JPH10304875A - New lipase protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new variant type lipase protein utilizable for improvement of oils and fats and production of raw materials for medicines and optically active group and capable of producing in industrially satisfiable amount by including a specific amino acid sequence. SOLUTION: This new protein comprises a lipase protein containing 1st to 617th amino acid sequence or a protein having one or more variations selected from substitution, loss, insertion and addition of one or several amino acid residues in the amino acid sequence and containing a sequence having >=70% homology to the above amino acid sequence and having activity which is substantially equal to the lipase protein. Furthermore, the lipase protein is obtained by culturing a transformant of Escherichia coli, etc., obtained by integrating DNA molecule coding the protein into a vector plasmid, and a racemic type 3-(4-methoxyphenyl)glycidic acid methyl ester is preferably subjected to optical resolution by using the protein and soil separation bacterium providing DNA sequence coding the lipase protein is deposited as FERM P-15915.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel lipase protein, a DNA molecule encoding the same, a recombinant plasmid containing the DNA molecule, a transformant containing the recombinant plasmid, and a lipase using the transformant. The present invention relates to a method for producing a protein, and a use of the lipase protein.

[0002]

2. Description of the Related Art Conventionally, Geotricum Candy Dam (Ge
otrichum candidum), Candida cylindrace (C
and lipases derived from microorganisms such as P. andida cylindracea) and Pseudomonas fluorescens. In addition, using recombinant DNA technology for the production of lipase, Rhizopus delemer (Rhizopus d.
elemar) -derived esterase
-87175) or a method for producing a lipase derived from Serratia marcescens by self-cloning (Japanese Patent Publication No. H8-24585). In addition, these lipases improve fats and oils,
It is used in the production of pharmaceutical raw materials and optically active groups.

[0003]

Although there have been various reports on lipases as described above, Escherichia coli, which is best known for mass production by genetic recombination technology, is used.
There is no known method for producing lipase derived from Pseudomonas in an industrially satisfactory amount. Conventionally, large-scale expression of lipase derived from Pseudomonas bacteria has been attempted by self-cloning. However, Escherichia coli, whose genetic background has been well known since ancient times, has been widely used as a host for expressing large amounts of heterologous proteins, can be used for hydrolysis of useful substances, etc. Lipase derived from Pseudomonas bacteria can be industrially advantageously supplied.

[0004]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that racemic 3- (4-methoxyphenyl) glycidic acid methyl ester (hereinafter abbreviated as (±) -MPGM).
To give (2R, 3S) -3- (4
A soil isolate capable of preparing (-methoxyphenyl) glycidic acid methyl ester (hereinafter abbreviated as (-)-MPGM) was found. A gene encoding lipase was successfully isolated from this strain, and after integrating the gene into a vector plasmid, a transformant was prepared by transforming Escherichia coli with the obtained recombinant plasmid. The lipase production expressed as inclusion bodies in the transformant was compared with that of the original soil isolate by sodium dodecyl sulfate-polyacryl gel electrophoresis (SDS-PAGE).
In the transformant, the production amount was significantly increased. After recovering this inclusion body, it has been found that an active lipase can be produced industrially advantageously through a solubilization / rewinding step, and the present invention has been completed.

[0005] The present invention relates to a lipase protein comprising the amino acid sequence from position 1 to position 617 of SEQ ID NO: 2. In another aspect, the present invention provides a method comprising the steps of:
At least one selected from substitution, deletion, insertion and addition of one or several amino acid residues in the amino acid sequence at position 17;
The present invention relates to a lipase protein having two mutations and containing a sequence having homology of 70% or more with its amino acid sequence, and having substantially the same activity as the lipase protein. As used herein, the term “mutant lipase protein” has the above mutation in the amino acid sequence of the original protein, and / or can contain a chemically or biochemically modified or natural or unnatural amino acid. Modified protein means a protein whose function or activity is substantially the same as the lipase protein of the present invention.

[0006] In another aspect of the present invention, a DNA molecule encoding the protein of the present invention and a mutant lipase protein thereof, for example, from position 1 to position 18 of SEQ ID NO: 1
DNA molecule having the nucleotide sequence at position 51; DNA of the present invention
A recombinant plasmid having a molecule incorporated into a vector plasmid; a transformant containing the recombinant plasmid of the present invention, preferably the transformant which is Escherichia coli; a protein of the present invention produced by the transformant of the present invention; The present invention relates to a method for producing the lipase protein of the present invention, which comprises a step of culturing the transformant and a step of recovering the protein produced in the cells, and a method of producing, further comprising a step of recovering the activity.

In another aspect, the present invention provides a racemic 3-
In a method for optically resolving (4-methoxyphenyl) glycidic acid methyl ester, the (2S, 3R) form is hydrolyzed using the protein of the present invention to give (2R, 3R)
(2R, 3S)-characterized by isolating the (S) form.
The present invention relates to a method for producing methyl 3- (4-methoxyphenyl) glycidate.

[0008] In a further aspect, the present invention provides an accession number F
Pseudomonas sp. SHS2 deposited at the Institute of Biotechnology and Industrial Technology under ERM P-15915
317 strain bacteria.

The soil isolate providing the DNA sequence encoding the lipase protein of the present invention is Pseudomonas sp. Strain SHS2317.
It was deposited with the Biotechnology Research Institute on October 23, 1996 under the accession number FERMP-15915. <Pseudomonas sp. SH
Various properties of S2317 strain> 1. Morphology Gram-negative short rod bacillus, motility, and flagellated state is polar flagella (one or more). No spores are formed and there is no acid resistance. The cell size is 0.7-0.8 μm × 1.
5 to 2.0 μm.

[0010] 2. Culturing findings (1) Broth agar plate culture (30 ° C., 48 hours) Slow formation: colony formation in 24 hours Shape: circular Surface condition: smooth Periphery: whole edge Height: hill-like Color: bright yellow Gloss: wet light Optical properties: opaque

(2) Gravy agar slant culture (30 ° C., 48 hours) Quality of growth: vigorous Shape: spread color Color: light yellow Gloss: wet light Optical properties: opaque Viscosity: beef dairy

(3) Liquid culture of gravy (30 ° C., 48 hours) Surface growth state: thin film formation on liquid surface Turbidity: moderate Odor: slight odor Precipitation: viscous Precipitation amount: large amount

3. Physiological Various properties (1) Reduction of nitrate: Positive (2) MR test: Negative (3) VP test: Negative (4) Indole production of: Negative (5) H ₂ S production of: Negative (6) starch hydrolysis Degradation: Negative (7) Use of citric acid: Positive (8) Use of inorganic nitrogen source: Do not use nitrate or ammonium salt (9) Dye formation: Water-soluble fluorescent dye formation (10) Urease test: Negative ( 11) Oxidase: positive (12) Catalase test: positive (13) Growth temperature: able to grow at 4 ° C., not grow at 41 ° C. (14) Growth under anaerobic: negative (15) Attitude to oxygen: aerobic (16) ) OF test: oxidatively decomposes sugar (17) Arginine hydrolysis: positive (18) Esculin hydrolysis: negative (19) Gelatin hydrolysis: positive (20) DNase production: negative (21) P PG: negative (22) acylamidase: negative (23) lipase production: positive (24) Ornithine decarboxylase: Negative (25) Lysine decarboxylation: negative (26) Gas production from glucose: negative

(27) Carbohydrate utilization test: Positive: D-glucose, L-arabinose, D-mannose, D-mannitol, trehalose, sucrose,
D-xylose, ribose, erythritol, D-fructose, galactose, melibiose, N-acetylglucosamine, potassium gluconate, 2-ketogluconic acid, 5-ketogluconic acid, n-capric acid, malic acid,
Phenylacetic acid negative: maltose, sorbitol, adonitol, rhamnose, D-arabinose, L-xylose, L-sorbose, dulcitol, α-methyl-D-mannoside,
α-methyl-D-glucoside, cellobiose, lactose, D-raffinose, xylitol, adipic acid (28) G + C content (%): 62.1 (HPLC method)

[0015] From the above results, the taxonomic properties of this strain were determined using Burgy's Manual of Systematic Bacteriology volume.
1 (1984), it was identified as a strain belonging to the genus Pseudomonas, which is closely related to Pseudomonas fluorescens;
Pseudomonas sp. SHS
2317 strains.

