CN105349507B - A kind of lipase LIPDa6 and its encoding gene and application - Google Patents
A kind of lipase LIPDa6 and its encoding gene and application Download PDFInfo
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Abstract
The invention discloses a kind of lipase LIPDa6 and its encoding gene and applications.The present invention clones from cyst bacterium (Dactylosporangium aurantiacum subsp.Hamdenensis) NRRL 18085 and has obtained new lipase gene lipDa6, its nucleotide sequence is as shown in SEQ ID NO.1, overall length is 795bp, the amino acid sequence of its lipase LIPDa6 encoded includes 264 amino acid altogether as shown in SEQ ID NO.2.Lipase LIPDa6 can be catalyzed fractionation (R, S) methyl mandelate, and optical purity is prepared up to 99% or more (R) 1 methyl mandelate, has very big application value in biochemical industry and biomedicine field.
Description
Technical field
The invention belongs to biochemical industries and biotechnology, and in particular to a kind of lipase LIPDa6 and its encoding gene and
Using.
Background technology
Although chipal compounds atom composition is the same, their stereochemical structure mirror image each other, in biology and
It but differs greatly on pharmaceutical properties.For example l-menthol has a cooling taste, and D- menthols then mouldy taste；S- asparagines are sweet teas
, R- asparagines are bitter；" reaction stops " of R types is pregnant woman's antalgesic and anodyne, and " reaction stops " of S types to fetus
Then there is teratogenesis, once occurred stopping the event for leading to extensive newborn teratogenesis using reaction in history.It can be seen that synthesis of chiral medicine
During object, the optical purity of precursor raw material is important link.
The synthesis of chipal compounds mainly has：(1) chemical method, that is, utilize enantiomer physics and chemical property difference into
Row separation.Usually there are salting out method, inclusion method and Combinatorial resolution.The disadvantage is that the nature difference between requiring enantiomer is big,
Applicable compound is few；(2) synthetic method carries out the chemical synthesis of chipal compounds by designing reaction.This method lacks
Point is that reaction process is usual relatively more violent, expends energy, and a large amount of toxic organic solvents are used in reacting；(3) chromatography
It splits, this method is realized using the difference of the chiral enantiomer adsorption property of filler, the disadvantage is that equipment is expensive, is popularized
Property is poor.
Lipase (Lipase, EC 3.1.1.3), is called and makees triacylglycerol ester hydolyases, is that one kind can be by triacylglycerol water
Solution is the enzyme of glycerine and aliphatic acid, and source is very extensive, has presence in microorganism, plant and animal.Lipase is as life
Object catalyst majority can act on non-natural substrates, and detachable substrate is more, and stereoselectivity is high, does not need confactor,
It is a kind of important chiral catalyst.
R-MA and its derivative are important chiral precursor, are not only semi-synthetic penicillin, cephalosporin, resist and swell
The synthesis precursor of tumor medicine Goniothalamus styryllactones and slimming medicine Phenethanolamines, and
Also it is important chiral selectors.Therefore R-MA and its derivative have the very extensive market demand and application prospect.
But most of lipase applied in the industry are all derived from import, such as the fat of Novo Nordisk companies
Fat enzyme Novozym 435 (comes from Candida antarctica), the Lipase PS of Amano Pharmaceutical companies
(coming from Burkholderiacepacia), Lipase A (coming from Candida antarctica) of Fluka companies etc..These
Lipase is expensive, and production technology is restricted, therefore it is very necessary to develop the lipase with autonomous property right.
Invention content
The purpose of the present invention is the deficiencies that expensive, production technology is restricted for lipase in the prior art, carry
For a kind of new lipase LIPDa6 and its encoding gene and application.
The present invention is from cyst bacterium (Dactylosporangium aurantiacum subsp.Hamdenensis) NRRL
A kind of new lipase LIPDa6 and its encoding gene lipase gene lipDa6 is developed in 18085, is constructed containing fat
The recombinant expression carrier and genetic engineering bacterium of enzyme gene lipDa6 obtains lipase LIPDa6 after culturing gene engineering bacteria, can
(R)-methyl mandelate is prepared applied to chiral resolution (R, S)-methyl mandelate.
The first purpose of the invention is to provide a kind of lipase LIPDa6, amino acid sequence such as SEQ ID NO.2 institutes
Show.
Second object of the present invention is to provide the lipase gene lipDa6 of the lipase LIPDa6 described in coding a kind of.
It is preferred that the nucleotide sequence of the lipase gene lipDa6 is as shown in SEQ ID NO.1.
