CN105349507A

CN105349507A - Lipase LIPDa6 as well as encoding gene and application thereof

Info

Publication number: CN105349507A
Application number: CN201510938236.4A
Authority: CN
Inventors: 胡云峰; 邓盾; 张云; 孙爱君
Original assignee: South China Sea Institute of Oceanology of CAS
Current assignee: South China Sea Institute of Oceanology of CAS
Priority date: 2015-12-15
Filing date: 2015-12-15
Publication date: 2016-02-24
Anticipated expiration: 2035-12-15
Also published as: CN105349507B

Abstract

The invention discloses lipase LIPDa6 as well as an encoding gene and application thereof. The new lipase gene lipDa6 is obtained by cloning from Dactylosporangiumaurantiacum subsp. Hamdenensis NRRRL 18085, the nucleotide sequence is represented as SEQ ID NO.1 and the whole length is 795bp; the amino acid sequence of the encoded lipase LIPDa6 is represented as SEQ ID NO.2 and comprises 264 amino acids. The lipase LIPDa6 can be catalytically split into (R,S)-methyl mandelate, and (R)-1-methyl mandelate with the optical purity which can be 99 percent or more is prepared; the (R)-1-methyl mandelate has a very great application value in the fields of biochemical engineering and biological medicines.

Description

A kind of lipase LIPDa6 and encoding gene thereof and application

Technical field

The invention belongs to biochemical industry and biological technical field, be specifically related to a kind of lipase LIPDa6 and encoding gene thereof and application.

Background technology

Although chipal compounds atom composition is the same, their three-dimensional arrangement mirror image each other, biology and pharmaceutical properties but differ greatly.Such as MENTHOL has cooling taste, and D-menthol is mouldy taste then; S-l-asparagine is sweet, and R-l-asparagine is bitter; " reaction stops " of R type is pregnant woman's anodyne and anodyne, and " reaction stops " of S type then has teratogenesis to fetus, once occurs in history using reaction to stop the event causing extensive newborn teratogenesis.In visible synthesis of chiral pharmaceutical procedures, the optical purity of precursor raw material is important step.

The synthesis of chipal compounds mainly contains: (1) chemical method, namely utilizes the difference of the physics and chemistry character of enantiomorph to be separated.Usually salting-out process, inclusion method and Combinatorial resolution is had.Its shortcoming is that the nature difference between requirement enantiomorph wants large, and the compound be suitable for is few; (2) synthesis method, carries out the chemosynthesis of chipal compounds by design reaction.This method shortcoming is that reaction process is usually relatively more violent, consumes energy, and uses organic solvent poisonous in a large number in reaction; (3) Chromatographic resolution, this method utilizes the difference of the chiral enantiomorph adsorption property of filler to realize, and its shortcoming is apparatus expensive, and popularization is poor.

Lipase (Lipase, EC3.1.1.3), is called triacylglycerol ester hydolyases again, is the enzyme that triacylglycerol can be hydrolyzed to glycerine and lipid acid by a class, and widely, there is existence in its source in microorganism, plant and animal.Lipase can act on non-natural substrates as biological catalyst majority, and detachable substrate is many, and stereoselectivity is high, does not need cofactor, is a kind of important chiral catalyst.

R-MA and its derivative are important chiral precursers, be not only the synthesis precursor of semi-synthetic penicillin, cynnematin, antitumor drug Goniothalamusstyryllactones and slimming medicine Phenethanolamines, but also be important chiral selectors.Therefore R-MA and its derivative have the market requirement and application prospect very widely.

But, the lipase that great majority are applied in the industry is all come from import, the lipase Novozym435 (from Candidaantarctica) of such as NovoNordisk company, the LipasePS (from Burkholderiacepacia) of AmanoPharmaceutical company, the LipaseA (from Candidaantarctica) etc. of Fluka company.These lipase are expensive, and production technology is restricted, and therefore develop the lipase with autonomous property right very necessary.

Summary of the invention

The object of the invention is the deficiency that, production technology expensive for lipase in prior art is restricted, a kind of new lipase LIPDa6 and encoding gene thereof and application are provided.

The present invention develops a kind of new lipase LIPDa6 and encoding gene lipase gene lipDa6 thereof from cyst bacterium (Dactylosporangiumaurantiacumsubsp.Hamdenensis) NRRL18085, construct the recombinant expression vector containing lipase gene lipDa6 and genetic engineering bacterium, lipase LIPDa6 is obtained after culturing gene engineering bacteria, it can be applicable to chiral separation (R, S)-methyl mandelate preparation (R)-methyl mandelate.