[0016]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a Pseudomonas sp.
p.) a method of incorporating the lipase gene from the SHS2317 strain into a vector plasmid used in an E. coli host / vector system, and producing a lipase protein using a recombinant host cell transformed with the resulting plasmid. This will be described in the following steps. However, it is recognized by those skilled in the art that some of these steps can be omitted or simplified after the DNA sequence encoding the lipase derived from the SHS2317 strain has been revealed by the present invention.

Obtaining Chromosomal DNA Chromosomal DNA containing a gene encoding a lipase is prepared by lysing microorganism cells with a surfactant such as sodium dodecyl sulfate (SDS) or sodium N-lauroyl sarcosinate and then cetyltrimethylammonium bromide. A protein is removed by extraction with an organic solvent such as phenol or chloroform after treatment, etc., and the DNA is purified by cesium chloride-ethidium bromide equilibrium density gradient ultracentrifugation (Current Pr.
otocolsin Molecular Biology volume 1, 2.4.3, John
Wiley & Sons Inc. (1994).) And the method of Marmur (J.
Mol. Biol., 3 , 208 (1961)) and the like.

The vector for cloning the gene encoding lipase is not particularly limited as long as it is a vector that can be replicated in Escherichia coli. For example, it has a marker gene resistant to drugs such as ampicillin, kanamycin, chloramphenicol and tetracycline. And la
Those having an appropriate promoter such as c, tac, trp, and trc are desirable. Such plasmid vectors include, for example, pBR322, pUC18 / 19, pUC
118/119, pACYC177, pACYC184
And the like. Furthermore, λgt10, λZAPII
Phage vectors such as

Escherichia coli which can be used as a host for cloning and expression includes Escherichia coli K-
12 strains, HB101 strain, JM105 strain, JM1
09 strain, MV1184 strain, DH5 strain, DH5α strain and the like. As these vectors and hosts, commercially available vectors can be suitably used. A host other than Escherichia coli can be used if an appropriate vector corresponding to the host is used. Hosts other than E. coli include Pseudomonas, Bacillus, yeast, mold and animal cells.

As a means for obtaining the lipase gene derived from SHS2317 strain, a shotgun cloning method (Current
Protocols in Molecular Biology volume 1, 5.0.1, Jo
hn Wiley & Sons Inc. (1996)), and a hybridization method using a DNA probe.

Preparation of DNA Primer Pseudomonas fluorescens B52 strain which is considered to be closely related to SHS2317 strain (Appl. Environ. Microbiol.,
58 , 1402 (1992).), Pseudomonas fluorescens SIK W1 strain (Agric. Biol. Chem., 55 , 2359 (1992)).
Since the nucleotide sequence of the lipase gene derived from 1).) Has been clarified, DNA that can be prepared from DNA sequence homology
Probe-based methods may be used. That is, commercially available D
The DNA sequence of the lipase derived from the above two strains was compared using an NA analysis system, and a region having high homology was searched for.
Using this homologous region, primers for polymerase chain reaction (PCR) are synthesized by a DNA synthesizer. When PCR amplification is performed using the SHS2317 strain chromosome as a template, a gene fragment can be obtained with an appropriate combination of primers. If it can be confirmed that this gene fragment is not a completely different gene, this fragment can be used as a probe for colony hybridization. it can.

Production of Lipase Protein The recombinant plasmid consisting of the chromosomal DNA derived from the SHS2317 strain and a vector is subjected to chromosomal DNA by appropriate restriction enzymes.
It is obtained by cutting the strand and the DNA strand of the vector, and then randomly linking the DNA strands with DNA ligase. The obtained recombinant plasmid is transformed into E. coli, for example, J.
M109 strain, etc., and the above probe (labeled PCR
By performing colony hybridization using the amplified fragment), a gene fragment containing the lipase gene derived from the SHS2317 strain can be obtained. Note that a radioactive isotope label with ³² P or a non-radioactive label such as biotin, fluorescein, digoxigenin, etc. can be used for labeling the DNA probe.

Expression plasmids for efficiently expressing the lipase gene in Escherichia coli include suitable promoters lac, tac, trc, trp,
pKK223-3, pPL-Lambda having a λPL or the like and a Shine-Dalgarno (SD) sequence,
In addition, pKK233- with the translation initiation codon ATG
2. It can be prepared by inserting a DNA fragment containing a lipase gene into pTrc99A or the like. This expression plasmid is transferred to a suitable host, for example, E. coli JM109 strain, HB101 strain, D
Introduction into H5 strain, DH5α strain, etc. enables efficient expression.

Recovery of the lipase protein The resulting transformant can be cultured in a medium having any composition as long as it can grow Escherichia coli. The culture can be performed under the usual conditions of temperature, aeration and stirring. If the lipase produced by the recombinant Escherichia coli is obtained as an inclusion body,
The cells are crushed by French press, physical crushing such as ultrasonic waves, or enzymatic treatment such as lysozyme,
The inclusion bodies can be recovered by centrifugation.

The activity recovery then the lipase protein, urea solution, preferably to denature the lipase protein by solubilizing with 8M or more urea solution. To recover the activity of the lipase protein, any method can be used as long as the urea concentration in the solubilized enzyme solution can be reduced.For example, the urea concentration is reduced by replacing the buffer solution by dialysis or ultrafiltration, and Rewinding (recovery of activity) may be performed (Protein Purification: Mi
cro to Macro, p429-442, Alan R. Liss, Inc. (198
7).). In this case, high-purity lipase can be obtained in high yield. The buffer used in the buffer exchange by dialysis or ultrafiltration contains about 1 mM of calcium ion essential for lipase activity, and dithiothreitol (DTT) or 2-mercaptoethanol as an antioxidant. If necessary, a protease inhibitor or an antibacterial agent may be added.

The lipase protein whose activity has been restored exhibits the following properties. (1) Molecular weight 64,000 molecular weight (SDS-PAGE) (2) Optimal temperature The optimal reaction temperature was around 42 ° C. (3) Thermal stability The residual activity of 90% was maintained at 48 ° C for 60 minutes. (4) Optimum pH The optimum pH was 8.5 to 9.5. (5) pH stability After contacting with a buffer solution having a pH of 4 to 11 for 2 hours, 90% or more of the residual activity was maintained. (6) Solvent resistance In contact for 2 hours, 1-butanol had 8% residual activity, but normal hexane, cyclohexane, diisopropyl ether, and toluene retained 80 to 90% residual activity.