The present invention also provides a kind of recombinant expression carriers containing the lipase gene lipDa6.The expression carries
Body, preferably pET28a (+) carrier.
The present invention also provides a kind of genetic engineering bacteriums containing the lipase gene lipDa6.The genetic engineering
Bacterium, preferably e. coli bl21 (DE3).
Third object of the present invention is to provide the lipase LIPDa6 in preparation (R) -1- methyl mandelates
Using.
It is preferred that step is：It takes lipase LIPDa6 in the buffer solution that pH is 5.0-9.0, adds (R, S)-almond
Sour methyl esters is reacted, and (R) -1- methyl mandelates are obtained.
The buffer solution, preferably Acetic acid-sodium acetate, Na2HPO4/NaH2PO4With one in Tris-HCl buffer solutions
Kind.
Fourth object of the present invention is to provide the lipase LIPDa6 in tolerance Ca2+、Li+、Fe2+、Mg2+, methanol
Or the application being catalyzed under alcoholic environment.
The lipase LIPDa6 of the present invention derives from cyst bacterium (D.aurantiacum subsp.Hamdenensis) NRRL
18085, it is stored in Chinese Academy of Science Nanhai Ocean Research Institute.The method that the present invention utilizes bioinformatic analysis, from genome
Clone obtains lipase gene in cyst bacterium (D.aurantiacum subsp.Hamdenensis) NRRL 18085 of sequencing
LipDa6, overall length are 795bp (from initiation codon to terminator codon), the lipase LIPDa6 of coding, include 264 altogether
Amino acid；The gene is a completely new lipase gene.It is carried by cloning lipase gene lipDa6 and connecting expression
Body pET-28a (+) converts e. coli bl21 (DE3) afterwards, cultivates and after induced expression, the lipase recombinantly expressed
LIPDa6.Lipase LIPDa6 can be catalyzed fractionation (R, S)-methyl mandelate and optical purity is prepared under optimal conditions
Up to 99% (R) -1- methyl mandelates.Lipase LIPDa6 has the advantages that stability height, high catalytic efficiency, in biochemical industry
There is very big application value with biomedicine field.
Cyst bacterium (Dactylosporangium aurantiacum subsp.Hamdenensis) NRRL of the present invention
18085 are recorded in United States Patent (USP) herein before disclosing, in the patent of Patent No. US4918174, the Shen of the patent
Day it please be September in 1986 26；According to the record of the patent document, Dactylosporangium aurantiacum
Subsp.hamdenensis NRRL18085 are preserved in american agriculture research Culture Collection Center (Agricultural
Research Service Culture Collection, write a Chinese character in simplified form：NRRL), accession number is NRRL 18085.
Description of the drawings
Fig. 1 is influence of the p-nitrophenyl phenolic ester of different side chain lengths to lipase LIPDa6 enzyme activity.
Fig. 2 is the optimal pH and pH stability of lipase LIPDa6, and A is optimal pH curve, and B is pH stability curves.
Fig. 3 is the optimal reactive temperature and temperature stability of lipase LIPDa6, and A is optimal reactive temperature curve, and B is temperature
Spend stability curve.
Fig. 4 is the GC figures that lipase LIPDa6 splits (R, S) -1- methyl mandelates, A be lipase LIPDa6 hydrolysis (R,
S)-methyl mandelate reacts the GC figures after 3h；B is lipase LIPDa6 hydrolysis (R, S)-methyl mandelates, reacts the GC after 5h
Figure；C is methyl mandelate and the mixed GC figures of equimolar (R)-methyl mandelate.
Fig. 5 is the protein expression and purification situation of lipase LIPDa6, and 1 is to contain pET-28a (+) plasmid after IPTG is induced
E. coli bl21 (DE3) whole-cell protein；2 be albumen Marker；3 contain pET-28a (+)-for what is induced without IPTG
E. coli bl21 (DE3) whole-cell protein of LipDa6；4 be the large intestine containing pET-28a (+)-LipDa6 after IPTG inductions
Bacillus BL21 (DE3) whole-cell protein；5 be the recombinant lipase LIPDa6 after Ni column purifications.
Specific implementation mode
The following examples are further illustrations of the invention, rather than limiting the invention.
