JP2639677B2 - DNA having lipase genetic information - Google Patents

Description

The present invention relates to a DNA having lipase genetic information, and more specifically, a DNA having lipase genetic information containing a nucleotide sequence encoding a 319 amino acid sequence. And a vector having the same, a transformant carrying the same, a polypeptide having the lipase activity having the amino acid sequence, and a method for producing the polypeptide.

[Problems to be solved by conventional technology and invention]

Lipase is an enzyme that catalyzes the reaction of hydrolyzing triglyceride or diglyceride into glycerin and fatty acids or catalyzes the reversible reaction.It is widely used for digestive medicines, clinical test reagents, production of various fatty acids, or modification of glycerides. Is used in various fields. As the origin of lipase, pancreatic juice and gastric juice of animals, seeds of plants, microorganisms such as yeast and bacteria are known, and lipases derived from microorganisms are widely used from the viewpoint of supply stability.

By the way, properties required for lipases used in reactions such as clinical test reagents and fatty acid production include high stability against pH, heat, surfactants and the like.
In order to obtain a lipase having such properties, a search for a new microorganism (eg, Japanese Patent Publication No. 46-29787) has been conducted.

By the way, a number of DNAs encoding various physiologically active polypeptides have been reported with the recent advances in genetic recombination techniques, and as lipase-related genetic engineering, DNA from lipase-producing bacteria belonging to the genus Pseudomonas and Bacillus, And a method for producing lipase using the same (JP-A-62-228279), a DNA from a lipase-producing bacterium belonging to the genus Aspergillus, and a method for producing lipase using the same (JP-A-62-272988). ing.

[Means for solving the problem]

Thus, the present inventors have conducted intensive studies to obtain DNAs encoding such useful lipases by genetic engineering techniques, and as a result, have prepared recombinant DNAs using DNAs isolated from lipase-producing bacteria belonging to the genus Chromobacterium. However, it has been found that a polypeptide having a conventional lipase activity can be obtained as a base sequence encoding the amino acid sequence thereof, a novel DNA having a significantly different amino acid sequence, and a polypeptide exhibiting stability. Was completed.

That is, the present invention provides a DNA having lipase genetic information, comprising a nucleotide sequence encoding the amino acid sequence from the N-terminal side to the amino acid sequence from the 1st (Ala) to the 319th (Val) shown in FIG. A vector having the same, a transformant carrying the same, a polypeptide having the amino acid sequence and exhibiting lipase activity, and a method for producing the polypeptide.

The DNA of the present invention is produced, for example, using a gene recombination technique as follows. That is, after separating and purifying DNA from a microorganism that is a donor of a lipase gene having lipase-producing ability, for example, a lipase-producing bacterium belonging to the genus Chromobacterium, ultrasonically, DN cut with a restriction enzyme or the like.
The A fragment and the linearly cut expression vector are ligated and closed with DNA ligase or the like at the blunt or cohesive ends of both DNAs, and the resulting recombinant DNA vector is transferred to a replicable host microorganism, A microorganism holding the recombinant DNA vector obtained by cleaning using the marker of the vector and the activity of the lipase as an index is cultured, the recombinant DNA vector is separated and purified from the cultured cells, and then the recombinant vector is purified from the recombinant vector. It is produced by collecting the DNA of the present invention having lipase genetic information.

Microorganisms that are donors of the lipase gene include Chromobacterium viscosum varietas paralipolitic (Chromobacterium viscosum var.paralipolitic).
um).

In addition, a transformed microorganism to which lipase-producing ability has been imparted by a gene recombination technique may be used as a lipase gene donor.

DNA derived from a microorganism as a gene donor is collected as follows. That is, any of the above-mentioned bacteria that are donor microorganisms, for example, aerated and stirred for about 1 to 3 days in a liquid medium, and the resulting culture is collected by centrifugation,
Then, the lysate is lysed to prepare a lysate containing the lipase gene. As a lysis method, for example, treatment with a cell wall lysing enzyme such as lysozyme or β-glucanase is performed, and other enzymes such as a protease or a surfactant such as sodium lauryl sulfate are used in combination, if necessary. May be performed in combination with the lysis method described above.