(7) Substrate Specificity The activity was measured using fatty acid methyl esters having various chain lengths and triglycerides as substrates. As a result, methyl caprylate (having 8 carbon atoms) was used for fatty acid methyl esters, and tributyrin (having 4 carbon atoms) was used for triglycerides. ) Had high activity. (8) Effects of metal ions and inhibitors As a result of contacting the enzyme with 1 mM of metal ions and inhibitors at room temperature for 2 hours and measuring the residual activity, the metal ions were iron divalent ions, iron trivalent ions, lead ions, and zinc ions. Inhibited and activated by calcium and manganese ions. In the inhibitor, ethylenediaminetetraacetic acid (EDTA) almost lost activity, and SDS and phenylmethylsulfonyl fluoride (PMSF) were not affected. The activity of the enzyme was not affected by nickel ions and cobalt ions. This is a feature different from the lipase from Serratia marcescens.

Production of Mutant Lipase Protein The mutant lipase protein of the present invention can be prepared by a gene recombination technique known to those skilled in the art (Sambrook et al., Molecular Cloni).
ng: A Laboratory Manual 2nd edition, Cold SpringHarbor L
aboratory, New York (1989)).

Effect of the protein of the present invention The lipase protein of the present invention can be applied to organic synthesis reactions because it can cleave ester bonds to alcohol esters of organic carboxylic acids and triglycerides, for example, for optical resolution of racemic compounds. Can be used.

Hereinafter, the present invention will be described in more detail by way of Examples, but these are not intended to limit the present invention. In the following examples, the digestion reaction with restriction enzymes and the use of genes and enzyme-related kits were performed according to the protocol of each manufacturer, and the transformation of Escherichia coli with the plasmid was carried out according to the method of Cohen et al.
l. Acad. Sci. USA, 69 , 2110 (1972).
General genetic manipulation is performed by the method of Sambrook et al. (Mole
cular Cloning: A Laboratory Manual 2nd edition (Cold Sp
ring Harbor Laboratory (New York), (1989)). Unless otherwise specified, the enzyme activity was measured using a lipase kit S (manufactured by Dainippon Pharmaceutical Co., Ltd.) using dimercaprol tributyrate as a substrate. The activity was 1 U per minute to generate 1 μmole of a free SH group. did. The amount of protein was measured using a Bio-Rad protein assay kit.

[0031]

Example 1 Screening of lipase-producing strains Racemic 3- (4-methoxyphenyl) glycidic acid methyl ester ((±) -MPGM) is hydrolyzed to give (2R, 3S) -3- ( 4-methoxyphenyl) glycidic acid methyl ester ((-)-MPGM)
In order to find an enzyme that produces E. coli, the enzyme was screened from strains collected from soil. First, the soil is appropriately diluted with physiological saline, and a lipase detection medium (Nutrient Agar (manufactured by Difco), 1% tributyrin)
After culturing at 28 ° C. overnight, a strain having a clear zone around the colony was collected. 1 in test tube
0 ml of screening medium (0.5% glucose,
1.25% yeast extract, 1% peptone, 1% meat extract,
0.5% NaCl, 0.5% Tween80, pH
7.0), inoculate one loopful of each strain and incubate overnight at 28 ° C. with shaking. After that, 0.9 ml of culture solution, 0.1 ml of 1 M Tris-HCl buffer (pH 8.0) and 1% (±) -MPGM
Was placed in a screw-neck test tube, suspended in a blender, sealed, and allowed to shake at 28 ° C. overnight. Separate the toluene layer and use a chiral cell OJ column (φ
As a result of analyzing the decomposition amount of the substrate by HPLC using 4.6 × 250 mm (manufactured by Daicel Chemical Industries, Ltd.), (+)-MPGM was efficiently obtained from the soil collected in Amagasaki City, Hyogo Prefecture.
Of Pseudomonas sp. (Pseu), a strain producing an enzyme that selectively hydrolyzes and leaves (-)-MPGM.
domonas sp.) SHS2317 strain was found.

This Pseudomonas sp.
nas sp.) SHS2317 strain was obtained from the Biotechnology Industrial Technology Research Institute on October 23, 1996 (accession number FE).
Deposited under RMP-15915.

Example 2 Pseudomonas sp. SHS
Cloning of lipase gene derived from strain 2317 (1) Chromosomal DNA containing gene encoding lipase
Of Pseudomonas sp. Obtained in Example 1
s sp.) 100 ml of nutrient broth (manufactured by Difco) containing 0.1% glucose
The cells were cultured aerobically at 28 ° C. overnight with shaking, and collected by centrifugation. The cells were washed with 9.5 ml of 10 mM Tris-HCl.
1 mM EDTA · 2Na buffer (pH 7.4) (TE
Buffer solution), 0.5 ml of 10% SDS was added, and the mixture was shaken at 37 ° C. for 1 hour to lyse the cells. Then 1.8m
l of 5M NaCl and 1.5 ml of 10% cetyltrimethylammonium bromide (CTAB) /0.7M
A NaCl solution was added, and the mixture was kept at 65 ° C. for 20 minutes. Protein and polysaccharide were removed by extraction with an equal volume of chloroform: isoamyl alcohol (24: 1). 0.6 volume of isopropanol was added to the separated aqueous layer, and 1
The precipitate was collected by centrifugation at 000 rpm for 30 minutes.
The precipitate was washed with a 50 mM Tris-50 mM EDTA solution (p
H8.0) overnight and the solution was centrifuged with a cesium chloride-ethidium bromide equilibrium density gradient ultracentrifuge (38,0.
(00 rpm, 40 hours) to obtain 1.8 mg of purified chromosomal DNA.

(2) Preparation of DNA primer Pseudomonas sp. SHS
Pseudomonas fluorescens B52 strain which is considered to be closely related to 2317 strain (Appl. Environ. Microbiol., 58 , 1
402 (1992).) And Pseudomonas fluorescens SIK W1 strain (Agric. Biol. Chem., 55 , 2359 (199).
Since the nucleotide sequences of the lipase genes produced by the two strains 1) and 2) were clarified, a colony hybridization method using a DNA probe was selected as a means for cloning the lipase gene derived from the strain. That is, using the DNA analysis system DNASIS ^™ (manufactured by Takara Shuzo Co., Ltd.), DN of the lipase gene derived from the above two strains was used.
When comparing the A sequences, there is a region with high homology,
Utilizing this homologous region, the following oligonucleotide is
It was synthesized with an NA synthesizer (Pharmacia).

Primer on the sense side S-1: 5'-ATGGCATCT TTAGCTAT-3 'S-2: 5'-ATCACGTTGT ATTCCTATCA-3' S-3: 5'-CTGGCGGTCA ACAGCATG-3 'S-4: 5'-TTCTACAAGG ACGCCAACTA-3 ' Antisense primer S-1R: 5'-TAGTTGGCGT CCTTGTAGAA-3' S-2R: 5'-TTGCCGTCGCCGTCGATG-3 'S-3R: 5'-TCGTTGAGGCTGACGATGTT-3'S-4R: -GCGTAGTGGT CGTTGAAG-3'S-5R: 5'-GCTGGAACAAAAAGGTGTTTGT-3 'After completion of synthesis, the oligonucleotide was purified and recovered,
The amount of DNA was calculated from the absorbance at 0 nm, and 10 pmole
/ Μl.