Embodiment 1：LipDa6 design of primers and open reading frame boundary determine
Extract the genomic DNA of cyst bacterium (D.aurantiacum subsp.Hamdenensis) NRRL 18085, warp
After 16S rRNA verifications are errorless, hand over to the Shanghai bio tech ltd Mei Ji and be sequenced.Using bioinformatics means to gene
Group is annotated, and lipase gene therein is analyzed, it is determined that the wherein open reading frame of lipase gene lipDa6 passes through letter
Number analysis tool http://www.cbs.dtu.dk/services/SignalP/, analysis are found without signal peptide.The fat
Fat enzyme gene lipDa6 is a completely new lipase gene, and as shown in SEQ ID NO.1, overall length is nucleotide sequence
795bp (from initiation codon to terminator codon), the amino acid sequence such as SEQ ID NO.2 of the lipase LIPDa6 of coding
It is shown, totally 264 amino acid.
According to the lipase gene lipDa6 sequences that analysis obtains, design overall length amplimer is as follows：Forward primer：5’-
CACGAATTCGTGACCTTCCATCCCGTGCCAG-3 ', underscore are EcoRI restriction enzyme sites；Reverse primer：5’-
CCCAAGCTTTTAGCGCAGGACGTCGTCGAG-3 ', underscore are III restriction enzyme sites of Hind.
Embodiment 2：The clone of lipase gene lipDa6 and vector construction
2.1PCR amplification
Embodiment 1 is designed primer (forward primer 5 '-CACGAATTCGTGACCTTCCATCCCGTGCCAG-3 ', instead
To primer 5 '-CCCAAGCTTTTAGCGCAGGACGTCGTCGAG-3 ') supreme marine growth Engineering Co., Ltd synthetic primer is sent,
The primer of synthesis is diluted to 10 μM using TE, and extraction (uses the Gene Jet Genomic DNA of Thermo companies
Purtification Kit carry out extracting genome DNA purifying) cyst bacterium (D.aurantiacum subsp.Hamden
Ensis) total DNA of NRRL 18085 establishes reaction system as shown in table 1 as DNA profiling：
1 PCR reaction systems of table
Use following PCR amplification program amplification lipase gene lipDa6：A.95 DEG C denaturation 5min；B.95 DEG C denaturation
1min, 60 DEG C of annealing 0.5min, 72 DEG C of extension 1min 20s carry out 30 cycles；C.72 DEG C extension 10min, is cooled to 10 DEG C.
By PCR product in 1% Ago-Gel, electrophoresis 20min, is placed in gel imaging system and sees under 120V voltages
It examines.Recycle the band of 800bp or so.PCR product is recycled according to the method for plastic recovery kit, uses the 20 sterile washings of μ L
It is de-, obtain the PCR product of purifying recycling.
2.2 digestion
PCR product carries out double digestion, digestion time 1h using following system；Digestion system is：III 1 μ of EcoRI1 μ L, Hind
L, PCR product<The distilled water of 0.3 μ g, sterilizing add to 30 μ L.Purifying recycling obtains the PCR product by double digestion after digestion.
The double digestion of plasmid pET-28a (+)：Picking contains the bacillus coli DH 5 alpha single bacterium colony of the plasmid, is incubated overnight.Make
Plasmid is extracted with plasmid extraction kit, following system double digestion digestion, digestion time 1h are pressed with EcoRI and Hind III；Digestion
System is：1 III 1 μ L of μ L, Hind of EcoRI, Plasmid DNA<The distilled water of 1 μ g, sterilizing add to 20 μ L.Purifying is recycled after digestion
To pET-28a (+) carrier by double digestion.
The restriction enzyme that above-mentioned double digestion uses is the quick restriction endonuclease of Thermo companies production, the purifying after digestion
Recycling uses nucleic acid purification QIAquick Gel Extraction Kit (Magen, Hipure Gel Pure DNA Micro Kit), plasmid extraction reagent
Box is the Plasmid Miniprep Kit of Shanghai Jierui Biology Engineering Co., Ltd, and operating method presses its operation instructions.
2.3 connection
It is attached according to 3: 1 molar ratio by double digestion PCR product and pET-28a (+) carrier.What connection used
T4 ligases are purchased from Beijing Quan Shijin biotech companies, and it is 5U/5 μ L linked systems to connect the enzyme amount used, and connection temperature is
22 DEG C, Connection Time 20min.
2.4 conversions and screening
It takes in 5 μ L connection products and 50 μ L escherichia coli DH5a competent cells, ice bath 30min, in 42 DEG C of water-bath heat
Swash 90s, 500 μ L LB liquid mediums are added after ice bath 2min, 37 DEG C of 200rpm cultivate 1h.Culture 4000rpm centrifuges 1min
Afterwards, supernatant 400 μ L, remaining 100 μ L are abandoned and is coated on the LB tablets containing 50 μ L/mL kanamycins, single bacterium is selected after cultivating 20h
It falls.Single bacterium is fallen within be incubated overnight in 5mL LB culture mediums after extract plasmid, carry out double digestion verification, endonuclease bamhi and gene size
Identical is positive colony.
