 90 100 110 120 GACAAGTTCGCGAACGTGGTGGACTATTGGTACGGAATCCAGAGCGATCTGCAATCGCAT 140 150 160 170 180 GGCGCGAAGGTGTACGTCGCGAATCTCTCGGGATTCCAGAGCGACGACGGGCCGAACGGC 200 210 220 230 240 CGCGGCGAGCAGCTGCTCGCCTACGTGAAGCAGGTGCTCGCGGCCACCGGCGCGACCAAG 250 260 270 280 290 300 GTGAACCTGATCGGCCACAGCCAGGGCGGCCTGACCTCGCGCTACGTCGCGGCCGTCGCG 310 320 330 340 350 360 CCGCAACTGGTGGCCTCGGTGACGACGATCGGCACGCCGCATCGCGGCTCCGAGTTCGCC 370 380 390 400 410 420 GACTTCGTGCAGGACGTGCTGAAGACCGATCCGACCGGGCTCTCGTCGACGGTGATCGCC 430 440 450 460 470 480 GCCTTCGTCAACGTGTTCGGCACGCTCGTCAGCAGCTCGCACAACACCGACCAGGACGCG 490 500 510 520 530 540 CTCGCGGCGCTGCGCACGCTCACCACCGCGCAGACCGCCACCTACAACCGGAACTTCCCG 550 560 570 580 590 600 AGCGCGGGCCTGGGCGCGCCCGGTTCGTGCCAGACGGGCGCCGCGACCGAAACCGTCGGC 610 620 630 640 650 660 GGCAGCCAGCACCTGCTCTATTCGTGGGGCGGCACCGCGATCCAGCCCACCTCCACCGTG 670 680 690 700 710 720 CTCGGCGTGACCGGCGCGACCGACACCAGCACCGGCACGCTCGACGTCGCGAACGTGACC 730 740 750 760 770 780 GACCCGTCCACGCTCGCGCTGCTCGCCACCGGCGCGGTGATGATCAATCGCGCCTCGGGG 790 800 810 820 830 840 CAGAACGACGGGCTCGTCTCGCGCTGCAGCTCGCTGTTCGGGCAGGTGATCAGCACCAGC 850 860 870 880 890 900 TACCACTGGAACCATCTCGACGAGATCAACCAGCTGCTCGGCGTGCGCGGCGCCAACGCG 910 920 930 940 950 GAAGATCCGGTCGCGGTGATCCGCACGCACGTGAACCGGCTCAAGCTGCAGGGCGTG (II)

3. A vector comprising DNA as defined in Claim 1.

h

4. The vector according to Claim 3. which vector is plasmid pLIP1 or pLIP10.

5. A transformant having DNA which is foreign with respect to the host microorganism and is defined in Claim 1.

6. The transformant according to Claim 5. wherein the host microorganism is a microorganism belonging to the strain Escherichia coli.

7. A process for preparing a polypeptide comprising:

culturing a transformant having DNA which is foreign with respect to the host microorganism and is defined in Claim 1 to cause the transformant to express the genetic information of said DNA, and collecting the polypeptide having lipase activity from the culture broth.

8. A process according to Claim 7, wherein said host microorganism is a microorganism belonging to the strain Escherichla col!.

9. 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 through the 319th amino acid (Val):

AlaAspThrTyrAlaAlaThrArgTyrProValIleLeuValHisGlyLeuAlaGlyThr AspLysPheAlaAsnVa1ValAspTyrTrpTyrGlyIleGlnSerAspLeuGlnSerH!s GlyAlaLysValTyrValAlaAsnLeuSerGlyPheGlnSerAspAspGlyProAsnGly 31 ArgGlyGluGlnLeuLeuAlaTyrValLysGlnValLeuAlaAlaThrGlyAlaThrLys ValAsnLeuIleGlyHisSerGlnGlyGlyLeuThrSerArgTyrValAlaAlaValAla ProGlnLeuValAlaSerValThrThrIleGlyThrProHisArgGlySerGluPheAla AspPheValGlnAspValLeuLysThrAspProThrGlyLeuSerSerThrValIleAla AlaPheValAsnValPheGlyThrLeuValSerSerSerHisAsnThrAspGlnAspAla LeuAlaAlaLeuArgThrLeuThrThrAlaGlnThrAlaThrTyrAsnArgAsnPhePro SerAlaGlyLeuGlyAlaProGlySerCysGlnThrGlyAlaAlaThrGluThrValGly GlySerGlnHIsLeuLeuTyrSerTrpGlyGlyThrAlaIleGlnProThrSerThrVa1 LeuGlyValThrGlyAlaThrAspThrSerThrGlyThrLeuAspValAlaAsnValThr AspProSerThrLeuAlaLeuLeuAlaThrGlyAlaValMetIleAsnArgAlaSerGly GlnAsnAspGlyLeuValSerArgCysSerSerLeuPheGlyGlnValIleSerThrSer TyrHisTrpAsnHisLeuAspGluIleAsnGlnLeuLeuGlyValArgGlyAlaAsnAla GluAspProValAlaValIleArgThrHisValAsnArgLeuLysLeuGlnGlyVa1 (I) 1 Published 1989 atThe Patent Office, State House, 66.71 High HolborTLLondon WClR 4TP. Further copies maybe obtainedfrom The Patent Office. Wes Branch, St Mary Cray, Orpington, Kent BR5 3RD. Printed by Multiplex techniques ltd, St Mary Cray, Kent, Con. 1187