First object of the present invention is to provide a kind of lipase LIPDa6, and its aminoacid sequence is as shown in SEQIDNO.2.

Second object of the present invention is to provide the lipase gene lipDa6 of a kind of lipase LIPDa6 encoding described.

Preferably, the nucleotide sequence of described lipase gene lipDa6 is as shown in SEQIDNO.1.

The present invention also provides a kind of recombinant expression vector containing described lipase gene lipDa6.Described expression vector, preferred pET28a (+) carrier.

The present invention also provides a kind of genetic engineering bacterium containing described lipase gene lipDa6.Described genetic engineering bacterium, preferred e. coli bl21 (DE3).

3rd object of the present invention is to provide the application of described lipase LIPDa6 in preparation (R)-1-methyl mandelate.

Preferably, the steps include: that getting lipase LIPDa6 is in the damping fluid of 5.0-9.0 in pH, then add (R, S)-methyl mandelate and react, obtain (R)-1-methyl mandelate.

Described damping fluid, is preferably Acetic acid-sodium acetate, Na ₂hPO ₄/ NaH ₂pO ₄with the one in Tris-HCl damping fluid.

4th object of the present invention is to provide described lipase LIPDa6 at tolerance Ca ²⁺, Li ²⁺, Fe ²⁺, Mg ²⁺, carry out the application of catalysis under methyl alcohol or alcoholic environment.

Lipase LIPDa6 of the present invention derives from cyst bacterium (D.aurantiacumsubsp.Hamdenensis) NRRL18085, is kept at Chinese Academy of Science Nanhai Ocean Research Institute.The present invention utilizes the method for bioinformatic analysis, from cyst bacterium (D.aurantiacumsubsp.Hamdenensis) NRRL18085 of gene order-checking, clone obtains lipase gene lipDa6, total length is 795bp (from initiator codon to terminator codon), the lipase LIPDa6 of its coding, comprises 264 amino acid altogether; This gene is a brand-new lipase gene.By cloning lipase gene lipDa6 and connecting expression vector pET-28a (+) transformation of E. coli BL21 (DE3) afterwards, cultivate and after abduction delivering, obtain recombinant expressed lipase LIPDa6.Lipase LIPDa6 catalysis can split (R, S)-methyl mandelate, under optimal conditions, prepares (R)-1-methyl mandelate that optical purity reaches 99%.Lipase LIPDa6 has the advantage that stability is high, catalytic efficiency is high, has very large using value in biochemical industry and biomedicine field.

Cyst bacterium (Dactylosporangiumaurantiacumsubsp.Hamdenensis) NRRL18085 of the present invention was openly recorded in United States Patent (USP) before the application, its patent No. is in the patent of US4918174, and the applying date of this patent is on September 26th, 1986; According to the record of this patent documentation, Dactylosporangiumaurantiacumsubsp.hamdenensisNRRL18085 is preserved in american agriculture research DSMZ (AgriculturalResearchServiceCultureCollection, write a Chinese character in simplified form: NRRL), its accession number is NRRL18085.

Accompanying drawing explanation

Fig. 1 is the impact that the p-nitrophenyl phenolic ester of different side chain lengths is lived on lipase LIPDa6 enzyme.

Fig. 2 is optimal pH and the pH stability of lipase LIPDa6, and A is optimal pH curve, and B is pH beta stability line.

Fig. 3 is optimal reactive temperature and the temperature stability of lipase LIPDa6, and A is optimal reactive temperature curve, and B is temperature-stable linearity curve.

Fig. 4 is the GC figure that lipase LIPDa6 splits (R, S)-1-methyl mandelate, A is that lipase LIPDa6 is hydrolyzed (R, S)-methyl mandelate, the GC figure after reaction 3h; B is that lipase LIPDa6 is hydrolyzed (R, S)-methyl mandelate, the GC figure after reaction 5h; C is methyl mandelate and waits the mixed GC figure of mole (R)-methyl mandelate.

Fig. 5 is the protein expression and purification situation of lipase LIPDa6, and 1 is e. coli bl21 (DE3) whole-cell protein containing pET-28a (+) plasmid after IPTG induction; 2 is albumen Marker; 3 is e. coli bl21 (DE3) whole-cell protein containing pET-28a (+)-LipDa6 without IPTG induction; 4 is e. coli bl21 (DE3) whole-cell protein containing pET-28a (+)-LipDa6 after IPTG induction; 5 is the recombinant lipase LIPDa6 after Ni column purification.