2.5 gene nucleotide series measure
The correct positive colony of acquisition is sent to Shanghai Mei Ji biological medicines Co., Ltd and is sequenced, sequencing result and fat
The nucleotide sequence of fat enzyme gene lipDa6 is compared, and confirmation is by lipase gene lipDa6 (its nucleotide sequence such as SEQ
Shown in ID NO.1) it is inserted into pET-28a (+) plasmid, as a result completely correct rear confirmation obtains carrying lipase gene lipDa6
PET-28a (+) plasmid (being named as pET-28a (+)-lipDa6), can be used for carry out next step experiment.
Embodiment 3：High efficient expressions of the lipase LIPDa6 in e. coli bl21 (DE3)
It is prepared by 3.1 e. coli bl21s (DE3) competent cell.
A, e. coli bl21 (DE3) is accessed in 5mL LB liquid mediums, 37 DEG C are shaken training, 250rpm overnight；
B, e. coli bl21 (DE3) bacterium solution after shaking training overnight is inoculated into LB shaking flasks by the rate of vaccination of 1% volume ratio
In, 37 DEG C are shaken training 3h (>=300rpm), obtain stock culture；
C, shaking flask is rapidly cooled to 0 DEG C in ice water, stock culture is dispensed to the centrifuge tube (50mL) being pre-chilled to ice, ice is set
Several minutes；
D, 4 DEG C, 4000rpm centrifuges 10min and recycles cell, and residual liquid air is done (rapid)；
E, the CaCl of the 10mL 0.1M of ice precooling2Cell is resuspended, 4 DEG C, 4000rpm centrifuges 10min and recycles cell；
F, the CaCl of 10mL 0.1M2Cell, ice bath 1h or more is resuspended；
G, 4 DEG C, 4000rpm centrifuges 10min and recycles cell；
H, the recycling cell 2mL obtained per 50mL stock cultures contains the CaCl of 15% glycerine2It is resuspended, is sub-packed in
1.5mL centrifuge tubes, 200 μ L are often managed.- 80 DEG C of preservations.Thus e. coli bl21 (DE3) competent cell is obtained.
3.2 conversion
0.5~1 μ L of pET-28a (+)-lipDa6 plasmids and 50 μ L e. coli bl21s (DE3) obtained in Example 2
Competent cell mixes, and 500 μ L LB Liquid Cultures are added in ice bath 30min after 42 DEG C of water-baths heat shock 90s, ice bath 2min
Base, 37 DEG C of 200rpm cultivate 1h.LB tablet of the coating containing 50 μ L/mL kanamycins, list is selected after cultivating 20h after culture centrifugation
Bacterium.Thus the e. coli bl21 (DE3) containing pET-28a (+)-lipDa6 is obtained.
Embodiment 4：The expression and purifying of lipase LIPDa6
4.1 protein induced
It is 0.5 that e. coli bl21 (DE3) containing pET-28a (+)-lipDa6, which is cultivated in LB culture mediums to OD600,
Left and right adds IPTG to concentration 0.5mM, 22 DEG C of 16 hours of culture.300mL bacterium solutions 4000rpm, 4 DEG C of centrifugation 10min collect bacterium
Body, with 30mL (50mM, pH 7.2) NaH2PO4/Na2HPO4Thalline is resuspended in buffer solution, and ultrasonic 400w, super 4s stop 6s, is crushed
10min minutes, supernatant was collected in centrifugation.
The purifying of 4.2 lipase
Purified to the supernatant collected in step 4.1 with nickel ion affinity chromatograph column, the lipase purified
LIPDa6 enzyme solutions (Fig. 5), the albumen size about 30kD of purifying, meet theory expectation.Specific embodiment is as follows：Use 5mM's
Imidazoles elutes 5 column volumes, and 20~40mM imidazoles elutes 10 column volumes, finally 300mM imidazoles is used to elute 5 column volumes,
Collect intermediate 3.5mL.Desalination is carried out to above-mentioned lipase with desalting column SephadexG25, concrete operation method is with reference to GE companies
Operation manual carry out.