(3) Partial cloning of lipase gene by PCR amplification Using the above primers, polymerase chain reaction (PC
R) Amplification was performed using the LA PCR kit (Takara Shuzo). 1 μl of sense primer and 1 μl of antisense primer obtained in (2), 8
μl of a dNTP mixed solution (2.5 mM each) and one grain of Ampli Wax ^™ (manufactured by Perkin Elmer) were added, heated at 77 ° C. for 7 minutes, and cooled at 25 ° C. for 3 minutes. Then, 0.5 μl of Taq DNA polymerase (5
U / μl) and 0.5 μl of chromosomal DNA (0.75 μg) derived from SHS2317 strain, and the volume was increased to 90 μl with distilled water.
μl was added. Next, it was set in a DNA thermal cycler (Model PJ2000: manufactured by Takara Shuzo Co., Ltd.) and subjected to PCR amplification. One cycle of the reaction was set at 94 ° C. for 1 minute, 55 ° C. for 1.5 minutes, and 72 ° C. for 2 minutes, and this was repeated 25 times. Finally, after heating at 72 ° C. for 7 minutes, the mixture was placed on ice, and 5 μl was dispensed from the aqueous layer below the solidified wax layer.
4% agarose gel electrophoresis was performed. As a result, two of the combinations of various primers (S-3 and S-2
R and S-3 and S-4R), DNA fragments close to the lengths expected from the nucleotide sequences of Pseudomonas fluorescens B52 strain and Pseudomonas fluorescens SIK W1 strain are amplified. The L6 fragment (= 0.45 kb) and the L8 fragment (= 0.
22 kb). 16 μl was dispensed from each PCR reaction completion solution, and cloned into the SmaI site of pUC18 (Takara Shuzo) using SureClone ^™ Ligation Kit (Pharmacia). 2 obtained
As a result of analyzing the base sequences of the two fragments, Pseudomonas
There is homology with the lipase gene from fluorescens,
It was presumed to be part of the lipase gene derived from SHS2317 strain. Since the L8 fragment is included in the L6 fragment, the following procedure used the L6 fragment.

(4) Cloning of lipase full-length gene by colony hybridization Using the DNA fragment (0.45 kb L6 fragment) obtained in (3) as a probe, a non-radioactive DNA labeling / detection kit (Boehringer Mannheim) ), SHS
Southern hybridization was performed using chromosomal DNA of strain 2317 as a template. First, 16 μg of the pUC18 plasmid into which the L6 fragment obtained in (3) was inserted was added to EcoR
I and HindIII (100 U each) and DNA recovery system S from agarose gel electrophoresis
A 0.45 kb L6 fragment was recovered using pinBind ^™ (Takara Shuzo). 1 μg of the solution was dissolved in 10 μl of distilled water, denatured by heating at 100 ° C. for 10 minutes, rapidly cooled in ice, and then subjected to a digoxigenin labeling reaction at 37 ° C. for 1.5 hours according to the protocol of the kit. The reaction-terminated liquid is precipitated with ethanol, and the precipitate is washed with 50 μl of TE.
Dissolved in buffer.

On the other hand, 15 μg of the chromosomal DNA of the SHS2317 strain was isolated from BamHI, EcoRI, HindIII, Sam
Various restriction enzymes of acI, SalI or XbaI were
After digestion with 0 to 200 U, electrophoresis was performed on a 1% agarose gel, followed by a conventional method (Molecular Cloning 2nd edition: A Laborator).
y Manual, 9.31, Cold Spring Harbor Laboratory (New
York) (1989).) And a nylon membrane (Boehringer
The DNA was transcribed and immobilized to Mannheim. Less than,
According to the protocol of the non-radioactive DNA labeling / detection kit, 20 ml of hybridization buffer (containing 50% formamide) per 100 cm ^{2 of} nylon membrane
Pre-hybridization at 42 ° C. for 1 hour at 42 ° C. for 18 hours at 42 ° C. for 18 hours with 2.5 ml of a hybridization buffer (containing 12.5 μl of the above labeled probe) per 100 cm ^{2 of} nylon membrane. Was. At room temperature, 50 ml of the washing solution (2 × SSC [SSC = 150 mM) per 100 cm ² of the nylon membrane
NaCl, 15 mM sodium citrate, pH7.
0], 0.1% SDS) for 5 minutes, and then twice at 68 ° C. for 15 minutes with a washing solution containing 0.1 × SSC and 0.1% SDS. Finally, a color reaction was performed with a detection reagent attached to the kit, and as a result, a band hybridizing with the L6 fragment was detected. From the comparison with the length of the base sequence of Pseudomonas fluorescens lipase, it was determined that the 7 kb EcoRI fragment contained the full-length lipase gene. Therefore, the lipase full-length gene was obtained by colony hybridization using EcoRI. Was cloned.

Chromosomal DNA 10 μm derived from SHS2317 strain
g was digested with 200 U of EcoRI, and then subjected to agarose gel electrophoresis.
A gel around 20 kb was cut out and a DNA fragment was recovered by SpinBind ^™ . After digestion with 2 μg of this EcoRI fragment and EcoRI, 0.5 μg of pUC119 (manufactured by Takara Shuzo) whose terminal was dephosphorylated with alkaline phosphatase was ligated with Ready-to-Go ^™ T4 DNA ligase (manufactured by Pharmacia), and E. coli DH5 The strain (manufactured by Toyobo) was transformed. 50 μg of transformant
Nylon membrane (Colony / PlaqueScreen) on an L agar plate containing 1 / ml ampicillin (1% bactotripton, 0.5% yeast extract (manufactured by Difco), 0.5% NaCl, 1.5% agar)
n: manufactured by Daiichi Kagaku) and cultured overnight at 37 ° C. Take a replica of this nylon membrane and take an L agar plate (5
0 μg / ml ampicillin), and cultured at 37 ° C. to immobilize the colony-formed nylon membrane by a method well known to those skilled in the art. Colony hybridization was performed using the digoxigenin-labeled L6 fragment probe in the same manner as in (4), and as a result of the color-forming reaction,
The strain has developed color. The plasmids of these clones were analyzed for restriction enzyme fragments, and one positive clone into which a 7 kb fragment was inserted was arbitrarily selected. This positive clone formed a clear zone on the tributyrin plate, indicating that the lipase gene could be obtained. The recombinant plasmid extracted from the positive clone was pUMP98
It was named. FIG. 1 shows a restriction map of pUMP98.

Example 3 Miniaturization of Recombinant Plasmid In order to remove the region unrelated to the lipase gene from the cloned fragment obtained in Example 2, the recombinant plasmid pU
MP98 was downsized. First, 10 μg of pUMP
98 was digested with 150 U of EcoRI, and then subjected to agarose gel electrophoresis.
Collected by ^TM . Then, judging from the restriction enzyme fragment map,
After digestion with 00U PuvII, a 3 kb EcoRI-PuvII fragment containing the lipase gene was recovered from the agarose gel. pUC118 and pUC119 (both manufactured by Takara Shuzo) were double-digested with EcoRI and HincII, and 0.5 μg of the digest and 0.5 μg of the recovered DNA fragment were digested.
g was ligated with Ready-to-Go ^™ T4 DNA ligase to transform Escherichia coli DH5 strain.
As a result, the 3 kb EcoRI-PvuII fragment was converted to pU
EcoRI, HincI of C119 and pUC118.
The plasmids inserted between I and I were obtained,
They were named EPv198 and pUEPv298. In the former, the direction of the lipase structural gene was forward with respect to the lac promoter, and in the latter, the direction was reverse. FIG. 2 shows pUEPv19.
8 shows a restriction map of pUEPv298.