Embodiment

Following examples further illustrate of the present invention, instead of limitation of the present invention.

Embodiment 1:LipDa6 design of primers and open reading frame border are determined

Extract the genomic dna of cyst bacterium (D.aurantiacumsubsp.Hamdenensis) NRRL18085, after 16SrRNA checking is errorless, hands over and check order to Mei Ji bio tech ltd, Shanghai.Information biology means are utilized to annotate genome, analyze lipase gene wherein, determine the open reading frame of wherein lipase gene lipDa6, by signal analysis instrument http://www.cbs.dtu.dk/services/SignalP/, analyze and find wherein there is no signal peptide.This lipase gene lipDa6 is a brand-new lipase gene, its nucleotide sequence is as shown in SEQIDNO.1, total length is 795bp (from initiator codon to terminator codon), the aminoacid sequence of lipase LIPDa6 of its coding as shown in SEQIDNO.2, totally 264 amino acid.

According to analyzing the lipase gene lipDa6 sequence obtained, design total length amplimer is as follows: forward primer: 5 '-CAC gAATTCgTGACCTTCCATCCCGTGCCAG-3 ', underscore is EcoRI restriction enzyme site; Reverse primer: 5 '-CCC aAGCTTtTAGCGCAGGACGTCGTCGAG-3 ', underscore is Hind III restriction enzyme site.

Embodiment 2: the clone of lipase gene lipDa6 and vector construction

2.1PCR amplification

Primer (the forward primer 5 '-CACGAATTCGTGACCTTCCATCCCGTGCCAG-3 ' that embodiment 1 is designed, reverse primer 5 '-CCCAAGCTTTTAGCGCAGGACGTCGTCGAG-3 ') deliver to Shanghai biotechnology company limited synthetic primer, the primer of synthesis uses TE to be diluted to 10 μMs, extract the STb gene of (adopting the GeneJetGenomicDNAPurtificationKit of Thermo company to carry out extracting genome DNA purifying) cyst bacterium (D.aurantiacumsubsp.Hamdenensis) NRRL18085 as DNA profiling, set up reaction system as shown in table 1:

Table 1PCR reaction system

Use following pcr amplification program amplification lipase gene lipDa6:a.95 DEG C of sex change 5min; B.95 DEG C sex change 1min, 60 DEG C of annealing 0.5min, 72 DEG C extend 1min20s, carry out 30 circulations; C.72 DEG C extension 10min, is cooled to 10 DEG C.

By PCR primer in 1% sepharose, electrophoresis 20min under 120V voltage, is placed in gel imaging system and observes.Reclaim the band of about 800bp.The method that PCR primer reclaims test kit according to glue reclaims, and uses 20 μ L sterilized water wash-outs, obtains the PCR primer that purifying reclaims.

2.2 enzymes are cut

PCR primer uses following system to carry out double digestion, and enzyme cuts time 1h; The enzyme system of cutting is: EcoRI1 μ L, Hind III 1 μ L, PCR primer <0.3 μ g, the distilled water of sterilizing adds to 30 μ L.Enzyme is cut rear purifying and is reclaimed the PCR primer obtained through double digestion.

The double digestion of plasmid pET-28a (+): picking contains the bacillus coli DH 5 alpha list bacterium colony of this plasmid, incubated overnight.Use plasmid extraction kit to extract plasmid, cut by following system double digestion enzyme with EcoRI and Hind III, enzyme cuts time 1h; The enzyme system of cutting is: EcoRI1 μ L, Hind III 1 μ L, plasmid DNA <1 μ g, the distilled water of sterilizing adds to 20 μ L.Enzyme is cut rear purifying and is reclaimed pET-28a (+) carrier obtained through double digestion.

The quick restriction endonuclease that the restriction enzyme that above-mentioned double digestion uses is produced for Thermo company, enzyme cut after purifying reclaim and use nucleic acid purification to reclaim test kit (Magen, HipureGelPureDNAMicroKit), plasmid extraction kit is the Plasmid Miniprep Kit of Shanghai Jierui Biology Engineering Co., Ltd, and working method is by its working instructions.

2.3 connect

Through double digestion PCR primer and pET-28a (+) carrier according to 3: 1 molar ratio be connected.Connect the T4 ligase enzyme of use purchased from Beijing Quan Shijin biotech company, connecting the enzyme amount used is 5U/5 μ L linked system, and connecting temperature is 22 DEG C, tie-time 20min.