In order to separate and purify DNA from the lysate obtained in this manner, methods such as protein removal treatment by phenol extraction, protease treatment, ribonuclease treatment, alcohol precipitation, and centrifugation are appropriately combined according to a conventional method. It does by doing.

The method of cutting the separated and purified microbial DNA can be performed, for example, by sonication, treatment with a restriction enzyme, and the like.However, in order to facilitate the binding of the obtained DNA fragment to the vector DNA, a restriction enzyme, particularly a specific enzyme, is used. Suitable are type II restriction enzymes which act on the nucleotide sequence, such as, for example, EcoRI, HindIII, BamHI.

Further, the vector to be used is preferably one constructed by artificially treating a phage or a plasmid DNA capable of autonomous growth in the cells of a host microorganism, so as to be suitable for a gene recombinant vector.

Examples of phages include Escherichia coli (Es
cherichia coli) is used as the host microorganism.
λC, λgt and λB can be used.

Examples of plasmids include, for example, Escherichia
When E. coli is used as the host microorganism, pBR322, pBR325, pACYC1
84, pUC12, pUC13, pUC18, pUC19, etc. can be used.
When subtilis (Bacillus subtilis) is used as the host microorganism, pUB110, pC194 and the like can be used. When Saccharomyces cerevisiae is used as the host microorganism, YRp7, pYC1, YEp13, pJDB, Y
Ip1 etc. can be used. In addition, shuttle vectors that can autonomously propagate in cells of two or more host microorganisms such as Escherichia coli and Saccharomyces cerevisiae can also be used. Such a vector is preferably cleaved with the same restriction enzymes used for cutting the above-described microorganism DNA as a lipase gene donor to obtain a vector fragment.

The method of binding the microbial DNA fragment to the vector fragment may be a method using a known DNA ligase.For example, after annealing the cohesive end of the microbial DNA fragment and the cohesive end of the vector fragment, a suitable DNA ligase is used. By the action, a recombinant DNA of the microorganism DNA fragment and the vector fragment is prepared. If necessary, after annealing, the DNA can be transferred to a host microorganism, and recombinant DNA can be prepared using in-vivo DNA ligase.

As the host microorganism, any recombinant DNA can be stably and autonomously proliferated, as long as it can express the trait of the exogenous DNA.For example, when the host microorganism is Escherichia coli, Escherichia coli DH1, Escherichia Koli
HB101, Escherichia coli W3110, Escherichia coli C6
00 and the like, and when the host microorganism is Bacillus subtilis, Bacillus subtilis 207-25 [Gene
34, pages 1-8, 1985], Bacillus subtilis
207-21 [Journal of Biochemistry (Jou
rnal of Biochemistory) Vol. 95, pp. 87-93, 1984
Years], Bacillus subtilis BD170 [Natur
e) p. 293, p. 481-483, 1981], Bacillus subtilis M strain (ATCC6051), etc., and when the host microorganism is Saccharomyces cerevisiae, Saccharomyces cerevisiae AH-22 [Gene, Gene. Volume 39, Vol. 117-1
20, 1985), Satsukaromyces cerevisiae BWG1-
7A [Molecular & Cellular Biology, Volume 6, 355-367
1986] can be used.

As a method for transferring the recombinant DNA to the host microorganism, for example, when the host microorganism is a microorganism belonging to the genus Escherichia, transfer the recombinant DNA in the presence of calcium ions, the host microorganism is a microorganism belonging to the genus Bacillus In the case of, the competent cell method or the protoplast method can be adopted, and the microinjection method may be further used.If the host microorganism is a microorganism belonging to the genus Saccharomyces, the protoplast method, or A lithium acetate method or the like may be used.

Selection of the presence or absence of transfer of the target recombinant DNA to the host microorganism may be performed by searching for a microorganism capable of simultaneously expressing the drug resistance marker and the lipase of the vector holding the target recombinant DNA, for example, based on the drug resistance marker. A microorganism that grows on a selection medium and produces lipase may be selected.