Example 4 Determination of base sequence of lipase gene Miniaturized plasmid pUEPv198 obtained in Example 3
The nucleotide sequence of a lipase structural gene derived from SHS2317 strain was determined using the method of Sanger et al. (Proc. Natl. Aca).
d. Sci. USA, 74 , 5463 (1977).) using a commercially available kit. In the portion where the GC content was high, band compression was observed due to the secondary structure, and thus 7-deaza was observed.
-Use of dGTP or Sequencing P
RO (manufactured by Toyobo) and BcaBEST (manufactured by Takara Shuzo) were used in combination. As a result of determining the base sequence of the lipase gene,
An open reading frame consisting of 1851 bp was found beginning with ATG and ending with GCC (SEQ ID NO: 1). The number of encoded amino acid residues was 617, and the theoretical molecular weight of the protein was 64,796.

[0042] no manufacturing lacI ^q Example 5 for E. coli expression plasmid pKK233-2 and lacI ^q
Of pTrc99A (above, manufactured by Pharmacia)
A lipase gene derived from the SHS2317 strain was introduced into the seed expression vector to prepare an expression plasmid. First, pKK
233-2 was digested with 5 U of HindIII, repaired to a blunt end with a blunting kit (Takara Shuzo), and then Nco present at the start codon ATG.
The I site was cut with 40 U of NcoI. Also, lacI
1 μg of the expression vector pTrc99A carrying ^q
Digestion was performed with 0 U of NcoI and 20 U of SmaI. On the other hand, the inserted fragment contained 15 μg of pUEPv198 in 100 U
Of NcoI and 70U of Eco47III,
2.3 kb Nc containing SHS2317 lipase gene
The oI-Eco47III fragment was recovered by 1% agarose gel electrophoresis. This fragment was treated with pKK233-2 or pTrc99A and Ready-to-Go ^™
After ligation with T4 DNA ligase, E. coli JM109
The strain was transformed. The expression plasmids obtained from the transformants were pKLIP98NE for the former and pTrcLI for the latter.
It was named P98NE (FIG. 3). The two expression plasmid-bearing strains showed clear zone formation on L-agar plates containing 1% tributyrin.

Example 6 Preparation of lipase protein (1) Expression of lipase protein E. coli JM109 (pKLIP98) obtained in Example 5
NE) was inoculated into 200 ml of L medium (containing 40 μg / ml ampicillin) in a 500 ml flask.
Preculture was performed at 28 ° C. for 20 hours on a rotary shaker at 00 rpm. Next, 15 L of a main culture medium (4% glycerol, 2% casamino acid [acid digestion], 0.3% yeast extract, 0.6% Na ₂ ) was placed in a 30 L jar fermenter.
HPO ₄ , 0.3% KH ₂ PO ₄ , 0.05% NaC
1, 0.1% NH ₄ Cl, 0.024% MgSO ₄ ,
_{0.00147% CaCl 2 · 2H 2 O} , 0.02%
Anti-foaming agent, 40 μg / ml ampicillin)
0 ml was transplanted, and the culture was started at a controlled flow rate of 11.25 L / min, a stirring speed of 500 rpm, a temperature of 37 ° C. and a pH of 7.0. After 6 hours, the inducer isopropyl-β-D
Thiogalactopyranoside (IPTG) at a final concentration of 1 mM
And culture was continued for another 17 hours. At the end of the culture, the lipase derived from the SHS2317 strain was produced as an inclusion body in E. coli cells. From SDS-PAGE analysis, the lipase protein accounted for 30-40% of the intracellular protein.

This lipase inclusion body was recovered as follows. First, 13 kg of the culture solution was centrifuged by a solid ejecting centrifuge, and an equal volume of 0.1 was added to the obtained heavy liquid.
M phosphate buffer (pH 7.2) was added and centrifuged again to obtain 1.4 kg of heavy solution. The treatment with the pressurized cell disrupter was repeated three times, followed by centrifugation (10,000 × G,
0 min) to give 42 g of precipitate containing lipase inclusions.

(2) Examination of conditions for recovery (rewinding) of activity of lipase inclusion body (i) Treatment of activity recovery of lipase inclusion body The precipitate obtained in (1) was treated with 0.1 M Tris-HCl buffer (pH 8. (0: 1 mM DTT), and adjusted to 500 mg / ml. To 1 ml of the solution, 9 ml of a solubilizing solution (0.1 M Tris-HCl buffer, pH 8.0: 8 M urea) was added, and the mixture was solubilized at room temperature for 4 hours. Controls were diluted with 9 ml of 0.1 M Tris-HCl buffer (pH 8.0: 1 mM DTT) instead of lysate to determine the extent of activity recovery. Next, these enzyme solutions are applied to a dialysis tube seamless cellulose tubing 18/32 (manufactured by Visking).
And 1 L of dialysis buffer (0.1 M Tris-HCl buffer, pH 9.0: 1 mM CaCl ₂ , 1 mM DT
(T, 0.5% Tween 80) at room temperature overnight. After standing at room temperature for 6 days, the activity was measured.
The control was 112 U / ml (protein amount: 8.61 mg / m
l), whereas the unwound enzyme was 555
The activity was restored to 6 U / ml (protein amount: 7.18 mg / ml). (Ii) Effect of additive in dialysis buffer Effect of additive in dialysis buffer on rewinding of lipase derived from SHS2317 strain was measured by DTT (1 mM), Tween
n80 (0.5%) and CaCl ₂ (1 mM) were examined. The precipitate obtained in (1) was mixed with a 0.1 M Tris-HCl buffer (pH 8.0: 1 mM DTT) at 500 mg /
ml, of which 0.5 ml was taken and 49.5 m
l of solubilization solution (0.1 M Tris-HCl buffer, pH
8.0: 8 M urea, 1 mM DTT).
Thereafter, dialysis was performed overnight at room temperature against a dialysis buffer (0.1 M Tris-HCl buffer, pH 9.0) containing three kinds of additives in various combinations. After the enzyme solution was allowed to stand at room temperature for 4 days, the degree of activity recovery was examined. As shown in Table 1, CaCl ₂ was found to be essential for activity recovery.

[0046]

[Table 1]

(3) Preparation of Rewind Lipase (i) Recovery of Activity of Lipase Inclusion Body by Dialysis The precipitate obtained in (1) was treated with 0.1 M Tris-HCl buffer (pH 8.0: 1 mM DTT) at 500 mg / d and 495 ml of the lysate (0.1 ml).
M Tris-HCl buffer, pH 8.0: 8 M urea, 1 mM
DTT). 5 L of dialysis buffer (0.1 M
Tris-HCl buffer, pH 9.0: 1 mM CaCl ₂ ,
After dialysis against 1 mM DTT) overnight at room temperature, 5 L
Was dialyzed overnight to remove urea. After standing at room temperature for 8 days, the activity was measured to be 401 U /
ml (0.60 mg / ml protein).