4.3 lipase LIPDa6 enzyme activity determinations
Lipase LIPDa6 vitality tests use p-nitrophenyl phenolic ester, and the specific method is as follows：1. prepare 10mM to nitro
Phenol ester；2. 940 μ L Tris-HCl buffer (50mM, pH 8.0) are added in 1mL reaction systems, 40 μ L ethyl alcohol, 10 μ L
Lipase LIPDa6 enzyme solutions (2 μ g)；3. at 35 DEG C, after 3~5min, 410nm measures absorbance.
Enzyme-activity unit defines：P-nitrophenyl phenolic ester is hydrolyzed in 1min, discharges the enzyme amount definition needed for 1 μm of ol p-nitrophenol
For an enzyme-activity unit.
Embodiment 5：The zymologic property of lipase LIPDa6
The p-nitrophenyl phenolic ester of 5.1 hydrolysis different lengths
According to 4.3 determination condition, compare the p-nitrophenyl phenolic ester of lipase LIPDa6 effect different lengths, as a result as schemed
1, it is seen that lipase LIPDa6 cannot act on C14, illustrate to long-chain p-nitrophenyl phenolic ester poor specificity.And for middle short length
The function and effect of p-nitrophenyl phenolic ester are preferable, and best substrate is C6, i.e. p-nitrophenol capronate.
5.2 optimal pHs and pH stability
Different buffer solutions is prepared, these buffer solutions have different pH, and as shown in table 2, concentration is 50mM：
The pH of the different buffer systems of table 2
By determination condition (using p-nitrophenol capronate as substrate) in 4.3, by the buffer solution (Tris-HCl
Buffer it) is replaced according to the buffer solution in table 2, measures the enzyme activity of recombinant lipase LIPDa6 in the buffer solution of different pH
As a result power illustrates that (Fig. 2A) lipase LIPDa6 enzyme activity all has certain activity in the range of pH5-9, when pH is 7.5
Maximum, with 50mM Na2HPO4/NaH2PO4Buffer solution is best.
Recombinant lipase LIPDa6 is first placed in the buffer solution of different pH and handles 1h, by determination condition in 4.3 (with to nitro
Phenol capronate is as substrate) measure lipase LIPDa6 enzyme activity, enzyme activity of the lipase LIPDa6 in acid range decline compared with
Soon, the stability in alkaline range is preferable, the stability highest (Fig. 2 B) in the Tris-HCl buffer solutions that pH is 7.5.
5.3 optimum temperatures and temperature stability
Use the Na of pH7.52HPO4/NaH2PO4As buffer solution, the reaction prepared by determination condition in 4.3 is mixed
Liquid is placed at different temperature (using p-nitrophenol capronate as substrate) and handles 30min, and lipase LIPDa6 is then added,
Enzyme activity is measured, recombinant lipase LIPDa6 optimal reactive temperatures are at 45 DEG C or so, and higher than 55 DEG C enzyme activity will substantially reduce, 70 DEG C
Enzyme activity is essentially only 45 DEG C of 5% (Fig. 3 A).
Lipase LIPDa6 is placed in (25~70 DEG C) pretreatment 1h of different temperatures, at 45 DEG C, pH7.5, Na2HPO4/
NaH2PO4Buffer solution in, as a result the enzyme activity for measuring lipase LIPDa6 by 4.3 illustrates：Lipase LIPDa6 is at 30 DEG C
Stability is best, and as temperature increases, stability continuously decreases, with respect to the 8% (figure that enzyme activity is 30 DEG C of processing after 60 DEG C of processing 1h
3B).5.4 metal ions inhibit
Use the Na of 50mM, pH7.02HPO4/NaH2PO4Buffer different metal ions solution, per metal ion species
Concentration is 5mM, by lipase LIPDa6 enzyme solutions in the various metal ion solutions of 5mM, 12h is handled in 4 DEG C, to be not added with gold
Belong to the Na of 50mM, pH7.0 of ion2HPO4/NaH2PO4Buffer solution is control (control).According still further to the assay method in 4.3
(using p-nitrophenol capronate as substrate) measures enzyme activity, the results are shown in Table 3, it is seen that Ca2+、Li+And Fe2+To lipase LIPDa6
Enzyme activity has facilitation, and lipase LIPDa6 is to Mg2+Tolerance also relatively preferably, other metal ions are to lipase LIPDa6
Show inhibiting effect.