The thus-selected recombinant DNA having the lipase gene can be easily taken out from the transformed microorganism and transferred to another host microorganism. Also,
The lipase gene is cut out of the recombinant DNA holding the lipase gene by cutting with a restriction enzyme, etc.
The vector fragment obtained by cleavage by the same method as described above can be ligated and transferred to a host microorganism.

In the case of lipase mutein, which is an artificially mutated gene prepared by a genetic engineering technique from the lipase gene of the present invention having essentially lipase activity, first, the artificially mutated gene is amplified by using the various methods described above, Specifically, the lipase mutein can be produced by inserting the mutant gene into a vector to prepare a recombinant DNA and transferring it to a host microorganism.

The nucleotide sequence of the DNA of the present invention thus obtained is Science 21
4,1205-1210 (1981) and can be determined by the dideoxy method. For example, using a bacterium belonging to the genus Chromobacterium as a lipase gene donor,
The nucleotide sequence of the lipase gene in the plasmid obtained using Escherichia coli as the host microorganism is as shown in FIG.

In FIG. 4, the 5 'terminal upstream of the codon GCG representing Ala at position 1 at the N-terminus may be any codon that encodes an amino acid. It may have more than one, but preferably a polydeoxyribonucleic acid corresponding to ATG or a signal peptide. Further, the 3 ′ terminal side downstream of GTG representing C-terminal Val may be any translation termination codon or a codon encoding an amino acid,
Further, one or more codons encoding amino acids may be provided on the 3'-terminal side. In this case, it is preferable that the plurality of codons further have a translation termination codon at the 3'-terminal.

The amino acid sequence of a polypeptide produced by expressing the DNA of the present invention can be predicted and determined from the nucleotide sequence of the DNA. The partial amino acid sequence constituting the N-terminal part of the peptide can be determined as follows. That is, a lipase gene-donating microorganism having lipase-producing ability is cultured in a nutrient medium to produce and accumulate lipase in the cells.After completion of the culture, the cells are collected by means such as excess or centrifugation. Then, the cells are destroyed by a mechanical method or an enzymatic method such as lysozyme, and if necessary, EDTA and / or a suitable surfactant are added, so that the lipase is solubilized and separated and collected as an aqueous solution. Is done. The aqueous solution of the lipase thus obtained is concentrated or treated without concentration by ammonium sulfate fractionation, gel permeation, adsorption chromatography, ion-exchange chromatography to obtain a high-purity lipase. Using a purity lipase, a partial amino acid sequence constituting the N-terminal portion of the peptide which is a lipase is determined by a liquid-phase protein sequencer (BECKMAN System 890ME). It was also confirmed that at least the partial amino acid sequence was identical to the N-terminal partial amino acid sequence of lipase obtained by genetic manipulation. The amino acid sequence determined as described above from the base sequence shown in FIG. 4 is as shown in FIG. In the amino acid sequence of FIG. 3, the upstream amino acid residue represented by Ala at the N-terminus consists of one or more amino acids, and preferably includes a hydrogen atom, Met or a signal peptide. Further, the downstream one represented by C-terminal Val may be as it is or may be an acid amide,
It may be one or more amino acid residues.

The transformant thus obtained stably produces a polypeptide having a large amount of lipase activity when cultured in a nutrient medium.

The culture form of the host microorganism, which is a transformant, may be selected in consideration of the nutritional and physiological properties of the host.In most cases, the culture is usually performed in liquid culture or industrially by deep aeration and stirring culture. It is advantageous to do so. As the nutrient source of the medium, those commonly used for culturing microorganisms can be widely used. The carbon source may be any assimilable carbon compound, and for example, glucose, sucrose, lactose, maltose, fructose, molasses and the like are used.
Any available nitrogen compound may be used as the nitrogen source. For example, peptone, meat extract, yeast extract, casein hydrolyzate and the like are used. In addition, salts such as phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like are used as necessary.