(Ii) Treatment for Recovering Activity of Lipase Inclusion Body by Ultrafiltration Membrane The precipitate obtained in (1) was dissolved in the same solubilization solution as in (i) above, and allowed to stand at room temperature overnight. After removing the insoluble matter from the obtained enzyme solution by centrifugation, a hollow fiber type ultrafiltration system CL-100 (manufactured by Asahi Kasei Corporation: fractionation molecular weight 1)
The urea treatment of the solubilized enzyme solution was performed using 3,000 and an effective area of 0.2 m ² ). The volume of the solution reduced by ultrafiltration was reduced to 0.1 M Tris-HCl buffer (pH 9.0: 1 m
When the enzyme solution was replaced with a total of 5 times the volume of Tris-HCl buffer by supplementing with M CaCl ₂ , 1 mM DTT, the activity was restored to 443 U / ml (protein amount 0.59 mg / ml) after one day. .

Example 7 Mass Production of Lipase Protein E. coli JM109 (pTrcLIP) obtained in Example 5
98NE) in a 500 ml flask
Inoculate 150 ml of L medium containing 1 ampicillin,
Preculture was performed at 28 ° C. for 17 hours on a rotary shaker at 00 rpm. Next, in a 30 L jar fermenter, 15 L of a TB medium (4% glycerol, 1.2%
Tryptone, 2.4% yeast extract, 1.25% K ₂ HP
O ₄ , 0.23% KH ₂ PO ₄ , 0.02% defoamer, 4
0 μg / ml ampicillin), and agitated at 35
0 rpm, aeration rate 11.25 L / min, 37 ° C, pH
Culture was started under 7.2 control. Six hours later, 1 mM IPTG was added as an inducer, and then the cells were cultured for 17 hours. 14 kg of the culture solution was treated with a solid ejecting centrifuge, an equal volume of 0.1 M phosphate buffer (pH 7.2) was added to the obtained heavy solution, and the mixture was centrifuged again to obtain a heavy solution 2.5 g.
kg. This is crushed by treatment with a pressurized cell crusher (3 times), and then centrifuged (10,000 × G, 1 × G).
At 0 min), a precipitate containing lipase inclusion bodies was recovered.

12 L of a solubilizing solution (0.1 M Tris-HCl buffer, pH 8.0: 8 M urea, 1 mM DTT) was added to the whole amount of the obtained precipitate, and the mixture was solubilized overnight at room temperature. After insolubilized enzyme solution is removed by centrifugation,
Microfilter (Millipore: 0.45 pore size)
μm). Hollow fiber type ultrafiltration system (manufactured by Asahi Kasei Corporation: molecular weight cut off 13,000, effective area 1)
m ² ), and when the amount of the enzyme solution was halved, 0.1 M Tris-HCl buffer (pH 9.0: 1 mM Ca
Replenishment to the original volume with Cl ₂ (1 mM DTT) was repeated seven times to reduce the urea concentration. After 1 day, the activity was measured to be 1299 U / ml (protein amount 2.0
9 mg / ml).

Example 8 Properties of Lipase Protein The results of various properties examined using the lipase protein obtained in Example 6, (3) (i) are shown below. (1) Molecular weight The molecular weight measured by SDS-PAGE is 64,000.
It was 0. (2) Optimal reaction temperature and thermostability (Method) The optimal reaction temperature of lipase was measured by using a lipase kit S while changing the temperature during the enzyme reaction. The thermostability was measured by a lipase kit S after a shaking reaction in a 0.1 M Tris-HCl buffer (pH 8.0) at each temperature for 1 hour. (Result) The optimum reaction temperature was around 42 ° C. In addition, the thermal stability maintained a residual activity of 90% at 48 ° C.

(3) Optimum Reaction pH and pH Stability (Method) The following titration methods were used to measure the activity at the optimum reaction pH and pH stability. First, 8 ml of 0.1
M Tris-HCl buffer (pH 8.5: 1 mM CaCl
₂ 1 in 1 mM DTT, 0.5% Tween 80)
ml of methyl caprylate was added, and the mixture was sufficiently suspended with a homogenizer.
After a shaking reaction at 0 ° C. for a certain period of time, 10 ml of ethanol: acetone (1: 1) was added to terminate the reaction.
As a control, one reacted without adding an enzyme was used.
Phenolphthalein was added and titration was performed with a 0.05 N potassium hydroxide solution at the point where the color changed.
As for the activity, the amount of the enzyme that releases 1 μmole of fatty acid per minute was defined as 1 U. For the optimum reaction pH, the activity was measured by changing the pH. For the pH stability, the remaining activity was measured after shaking at room temperature for 2 hours in a 0.1 M buffer solution adjusted to each pH. The buffer for pH adjustment was McIlvine buffer (pH 3.0 to 8.0), 0.1 M Tris-HCl buffer (pH 7.5 to 9.0), 0.1 M glycine-NaC
1-NaOH buffer (pH 9.0-11.0) was used. (Results) The optimum reaction pH was 8.5 to 9.5, and the pH stability was stable at pH 4 to 10.5.

(4) Substrate Specificity (Method) The substrate was changed to fatty acid methyl ester having various chain lengths and triglyceride, and the activity was measured by the activity measuring method used in (3). When triglyceride was used as a substrate, 0.5 g of triglyceride was added to 8 ml of a 2% polyvinyl alcohol (polymerization degree: 2,000) solution.
After emulsifying with a homogenizer, 1 ml of 1M
Tris-HCl buffer (pH 8.5: 10 mM CaCl
₂ ) and 1 ml of the enzyme solution were added, followed by shaking at 30 ° C. for a certain period of time. The subsequent operation followed the activity measurement method of (3). (Results) As shown in Table 2, the fatty acid methyl ester had high activity with methyl caprylate (having 8 carbon atoms) and the triglyceride had high activity with tributyrin (having 4 carbon atoms).

[0054]

[Table 2]

(5) Influence of metal ion / inhibitor The enzyme was dissolved in 0.1 M Tris-HCl buffer (pH 8.0).
Contact with 1 mM metal ion or inhibitor for 2 hours at room temperature. Thereafter, the activity was measured by the titration method used in (3). As shown in Table 3, metal ions were inhibited by iron divalent and trivalent ions, lead ions, and zinc ions, and activated by calcium ions and manganese ions. Magnesium ion, potassium ion, copper divalent ion, cobalt ion and nickel ion did not affect the activity. Inhibitors almost lose activity in EDTA, and SDS and P
MSF was not affected.

[0056]

[Table 3]

(6) Solvent resistance 1 ml of the enzyme solution was brought into contact with 1 ml of each solvent (normal hexane, cyclohexane, 1-butanol, isopropyl ether, toluene) for 2 hours, and the solvent was separated. It was measured with Lipase Kit S. As a result, 1-butanol had 8% residual activity, but normal hexane, cyclohexane, diisopropyl ether, and toluene retained 80 to 90% residual activity.

Test Example 1 Preparation of (-)-MPGM It was examined whether the lipase protein of the present invention can optically resolve (±) -MPGM. (1) Optical resolution reaction of (±) -MPGM by lipase protein (±) -MPGM (manufactured by Midori Kagaku) in a 1 L beaker
50 g are dissolved in 250 ml of toluene and then 0.2 g
M Tris-HCl buffer (pH 9.0: 1 mM CaC
225 ml of (I ₂ , 1 mM DTT) was added, and the mixture was stirred at 300 rpm by a turbine-type stirring blade. When the mixing was stabilized, 25 ml (10,000 U) of the unwinding enzyme prepared in (i) of (6) of Example 6 was added. 2
After the reaction at room temperature while controlling the pH to 9 with an NNaOH solution, the decomposition state of (±) -MPGM was confirmed by HPLC. As a result, (+)-MPGM was completely decomposed in 3 hours.