2.4 transform and screening

Get 5 μ L to connect in product and 50 μ L escherichia coli DH5a competent cells, ice bath 30min, in 42 DEG C of water-bath heat shock 90s, adds 500 μ LLB liquid nutrient mediums after ice bath 2min, and 37 DEG C of 200rpm cultivate 1h.After the centrifugal 1min of culture 4000rpm, abandon supernatant 400 μ L, the LB that 100 remaining μ L coat containing 50 μ L/mL kantlex is dull and stereotyped, picking individual colonies after cultivation 20h.Single bacterium colony extracts plasmid after incubated overnight in 5mLLB substratum, and carry out double digestion checking, what endonuclease bamhi was identical with gene size is positive colony.

2.5 gene nucleotide series measure

The correct positive colony obtained is delivered to Shanghai Mei Ji biological medicine company limited check order, the nucleotide sequence of sequencing result and lipase gene lipDa6 is compared, be confirmed to be and lipase gene lipDa6 (its nucleotide sequence is as shown in SEQIDNO.1) is inserted in pET-28a (+) plasmid, the entirely true rear confirmation of result obtains pET-28a (+) plasmid (called after pET-28a (+)-lipDa6) with lipase gene lipDa6, can be used for carrying out next step test.

Embodiment 3: the high expression of lipase LIPDa6 in e. coli bl21 (DE3)

Prepared by 3.1 e. coli bl21s (DE3) competent cell.

A, access in 5mLLB liquid nutrient medium by e. coli bl21 (DE3), 37 DEG C are spent the night and shake training, 250rpm;

B, shake training by the rate of vaccination of 1% volume ratio by spending the night after e. coli bl21 (DE3) bacterium liquid be inoculated in LB shaking flask, 37 DEG C shake training 3h (>=300rpm), obtain stock culture;

C, shaking flask are cooled to rapidly 0 DEG C in frozen water, and stock culture is divided the centrifuge tube being filled to ice precooling (50mL), ice puts several minutes;

D, 4 DEG C, the centrifugal 10min of 4000rpm reclaims cell, will remain liquid air and do (rapidly);

The CaCl of the 10mL0.1M of e, ice precooling ₂re-suspended cell, 4 DEG C, the centrifugal 10min of 4000rpm reclaims cell;

The CaCl of f, 10mL0.1M ₂re-suspended cell, more than ice bath 1h;

G, 4 DEG C, the centrifugal 10min of 4000rpm reclaims cell;

The recovery cell 2mL that h, every 50mL stock culture obtain is containing the CaCl of 15% glycerine ₂come resuspended, be sub-packed in 1.5mL centrifuge tube, 200 μ L often manage.-80 DEG C of preservations.Obtain e. coli bl21 (DE3) competent cell thus.

3.2 transform

PET-28a (+)-lipDa6 plasmid 0.5 ~ 1 μ L obtained in Example 2 mixes with 50 μ L e. coli bl21 (DE3) competent cells, ice bath 30min, in 42 DEG C of water-bath heat shock 90s, add 500 μ LLB liquid nutrient mediums after ice bath 2min, 37 DEG C of 200rpm cultivate 1h.The centrifugal rear coating of culture is dull and stereotyped containing the LB of 50 μ L/mL kantlex, selects single bacterium after cultivating 20h.Obtain the e. coli bl21 (DE3) containing pET-28a (+)-lipDa6 thus.

Embodiment 4: the expression and purification of lipase LIPDa6

4.1 protein induced

It is about 0.5 that e. coli bl21 (DE3) containing pET-28a (+)-lipDa6 is cultured to OD600 in LB substratum, adds IPTG to concentration 0.5mM, cultivates 16 hours for 22 DEG C.300mL bacterium liquid 4000rpm, 4 DEG C of centrifugal 10min, collect thalline, with 30mL (50mM, pH7.2) NaH ₂pO ₄/ Na ₂hPO ₄the resuspended thalline of damping fluid, ultrasonic 400w, super 4s, stops 6s, broken 10min minute, centrifugal, collects supernatant.

The purifying of 4.2 lipase

Carried out purifying with nickel ion affinity chromatograph post to the supernatant collected in step 4.1, obtain lipase LIPDa6 enzyme liquid (Fig. 5) of purifying, the albumen size of purifying is about 30kD, and coincidence theory is expected.Specific embodiments is as follows: imidazoles wash-out 5 column volumes using 5mM, and 20 ~ 40mM imidazoles wash-out, 10 column volumes finally use 300mM imidazoles wash-out 5 column volumes, 3.5mL in the middle of collecting.Carry out desalination with desalting column SephadexG25 to above-mentioned lipase, concrete operation method carries out with reference to the operational manual of GE company.