The culture temperature can be appropriately changed within the range in which the bacteria grow and produce lipase.When the host microorganism is Escherichia coli, it is preferably about 20 to 42 ° C., and when the host microorganism is Bacillus subtilis, it is preferably used. The temperature is about 30 to 37 ° C, and preferably 25 to 35 ° C when the host microorganism is Saccharomyces cerevisiae. The cultivation time varies somewhat depending on the conditions, but it is sufficient to terminate the cultivation at an appropriate time in anticipation of the time when the lipase reaches the maximum yield, and usually about 12 to 48 hours when the host microorganism is Escherichia coli. And the host microorganism is Bacillus subtilis
It is about 18 to 42 hours, and about 24 to 48 hours when the host microorganism is Saccharomyces cerevisiae. The medium pH can be appropriately changed within the range in which the bacteria grow and produce lipase,
Particularly preferably, the pH is about 6 to 8 when the host microorganism is Escherichia coli, the pH is about 7 when the host microorganism is Bacillus subtilis, and the pH is about 5 to 7 when the host microorganism is Saccharomyces cerevisiae.

The lipase in the culture can be used by directly collecting and using the culture solution containing the cells, but generally, if the lipase is present in the culture solution, filtration, centrifugation, etc. Is used after separating the lipase-containing solution from the microbial cells. If lipase is present in the cells, the obtained culture is collected by filtration or centrifugation, and then the cells are collected.Then, the cells are disrupted by a mechanical method or an enzymatic method such as lysozyme. If necessary, a chelating agent such as EDTA and / or a surfactant are added to solubilize the lipase and separate and collect it as an aqueous solution.

The lipase-containing solution thus obtained is, for example,
Concentration under reduced pressure, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate or the like, or precipitation by a fractional precipitation method with a hydrophilic organic solvent such as methanol, ethanol, acetone or the like may be used. The precipitate can then be dissolved in water and dialyzed through a semi-permeable membrane to remove lower molecular weight impurities. Further, the solution was purified by gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, and the lipase-containing solution obtained using these means was purified by processes such as concentration under reduced pressure and freeze-drying. Lipase can be obtained.

The method for measuring the activity of the lipase thus obtained is as follows.

<Reaction solution composition> 0.2 M Tris-HCl buffer (pH 7.5) 0.2 (ml) 27.5 mM dilinoleoyl glyceride (15% Triton X-10
0) 0.05 50 mM CaCl ₂ 0.01 200 mM ATP 0.005 100 mM CoASH 0.005 Acyl CoA synthetase (50 U / ml) 0.01 Amount of water to 0.5 ml 0.5 ml of the reaction solution having the above composition is dispensed into a test tube.
After pre-warming for ~ 3 minutes, the enzyme solution 50μ (10mM PIPES-Na
OH buffer, pH 7.3, 0.1% bovine serum albumin)
The reaction was carried out at a temperature of 10 ° C. for 10 minutes.
Incubate at 37 ° C for 5 minutes. Next, the reaction is stopped by adding 1.5 ml of 0.5% sodium dodecyl sulfate, and colorimetrically determined at a wavelength of 550 nm. The activity of releasing 1 μmole of linoleic acid per minute is defined as 1 unit (1 U).

<Reagent composition (R-2)> 0.2 M PIPES-NaOH buffer (pH 7.3) 0.05 (ml) 0.3% 4-aminoantipyrine 0.05 0.3% TOOS ^{* )} 0.05 Peroxidase (45 U / ml) 0.05 Acyl CoA oxidase (500 U / ml) 0.02 0.2M ATP 0.01 water 0.27 *): N-ethyl-N- (2-hydroxy-3-sulfopropyl-meta-toluidine) Amino acids, peptides, nucleic acids, nucleic acid-related substances, etc. described in this specification Abbreviations for are based on their conventional abbreviations in the art, examples of which are listed below. In addition, all amino acids show L-form.

DNA: deoxyribonucleic acid RNA: ribonucleic acid A: adenine T: thymine G: guanine C: cytosine Ala: alanine Arg: arginine Asn: asparagine Asp: aspartic acid Cys: cysteine Gln: glutamine Glu: glutamic acid Gly: glycine His: histidine Ile: Isoleucine Leu: Leucine Lys: Lysine Met: Methionine Phe: Phenylalanine Pro: Proline Ser: Serine Thr: Threonine Trp: Tryptophan Tyr: Tyrosine Val: Valine (Example) Hereinafter, the present invention will be described in detail with reference to Examples. However, the present invention is not limited by these.