(2) Purification and isolation of (−)-MPGM To the reaction solution obtained in the above (1) was added 80 ml of an aqueous solution containing 0.5 g SDS and 15 g NaCl, and the mixture was transferred to a separating funnel. After stirring well, the mixture was allowed to stand overnight. After removing the aqueous layer, 250 ml of a 4.6% aqueous sodium bisulfite solution (pH 7.0) was added to the toluene layer, and the mixture was stirred well, and allowed to stand still to remove the aqueous layer. After performing this washing three times, a separation filter paper (manufactured by Advantech Toyosha: No. 2)
The remaining aqueous layer was removed in S), and the toluene layer was concentrated using an evaporator. The precipitated (-)-MPGM crude crystals were completely dissolved in 50 ml of isopropanol while heating to 60 ° C, and then crystallized at 0 ° C. After removing isopropanol by suction filtration, the crystals were washed twice with cold isopropanol. After drying the crystals,
20.15 g of (-)-MPGM was obtained. The optical purity of the obtained crystal was measured using an optically active column and a chiral cell OJ (φ
4.6 × 250 mm: H by Daicel Chemical Industries, Ltd.
It was 99.9% or more as measured by PLC.

[0060]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1851 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Pseudomonas sp.
p.) SHS2317 sequence ATG GGT GTG TAT GAC TAC AAA AAC CTC GGC ACA GAA GAT TCA AAG GCG 48 CTG TTT TCC GAT GCG CTG GCG ATC ACG CTG TAT TCC TAC CAC AAC CTC 96 GAC AAC GGC TTT TCC GCC GGT TAT CAG CAATAC GGC TTC GGT CTA GGC 144 CTA CCG GCG ACC CTG GTG ACG GCG CTG ATC GGC AGC AGC GAT TCG CAA 192 GGG GTG ATT CCC GGG CTG CCG TGG AAC CCC GAC TCG GAG AAA GCC GCG 240 CTG GAG GCG GTG CAA AAG GCC GGC TGG ACG CCG ATC GGC GCT TCC CAA 288 CTG GGC TAC GAC GGC AAG ACC GAC TTC AGG GGC ACC TAC TAC GGG GAA 336 AAG GCC GGC TAC ACC ACG GCC CAG GCC GAA GTC CTC GGC AAG TAC GAC 384 GAC CAG GGC CAC CTG GACC TTC ATC ATT GCC TTT CGC GGC ACC AGC 432 GGG CCT CGT GAA TCG CTG ATC AGC GAC TCC ATC GGC GAT GCG ATC AAC 480 GAC CTG ATG GCG GCG TTC GGG CCA AAG GAC TAC GCG AAA AAC TAC GCC 528 GGC GAA GCC TTC GGC AAA CTC GCC GAT GTC GCG GCC TTC GCC CAG 576 GCC AAC GGC CTG ACG GGC AAG GAC ATC CTG GTC AGC GGC CAC AGC CTT 624 GGC GGC CTG GCG GTC AAC AGC CTG GCG GAC TTG AGC GGC GAT CAC TGG 672 TCG GGG TTC TAC CAG GAC TCC AGC TAC GTC GCC TAC GCC TCG CCG ACC 720 CAG AGC GTG GGG GAC AAG GTG CTG AAT ATC GGC TAC GAA AAC GAC CCG 768 GTG TTC CGC GCC CTC GAT GGT TCC TCG TTC AAC CTG GCG ACC CTC GGC 816 GTG CAC ACG GCG CAG GAG TCG GCG ACC AAC AAC ATC GTC AGC TTC 864 AAC GAC TAT TAC GCC TCG ACG GCC TGG AAC CTG TTG CCG TTC TCC ATC 912 CTG AAT CTT CCG ACC TGG CTG TCG CAC ATG CCC ACC GGT TAC GGC GAC 960 GGC ACG CGG ATC CTC GAG TCG AAG TTC TAC GAC CTG ACG AGC AAG 1008 GAC TCG ACC ATC ATC GTC GCC AAC CTG TCG GAC CCG GCG CGG GCC ACC 1056 ACC TGG GTC CAG GAC CTC AAC CGC AAT GCC GAA ACC CAC AAG GGC AGC 1 TTC ATC ATC GGC ACC GAC AGC AAC GAC CTG ATC CAG GGC GGC CAG 1152 GGC AAC GAC TAC CTG GAA GGC CGC GCC GGC AAC GAC ACC TTC CGC GAT 1200 TCC GGC GGC TAC AAC ATC CTG CTG GGC GGG CAG GGC AAC AAC ACC CTG GAC CTG CAG CAG TCG GTG AAG AAC TTC AGC TTC GCC AAC GAT GGC GCC 1296 GGC ACC CTG TAT GTG CGC GAT GCC AAT GGC GGC ATC AGC ATC ACC CGC 1344 GAT ATC GGC GCG ATC ACC AGC AAG GAG CCG GGG TTC CTC TGG GGC CTG 1392 TTC AAG GAC GAC GTG ACC CAC AGC GTC ACC GAC AAG GGC CTG CTG GCG 1440 GGG AGC AAC CTG ACT CAG TAC GCC TCT TCG GTG AAG GGC GAC GCT GCG 1488 GCC AAT ACC CTG ACC GCT CAC GCC AGC GGC GGC TGG TTC GGC CTG 1536 GAC GGC GAT GAC CAC CTG ATC GGC GGC CGG GGC AAC GAT GTG CTG GTC 1584 GGT GGC GCG GGC AAC GAC CTG CTG GAG GCG GGC GGG GGC AGC AAT ACC 1632 TTC CTG TTC AGC GGC GAC TTC GGC CAGA GGT CTG GGC TAC CAG 1680 AGC AGC GAC AAA CTG GTG TTC CTC GGC GTC GAA GGC GTC GTC GCC AAC 1728 GAC GAC TTC CGC GCC CAC GCC AGC ACC CTG GGC AAC GAT ACC CTG CTG 1776 ACC TTC GGC AGC GAC TCG GTG ACC CTG GTA GTC AGC CTG GAC AGC 1824 CTC AAC GGC GCG AGC ATC ACC ATC GCC 1851