4.3 lipase LIPDa6 enzyme activity determinations

Lipase LIPDa6 vitality test adopts p-nitrophenyl phenolic ester, and concrete grammar is as follows: the p-nitrophenyl phenolic ester 1. preparing 10mM; 2. in 1mL reaction system, 940 μ LTris-HClbuffer (50mM, pH8.0) are added, 40 μ L ethanol, 10 μ L lipase LIPDa6 enzymes liquid (2 μ g); 3., at 35 DEG C, after 3 ~ 5min, 410nm measures absorbancy.

Enzyme is lived, and unit definition: 1min is interior is hydrolyzed p-nitrophenyl phenolic ester, and the enzyme amount discharged needed for 1 μm of ol p-NP is defined as a Ge Meihuo unit.

Embodiment 5: the zymologic property of lipase LIPDa6

The p-nitrophenyl phenolic ester of 5.1 hydrolysis different lengthss

According to the condition determination of 4.3, compare the p-nitrophenyl phenolic ester of lipase LIPDa6 effect different lengths, result is as Fig. 1, and visible fat enzyme LIPDa6 can not act on C14, illustrates long-chain p-nitrophenyl phenolic ester poor specificity.And better for the action effect of the p-nitrophenyl phenolic ester of short-and-medium length, best substrate is C6, i.e. p-NP capronate.

5.2 optimal pHs and pH stability

Prepare different buffered soln, these buffered soln have different pH, as shown in table 2, and its concentration is 50mM:

The pH of the different buffer system of table 2

By condition determination (using p-NP capronate as substrate) in 4.3, described damping fluid (Tris-HClbuffer) is replaced according to the buffered soln in table 2, measure the enzyme activity of recombinant lipase LIPDa6 in the buffered soln of different pH, result illustrates that the work of (Fig. 2 A) lipase LIPDa6 enzyme all has certain activity in the scope of pH5-9, maximum when pH is 7.5, with 50mMNa ₂hPO ₄/ NaH ₂pO ₄damping fluid is best.

The damping fluid that recombinant lipase LIPDa6 is first placed in different pH processes 1h, measure lipase LIPDa6 enzyme by condition determination (using p-NP capronate as substrate) in 4.3 to live, the enzyme of lipase LIPDa6 in acid range is lived and is declined comparatively fast, stability in alkaline range is better, is stability the highest (Fig. 2 B) in the Tris-HCl damping fluid of 7.5 at pH.

5.3 optimum temperutures and temperature stability

Use the Na of pH7.5 ₂hPO ₄/ NaH ₂pO ₄as buffered soln, 30min is processed under the reaction mixture (using p-NP capronate as substrate) prepared by condition determination in 4.3 is placed in different temperature, then lipase LIPDa6 is added, mensuration enzyme is lived, recombinant lipase LIPDa6 optimal reactive temperature is at about 45 DEG C, will greatly reduce higher than 55 DEG C of enzyme work, 70 DEG C of enzymes 5% (Fig. 3 A) being substantially only 45 DEG C alive.

Lipase LIPDa6 is placed in differing temps (25 ~ 70 DEG C) pre-treatment 1h, at 45 DEG C, pH7.5, Na ₂hPO ₄/ NaH ₂pO ₄buffered soln in, the enzyme measuring lipase LIPDa6 by 4.3 is lived, and result illustrates: lipase LIPDa6 is best the stability of 30 DEG C, along with temperature raises, stability reduces gradually, and after 60 DEG C of process 1h, enzyme work is 8% (Fig. 3 B) of 30 DEG C of process relatively.

5.4 metal ions suppress

Use the Na of 50mM, pH7.0 ₂hPO ₄/ NaH ₂pO ₄buffer different metal solion, every metal ion species concentration is 5mM, by lipase LIPDa6 enzyme liquid in the various metal ion solutions of 5mM, in 4 DEG C of process 12h, not add the Na of 50mM, pH7.0 of metal ion ₂hPO ₄/ NaH ₂pO ₄damping fluid is contrast (control).Measure enzyme according to the measuring method (using p-NP capronate as substrate) in 4.3 again to live, the results are shown in Table 3, visible Ca ²⁺, Li ²⁺and Fe ²⁺live to lipase LIPDa6 enzyme and have promoter action, lipase LIPDa6 is to Mg ²⁺tolerance also relatively better, other metal ion all shows restraining effect to lipase LIPDa6.