Example 1 [Separation of chromosomal DNA] Chromosomal DNA of Chromobacterium biscosum varietas paralipolyticum (JP-B-46-29787) was isolated by the following method. The strain was cultured with shaking overnight at 37 ° C. in 150 ml of normal broth medium (containing 0.5% sodium thiosulfate), and then collected by centrifugation (3,000 rpm, 10 minutes). Solution containing 10% sucrose, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA
The suspension was suspended in 5 ml, 1 ml of lysozyme solution (10 mg / ml) was added, the mixture was kept at 37 ° C. for 15 minutes, and then 1 ml of 10% SDS (sodium dodecyl sulfate) solution was added. An equal volume of a chloroform-phenol mixture (1: 1) was added to the suspension, mixed by stirring, separated into an aqueous layer and a solvent layer by centrifugation at 10,000 rpm for 3 minutes, and the aqueous layer was separated. The aqueous layer was gently overlaid with twice the volume of ethanol, and the DNA was sprinkled on the glass rod with gentle stirring with a glass rod to separate the DNA. This was dissolved in a solution (hereinafter abbreviated as TE) containing 10 ml of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. This was treated with an equal volume of a mixed solution of chloroform and phenol, the aqueous layer was separated by centrifugation, twice the amount of ethanol was added, the DNA was separated again by the above method, and dissolved with 2 ml of TE.

Example 2 [Isolation of pACYC184 plasmid DNA] Escherichia coli pM191 carrying pACYC184 (J. Bac
teriol 134 , 1141 (1981); ATCC37033) was cultured with shaking in 1 BHI medium (manufactured by Difco). When the turbidity grew to OD ₆₆₀ = 1.0, spectinomycin (final concentration 300 μg / m
l) was added and shaking was continued at 37 ° C. for more than 16 hours.

The cells were collected by centrifugation at 3,000 rpm for 10 minutes, and lysozyme was collected.
SDS method and cesium chloride-ethidium bromide method (Maniatis et al .: Molecular Cloning pp. 86-94 Cold Sprin
g Harbor (1982)).

Example 3 [Preparation of plasmid pLIP1 having lipase gene] (i) Chromosomal DNA, chromosome, biscosum, varietas, paralipolyticum prepared in Example 1
μ (about 0.5 μg) and 10 times concentration of H buffer (Molecule described above)
ar Cloning, pp104) 1μ, Bel II (Takara Shuzo 10unit / μ)
) 1μ and water 6μ were mixed and cut at 37 ° C for 1 hour.
Approximately 0.3 μg of the separately prepared plasmid pACYC184 DNA is digested with BamHI using the same method, and furthermore, alkaline phos-phatase (hereinafter abbreviated as BAP; manufactured by Takara Shuzo) 0.6
The unit was added and treated at 65 ° C. for 1 hour. These cut 2
The seed DNA solutions were mixed, 1/10 volume of 3M sodium acetate was added, and the mixture was treated with a chloroform-phenol mixed solution equivalent to the whole volume, and the aqueous layer was separated by centrifugation. Two volumes of ethanol was added to the aqueous layer, DNA was precipitated by centrifugation, and dried under reduced pressure. After dissolving in 89 μm of water, ligation buffer (0.5 M Tris-HCl (pH 7.6), 0.1% concentration) was added.
1M MgCl ₂ , 0.1M dithiothreitol, 10mM spermidine, 1
0mM ATP) 10μ and T4 DNA ligase 1μ (Takara Shuzo 350u)
nit) was added, mixed, and left at 4 ° C. overnight. This DNA solution was treated with chloroform-phenol, and the ethanol precipitate was collected and dried under reduced pressure, and then dissolved with 10 µ of TE.