SEQ ID NO: 2 Sequence length: 617 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Met Gly Val Tyr Asp Tyr Lys Asn Leu Gly Thr Glu Asp Ser Lys Ala 1 5 10 15 Leu Phe Ser Asp Ala Leu Ala Ile Thr Leu Tyr Ser Tyr His Asn Leu 20 25 30 Asp Asn Gly Phe Ser Ala Gly Tyr Gln Gln Tyr Gly Phe Gly Leu Gly 35 40 45 Leu Pro Ala Thr Leu Val Thr Ala Leu Ile Gly Ser Ser Asp Ser Gln 50 55 60 Gly Val Ile Pro Gly Leu Pro Trp Asn Pro Asp Ser Glu Lys Ala Ala 65 70 75 80 Leu Glu Ala Val Gln Lys Ala Gly Trp Thr Pro Ile Gly Ala Ser Gln 85 90 95 Leu Gly Tyr Asp Gly Lys Thr Asp Phe Arg Gly Thr Tyr Tyr Gly Glu 100 105 110 Lys Ala Gly Tyr Thr Thr Ala Gln Ala Glu Val Leu Gly Lys Tyr Asp 115 120 125 Asp Gln Gly His Leu Thr Ser Ile Gly Ile Ala Phe Arg Gly Thr Ser 130 135 140 Gly Pro Arg Glu Ser Leu Ile Ser Asp Ser Ile Gly Asp Ala Ile Asn 145 150 155 160 Asp Leu Met Ala Ala Phe Gly Pro Lys Asp Tyr Ala Lys Asn Tyr Ala 165 170 175 Gly Glu Ala Phe Gly Lys Leu Leu Ala Asp Val Ala Ala Phe Ala Gln 180 185 190 Ala Asn Gly Leu Thr Gly Lys Asp Ile Leu Val Ser Gly His Ser Leu 195 200 205 Gly Gly Leu Ala Val Asn Ser Leu Ala Asp Leu Ser Gly Asp His Trp 210 215 220 Ser Gly Phe Tyr Gln Asp Ser Ser Tyr Val Ala Tyr Ala Ser Pro Thr 225 230 235 240 Gln Ser Val Gly Asp Lys Val Leu Asn Ile Gly Tyr Glu Asn Asp Pro 245 250 255 Val Phe Arg Ala Leu Asp Gly Ser Ser Phe Asn Leu Ala Thr Leu Gly 260 265 270 270 Val His Asp Thr Ala Gln Glu Ser Ala Thr Asn Asn Ile Val Ser Phe 275 280 285 Asn Asp Tyr Tyr Ala Ser Thr Ala Trp Asn Leu Leu Pro Phe Ser Ile 290 295 300 Leu Asn Leu Pro Thr Trp Leu Ser His Met Pro Thr Gly Tyr Gly Asp 305 310 315 320 Gly Met Thr Arg Ile Leu Glu Ser Lys Phe Tyr Asp Leu Thr Ser Lys 325 330 335 Asp Ser Thr Ile Ile Val Ala Asn Leu Ser Asp Pro Ala Arg Ala Thr 340 345 350 Thr Trp Val Gln Asp Leu Asn Arg Asn Ala Glu Thr His Lys Gly Ser 355 360 365 Thr Phe Ile Ile Gly Thr Asp Ser Asn Asp Leu Ile Gln Gly Gly Gln 370 375 380 380 Gly Asn Asp Tyr Leu Glu Gly Arg Ala Gly Asn Asp Thr Phe Arg Asp 385 390 395 400 400 Ser Gly Gly Tyr Asn Ile Leu Leu Gly Gly Gln Gly Asn Asn Thr Leu 405 410 415 Asp Leu Gln Gln Ser Val Lys Asn Phe Ser Phe Ala Asn Asp Gly Ala 420 425 430 Gly Thr Leu Tyr Val Arg Asp Ala Asn Gly Gly Ile Ser Ile Thr Arg 435 440 445 Asp Ile Gly Ala Ile Thr Ser Lys Glu Pro Gly Phe Leu Trp Gly Leu 450 455 460 Phe Lys Asp Asp Val Thr His Ser Val Thr Asp Lys Gly Leu Leu Ala 465 470 475 480 Gly Ser Asn Leu Thr Gln Tyr Ala Ser Ser Val Lys Gly Asp Ala Ala 485 490 495 Ala Asn Thr Leu Thr Ala His Ala Ser Gly Asp Trp Leu Phe Gly Leu 500 505 510 Asp Gly Asp Asp His Leu Ile Gly Gly Arg Gly Asn Asp Val Leu Val 515 520 525 Gly Gly Gly Ala Gly Asn Asp Leu Leu Glu Ala Gly Gly Gly Gly Ser Asn Thr 530 535 540 Phe Leu Phe Ser Gly Asp Phe Gly Gln Asp Arg Val Leu Gly Tyr Gln 545 550 555 560 Ser Ser Asp Lys Leu Val Phe Leu Gly Val Glu Gly Val Val Ala Asn 565 570 575 Asp Asp Phe Arg Ala His Ala Ser Thr Leu Gly Asn Asp Thr Leu Leu 580 585 590 590 Thr Phe Gly Ser Asp Ser Val Thr Leu Val Gly Val Ser Leu Asp Ser 595 600 605 Leu Asn Gly Ala Ser Ile Thr Ile Ala 610 615

[Brief description of the drawings]

FIG. 1 is a restriction map of a recombinant plasmid pUMP98. E: EcoRI restriction site, Pv: PvuII restriction site, P _lac : lac promoter, lip _98-2 : SHS
Lipase structural gene derived from strain 2317, Amp ^r : ampicillin resistance gene.

FIG. 2. Plasmids pUEPv198 and pUEP
v298 each restriction map. E: EcoRI restriction site,
H: HindIII restriction sites, Pv: PvuII restriction sites, Hc: HincII restriction sites, N: NcoI restriction site, P _{lac: lac} promoter, lip _98-2: SHS
Lipase structural gene derived from strain 2317, Amp ^r : ampicillin resistance gene.

FIG. 3 is a restriction map of each of expression plasmids pKLIP98NE and pTrcLIP98NE. H: Hin
dIII restriction site, Sma: SmaI restriction site,
P _trc : trc promoter, lip _98-2 : SHS23
Lipase structural gene derived from 17 strains, Amp ^r : ampicillin resistance gene, rrnBT ₁ T ₂ : 5S rRNA T ₁
T ₂ terminator, lacI ^q: lac repressor mutant gene.

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁶ Identification symbol FI // (C12N 9/20 C12R 1:19) (C12N 1/21 C12R 1:19) (C12N 15/09 ZNA C12R 1:38 )

Claims (11)

[Claims] 1. A lipase protein comprising the amino acid sequence from position 1 to position 617 of SEQ ID NO: 2, or at least one selected from substitution, deletion, insertion and addition of one or several amino acid residues in the amino acid sequence. A mutated lipase protein having a single mutation and containing a sequence having at least 70% homology with its amino acid sequence, and having substantially the same activity as the lipase protein. 2. A DNA molecule encoding the protein according to claim 1. 3. The DNA molecule according to claim 2, which has the nucleotide sequence from position 1 to position 1851 of SEQ ID NO: 1. 4. A recombinant plasmid comprising the DNA molecule according to claim 2 incorporated in a vector plasmid. A transformant comprising the recombinant plasmid according to claim 4. 6. The transformant according to claim 5, which is Escherichia coli. 7. The lipase protein according to claim 1, which is produced by the transformant according to claim 5. 8. The method for producing a lipase protein according to claim 1, comprising a step of culturing the transformant according to claim 5, and a step of collecting the protein produced in the cells. 9. The method according to claim 8, further comprising a step of recovering the activity by dialysis or ultrafiltration. 10. A method for optically resolving racemic 3- (4-methoxyphenyl) glycidic acid methyl ester, wherein the protein (2)
A method for producing methyl (2R, 3S) -3- (4-methoxyphenyl) glycidate, comprising hydrolyzing the (S, 3R) form and isolating the (2R, 3S) form. 11. A bacterium deposited under the accession number FERM P-15915 at the Institute of Biotechnology and Industrial Technology.