Table 3 metal ion is on the impact of lipase LIPDa6 vigor

5.5 organic solvents are on the impact of lipase activity

Organic solvent (the volume fraction of respective concentration in table 4 is added in 1mL reaction system, contrast is the 50mM of equivalent, the Tris/HCl damping fluid of pH8.0), add the lipase LIPDa6 after 10 μ L purifying, in 4 DEG C of process 12h, measure residual enzyme work according to the measuring method (using p-NP capronate as substrate) of 4.3 later.Result is as shown in table 4, illustrate that lipase LIPDa6 has good tolerance to methyl alcohol and ethanol, after the methyl alcohol process of 10% (V/V), lipase LIPDa6 enzyme is lived as after 101.91 ± 1.13%, 10% (V/V) Ethanol Treatment of contrast, enzyme is lived as 90.60 ± 3.96% of contrast.

Table 4 organic solvent, denaturing agent and inhibitor are on the impact of lipase LIPDa6 activity

5.6 inhibitor and activator are on the impact of lipase LIPDa6 activity

In 1mL reaction system, adding the inhibitor of respective concentration in table 5 and activator, (concentration that the percentage ratio in table 5 represents is volume fraction, contrast is the 50mM of equivalent, the Tris/HCl damping fluid of pH8.0,), add the lipase LIPDa6 after 10 μ L purifying, in 4 DEG C of process 12h, measure residual enzyme work according to the measuring method (using p-NP capronate as substrate) of 4.3 later.The results are shown in Table 5, the reagent in visible table is all lived to lipase LIPDa6 enzyme and is created restraining effect, especially denaturing agent SDS, urea and Guanidinium hydrochloride.

Table 5 inhibitor and activator are on the impact of lipase LIPDa6 activity

Embodiment 6: lipase LIPDa6 is splitting the application in (R, S)-methyl mandelate

This law adopts hydrolysis (R, the S)-methyl mandelate in aqueous phase to obtain (R)-methyl mandelate.

Methyl mandelate is a kind of important medicine intermediate and pharmaceutical synthesis skeleton, and Enzymatic Resolution (R, S)-methyl mandelate is subject to pH value of solution, temperature of reaction, helps the impact of all many condition such as organic solvent kind, concentration of substrate and reaction times.Present invention optimizes above-mentioned parameter, achieve (R, S)-the chiral separation of methyl mandelate.Under optimal conditions, namely at the pH7.550mMNa of 500 μ L50mM ₂hPO ₄/ NaH ₂pO ₄in solution, add (R, the S)-methyl mandelate of 50 μ L lipase LIPDa6 enzymes liquid (10 μ g) and 10mmol, in 37 DEG C, under 200rpm condition, (the R)-methyl mandelate (Fig. 4) of 99% optical purity can be obtained in 5h.

Concrete analysis condition is: adopt welfare gas chromatograph, be furnished with chiral column (30m × 0.25mmCyclosilBchirlcolumn) and hydrogen ion flame detector.Instrumental conditions is set to: injector temperature 220 DEG C, detector temperature 250 DEG C, and carrier gas is N ₂, flow velocity 1.2mL/min, adopts gradient increased temperature to analyze: 100 DEG C keep 1min, 10 DEG C/min to 220 DEG C, keep 1min.

Claims

1. a lipase LIPDa6, is characterized in that, its aminoacid sequence is as shown in SEQIDNO.2.

2. the lipase gene lipDa6 of a coding lipase LIPDa6 according to claim 1.

3. lipase gene lipDa6 according to claim 2, is characterized in that, the nucleotide sequence of described lipase gene lipDa6 is as shown in SEQIDNO.1.

4. the application of lipase LIPDa6 according to claim 1 in preparation (R)-1-methyl mandelate.

5. application according to claim 4, is characterized in that, the steps include: that getting lipase LIPDa6 is in the damping fluid of 5.0-9.0 in pH, then adds (R, S)-methyl mandelate and react, and obtains (R)-1-methyl mandelate.

6. application according to claim 5, is characterized in that, described damping fluid is Acetic acid-sodium acetate, Na ₂hPO ₄/ NaH ₂pO ₄with the one in Tris-HCl damping fluid.

7. lipase LIPDa6 according to claim 1 is at tolerance Ca ²⁺, Li ²⁺, Fe ²⁺, Mg ²⁺, carry out the application of catalysis under methyl alcohol or alcoholic environment.