Influence of 3 metal ion of table to lipase LIPDa6 vigor
Influence of 5.5 organic solvents to lipase active
In 1mL reaction systems be added table 4 in respective concentration organic solvent (volume fraction compares the 50mM for equivalent,
The Tris/HCl buffer solutions of pH 8.0), the lipase LIPDa6 of 10 μ L after purification are added, 12h are handled in 4 DEG C, later according to 4.3
Assay method (using p-nitrophenol capronate as substrate) measure remaining enzyme activity.The results are shown in Table 4, illustrates lipase
LIPDa6 has a preferable tolerance to methanol and ethyl alcohol, and lipase LIPDa6 enzyme activity is control after the methyl alcohol process of 10% (V/V)
101.91 ± 1.13%, enzyme activity is the 90.60 ± 3.96% of control after 10% (V/V) alcohol treatment.
4 organic solvent of table, denaturant and inhibitor are on the active influences of lipase LIPDa6
5.6 inhibitor and activator are on the active influences of lipase LIPDa6
The inhibitor and activator of respective concentration in table 5 is added in 1mL reaction systems, and (percentage in table 5 indicates
A concentration of volume fraction, compares the 50mM for equivalent, the Tris/HCl buffer solutions of pH 8.0), the fat of 10 μ L after purification is added
Enzyme LIPDa6 handles 12h in 4 DEG C, is measured later according to 4.3 assay method (using p-nitrophenol capronate as substrate) surplus
Remaining enzyme activity.It the results are shown in Table 5, it is seen that the reagent in table produces inhibiting effect to lipase LIPDa6 enzyme activity, is especially denaturalized
Agent SDS, urea and guanidine hydrochloride.
5 inhibitor of table and activator are on the active influences of lipase LIPDa6
Embodiment 6：Applications of the lipase LIPDa6 in fractionation (R, S)-methyl mandelate
This law obtains (R)-methyl mandelate using the hydrolysis (R, S) in water phase-methyl mandelate.
Methyl mandelate is a kind of important medicine intermediate and pharmaceutical synthesis skeleton, Enzymatic Resolution (R, S)-mandelic acid first
Ester by pH value of solution, reaction temperature, helping organic solvent type, concentration of substrate and reaction time etc., all many conditions are influenced.This hair
It is bright to optimize above-mentioned parameter, realize the chiral resolution of (R, S)-methyl mandelate.Under optimal conditions, i.e., in 500 μ L
The pH7.550mMNa of 50mM2HPO4/NaH2PO4In solution, 50 μ L lipase LIPDa6 enzyme solutions (10 μ g) and 10mmol is added
Under the conditions of 200rpm, (R)-methyl mandelate of 99% optical purity can be obtained in 37 DEG C in (R, S)-methyl mandelate in 5h
(Fig. 4).
Concrete analysis condition is：Using welfare gas chromatograph, it are furnished with chiral column (30m × 0.25mm Cyclosil B
Chirl column) and hydrogen ion flame detector.Instrumental conditions are set as：220 DEG C of injector temperature, detector temperature
250 DEG C, carrier gas N2, flow velocity 1.2mL/min analyzed using gradient increased temperature：100 DEG C keep 1min, 10 DEG C/min to 220
DEG C, keep 1min.
Claims (7)
1. a kind of lipase LIPDa6, which is characterized in that its amino acid sequence is as shown in SEQ ID NO.2.
2. a kind of lipase gene of coding lipase LIPDa6 described in claim 1.
3. lipase gene according to claim 2, which is characterized in that the nucleotide sequence of the lipase gene is such as
Shown in SEQ ID NO.1.
4. applications of the lipase LIPDa6 described in claim 1 in preparation (R) -1- methyl mandelates.
5. application according to claim 4, which is characterized in that its step is：Take lipase LIPDa6 in pH be 5.0-9.0
Buffer solution in, add (R, S)-methyl mandelate and reacted, obtain (R) -1- methyl mandelates.
6. application according to claim 5, which is characterized in that the buffer solution is Acetic acid-sodium acetate, Na2HPO4/
NaH2PO4With one kind in Tris-HCl buffer solutions.
7. lipase LIPDa6 described in claim 1 is in tolerance Ca2+、Li+、Fe2+、Mg2+, catalytic water under methanol or alcoholic environment
Solution p-nitrophenol capronate or catalyzing hydrolysis (R, S)-methyl mandelate obtain the application in (R) -1- methyl mandelates.
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| CN201510938236.4A CN105349507B (en) | 2015-12-15 | 2015-12-15 | A kind of lipase LIPDa6 and its encoding gene and application |
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