(Ii) 100 ml of BHI medium (Brain Heart Infusion, Difco
E. coli DH1 strain in the logarithmic growth phase (stock number ME8569; ATCC3
3849] by centrifugation (10,000 rpm, 2 minutes) 40m
l of ice-cold 30 mM potassium acetate, 100 mM RbCl, 10 mM CaCl
₂ , a solution containing 50 mM MnCl ₂ and 15% glycerin (pH
5.8). After standing at 0 ° C. for 5 minutes, the mixture was centrifuged, the supernatant was discarded, and the suspension was further suspended with a solution (pH 6.5) containing 4 ml of 10 mM MOPS buffer (manufactured by Dotite), 75 mM CaCl ₂ , 10 mM RbCl, and 15% glycerin. It became turbid and left at 0 ° C. for 15 minutes to obtain competent cells.

(Iii) 200 μl of this E. coli suspension was prepared in (i)
10 μl of the DNA solution was added and left at 0 ° C. for 30 minutes. BHI medium
After adding 1 ml and keeping the mixture at 37 ° C for 90 minutes, 100 µl of this was spread on a BHI agar plate containing chloramphenicol (25 µg / ml) and cultured at 37 ° C overnight to obtain a transformant. This transformant was replicated on a plate of a Crosley agar medium (Eiken Chemical Co., Ltd.) and further cultured at 37 ° C. overnight.

When a transformant of about 50,000 colonies was examined, one strain in which the colony turned blue was obtained, and this strain was named Escherichia coli DH1 / pLIP1 strain.

Example 4 [Mapping of pLIP1 and determination of base sequence of lipase gene] From Escherichia coli DH1 / pLIP1 strain obtained in Example 3
Plasmid pLIP1 DNA was prepared in the same manner as for pACYC184.

pLIP1 DNA restriction enzymes BamH I, Cla I, EcoR V, Sal
Cut maps were prepared using I, Mlu I, Pst I, and Xho I (all manufactured by Takara Shuzo). The result is shown in FIG.

Example 5 Approximately 1.0 μg (8 μl) of plasmid pLIP1 DNA isolated from the Escherichia coli DH1 / pLIP1 strain by the method of Example 2 above.
) To a 10-fold concentration of M buffer (Maniatis et al.
Cloning p 104) 1μ and Cla I (Takara Shuzo 10unit / μ)
1 μm was added and the mixture was cut at 37 ° C. for 2 hours. About by electrophoresis
A 3.2 kb DNA fragment was separated, precipitated with phenol-treated ethanol, and dissolved in 20 µ of TE (10 mM Tris-HCl, 1 mM EDTA pH 8.0). Separately, about 0.2 μg (1 μ) of pBR322 plasmid DNA separated and purified by the method of Example 2 was added to a 10-fold concentration of M buffer solution 1
μ, 7 μ of water and 1 μ of Cla I (Takara Shuzo 10 units / μ), cut at 37 ° C. for 2 hours, and then 1M Tris-HCl (pH 8.
0) 5 μ, 80 μ of water and 5 μ of alkaline phosphatase (Takara Shuzo 0.5 u / μ) were added and reacted at 65 ° C. for 2 hours. After the reaction, the mixture was treated three times with an equal volume of a chloroform-phenol solution, and the DNA was recovered by ethanol precipitation.
Dissolved in TE. 5 μl of each of the two DNA solutions prepared as described above was mixed, and a 10-fold concentration ligation buffer 2 μm was mixed.
, Water 7μ and T4 DNA ligase (Takara Shuzo 175unit / μ)
1 μm was added and left at 4 ° C. overnight. This DNA solution was treated with chloroform and phenol, and precipitated with ethanol.
And transformed into competent Escherichia coli DH1 cells prepared in the same manner as in Example 3 (ii).
After spreading on a BHI agar medium containing ampicillin (50 μg / ml) and culturing at 37 ° C. overnight, the resulting colonies were subjected to Example 3 (ii).
Replicate was performed on Crosley agar medium in the same manner as in i) to obtain lipase-producing Escherichia coli. This is Escherichia coli DH1 / p
It was named LIP10 and deposited with the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology as Microbiological Laboratory Bacteria No. 9926 (FERM P-9926).

After pure isolation of the bacterium, the bacterium was cultured overnight at 37 ° C. in a BHI medium, and the lipase productivity was determined by the lipase activity measurement method described above. As a result, the lipase activity was about 0.01 U / ml.

The plasmid contained in this strain was isolated in the same manner as in Example 2 and contained the lipase gene.
The plasmid containing the 22 genes was named pLIP10.

Example 6 [Mapping of pLIP10 and Determination of Lipase Gene Nucleotide Sequence] pLIP10 plasmid DNA was prepared from Escherichia coli DH1 / pLIP10 strain in the same manner as for pACYC184.

For pLIP10 DNA, restriction enzymes BamH I, Cla I, Sal I,
Cut maps were prepared using Mlu I, Pst I, and Xho I (all manufactured by Takara Shuzo). The result is shown in FIG. In addition, the restriction enzyme map of the lipase gene of the present invention is disclosed in
This is clearly different from the restriction enzyme map of the DNA fragment containing the chromobacterium biscosan gene reported in Japanese Patent No. 72 (Hpa I and Bal II shown in FIGS. 7 and 8 of JP-A-60-188072). Are not present in the DNA of the gene of the present invention). The nucleotide sequence of the DNA containing the lipase gene was determined by the dideoxy method using M13 phage (Science 214 , 1205-1210 (1981)). The nucleotide sequence and amino acid sequence of the lipase structural gene are shown in FIGS. 4 and 3.

Example 7 [Manufacture of lipase] Escherichia coli DH1 / pLIP10 strain was incubated in 20 BHI medium (manufactured by Difco) at 37 ° C. for 18 hours using a jar amenter (30 minutes).
), And the culture was centrifuged at 5,000 rpm for 10 minutes to collect the cells. Then, the cells were added to physiological saline 2
After washing with 2, 0.1 M albumin phosphate buffer (13.61 g)
KH ₂ PO ₄ , 3.92 g NaOH, 1 g bovine serum albumin, 1 g NaN ₃ , water 1
, pH 8.0), lysozyme 1 mg / ml, EDTA-2Na
mM and Triton X-100 at 0.1%.
The supernatant was separated by centrifugation at 5,000 rpm for 10 minutes. The supernatant 1.9 was subjected to acetone precipitation (50% to 8%).
0%) and the precipitate was collected by centrifugation (5,000 rpm, 30 minutes). The precipitate was dissolved in 100 ml of albumin phosphate buffer, subjected to ion exchange chromatography using DEAE-Sepharose CL-6B, and the active fraction was collected, desalted, and freeze-dried to obtain a powder preparation. Was. As a result of measuring this enzyme preparation by the lipase activity measurement method described above, 350 u of lipase could be obtained.

〔The invention's effect〕

According to the present invention, a polypeptide having a highly stable lipase activity can be produced in large quantities. Such a lipase can be used as a reagent for producing fatty acids from triglyceride, a reagent for quantifying triglyceride, and the like, and is also useful as a material for modifying fats and oils using a transesterification reaction.

[Brief description of the drawings]

FIG. 1 is a drawing showing a restriction map of plasmid pLIP1. FIG. 2 is a drawing showing a restriction map of plasmid pLIP10. FIG. 3 is a drawing showing an amino acid sequence corresponding to the nucleotide sequence of the DNA obtained in Example 6. FIG. 4 is a drawing showing the nucleotide sequence of the DNA obtained in Example 6.

Continued on the front page (51) Int.Cl. ⁶ Identification number Reference number in the agency FI Technical indication (C12N 1/21 C12R 1:19) (C12N 9/20 C12R 1:19)

Claims (7)

(57) [Claims] 1. A DNA having lipase genetic information, comprising a nucleotide sequence encoding the amino acid sequence from the N-terminal side to the amino acid sequence from the 1st (Ala) to the 319th (Val) shown in FIG. . 2. The DNA according to claim 1, wherein the base sequence is 957 bases as shown in FIG. 4 from the 5 'end. [3] a vector having the DNA of [1]; 4. The vector is a plasmid pLIP1 or pLIP10.
The vector according to claim 3, which is [5] a transformant, which comprises the DNA of [1], which is exogenous to a host; 6. The transformant according to claim 5, wherein the host is a microorganism belonging to the genus Escherichia. 7. The transformant according to claim 6, wherein the microorganism belonging to the genus Escherichia is Escherichia coli.