CN105296446B

CN105296446B - A kind of lipase L-1 and its encoding gene and application

Info

Publication number: CN105296446B
Application number: CN201510760314.6A
Authority: CN
Inventors: 胡云峰; 王召贺; 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: 2015-11-09
Filing date: 2015-11-09
Publication date: 2018-06-19
Anticipated expiration: 2035-11-09
Also published as: CN105296446A

Abstract

The invention discloses a kind of lipase L 1 and its encoding gene and applications.The present invention clones from actinomyces (Streptomyces sp.) SCSIO 13580 and obtains lipase gene l 1, its nucleotide sequence is as shown in SEQ ID NO.1, overall length is 1005bp, the amino acid sequence of its lipase L 1 encoded is as shown in SEQ ID NO.2, altogether comprising 334 amino acid；E. coli bl21 (DE3) is converted afterwards by cloning lipase gene l 1 and connecting expression vector pET 28a (+), is cultivated and after induced expression, the lipase L 1 recombinantly expressed.Lipase L 1 is reacted as catalyst cinnamyl alcohol and acry radical donor, cinnamyl acetate is prepared, the cinnamyl acetate yield of acquisition is up to 48.3%.Lipase L 1 has the advantages that stability height, high catalytic efficiency, available for fields such as biological medicine, cosmetics and fine chemistry industries.

Description

A kind of lipase L-1 and its encoding gene and application

Technical field

The invention belongs to biochemical industries and biotechnology, and in particular to a kind of lipase L-1 and its encoding gene and should With.

Background technology

Cinnamyl acetate is one of fragrance component extracted from Chinese cassia tree, is fragrance workable for China's regulation.Mostly Number cinnamyl acetate chemically synthesizes, and is only extracted from natural plants on a small quantity.Chemical method wants condition It asks high, needs to complete at high temperature under high pressure, the consumption of equipment and high energy consumption, and there are many by-product productions for meeting in building-up process Raw, subsequent extracted complex process causes serious pollution to the environment.As biocatalyst enzyme have reaction condition is mild, specificity is high, The advantages that by-product is few, environmental pollution is small, the shortcomings that above-mentioned chemical method can be overcome to synthesize, be widely used at present chemical industry, The fields such as pharmacy, papermaking, leather, cosmetics, food.

Lipase (Lipase, EC 3.1.1.3), is called and makees triacylglycerol ester hydolyases, is that one kind can be by triacylglycerol water The enzyme for glycerine and aliphatic acid is solved, 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 to confactor, It is a kind of important chiral catalyst.To the initial stage of lipase research, source is mainly animals and plants, but the fat of plant and animal material Fat enzyme raw material sources are very limited, it is impossible to meet industrial needs, people more turn one's attention to microorganism, begin look for Microbe-derived lipase, to meet the needs of growing.The microbe species for being known to generate lipase are various, carefully There are many strains that can generate lipase in bacterium, mould, actinomyces, microbe-derived lipase is moved using pH, temperature ratio Plant origin will more extensively.1984, Zaks had found that enzyme also has catalytic activity in organic phase, changes that " enzyme can only be in water Be catalyzed in phase " traditional concept, open the epoch of Nonaqueous enzymology, and rapidly become using most living in enzymology Jump, field with the fastest developing speed, the application range of enzyme have also obtained very big extension.Ocean area is vast, is richly stored with Microbial resources also greatly extend the exploitation of ocean means and range that people obtain lipase.

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), the Lipase A (coming from Candida antarctica) of Fluka companies etc..This A little lipase are 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 being directed to the deficiency that lipase in the prior art is expensive, production technology is restricted, carry For a kind of new lipase L-1 and its encoding gene and application.

The present invention develops a kind of new lipase L-1 from actinomyces (Streptomyces sp.) SCSIO 13580 And its encoding gene lipase gene l-1, recombinant expression carrier and genetic engineering bacterium containing lipase gene l-1 are constructed, Lipase L-1 is obtained after culturing gene engineering bacteria, catalysis is can be applied to and prepares cinnamyl acetate.

First purpose of the present invention is to provide a kind of lipase L-1, and amino acid sequence is as shown in SEQ ID NO.2.

Second object of the present invention is to provide the lipase gene l-1 of the lipase L-1 described in coding a kind of.

It is preferred that the nucleotide sequence of the lipase gene l-1 is as shown in SEQ ID NO.1.

The present invention also provides a kind of recombinant expression carriers containing the lipase gene l-1.The expression vector, It is preferred that pET28a (+) carrier.

The present invention also provides a kind of genetic engineering bacteriums containing the lipase gene l-1.The genetic engineering bacterium, It is preferred that e. coli bl21 (DE3).

Third object of the present invention is to provide applications of the lipase L-1 in cinnamyl acetate is prepared.

It is preferred that step is：Lipase L-1 is taken in reaction medium, adds cinnamyl alcohol and acry radical donor, is carried out anti- Should, cinnamyl acetate is prepared.

The reaction medium, preferably Isosorbide-5-Nitrae-dioxane, tetrahydrofuran, the tert-butyl alcohol, isopropanol, dichloromethane, first One kind in benzene, hexamethylene, n-hexane, normal heptane and isooctane.

The acry radical donor, preferably vinyl acetate, methylvinyl acetate, acetic anhydride, acetic acid, sec-butyl acetate and One kind in ethyl acetate.

Fourth object of the present invention is to provide the lipase L-1 in tolerance short chain alcohol, hydro carbons, acetone, Tween- The application being catalyzed under 20 or TritonX-100 environment.

The short chain alcohol is methanol, ethyl alcohol, isopropanol, the tert-butyl alcohol, isobutanol, n-amyl alcohol or n-hexyl alcohol；The hydrocarbon Class is toluene, 1,4- dioxane, hexamethylene, normal heptane, normal octane or n-decane.

The lipase gene l-1 of the present invention comes from actinomyces (Streptomyces sp.) SCSIO 13580, in being stored in South Sea institute of oceanography of the academy of sciences of state.The method of present invention bioinformatic analysis, from the actinomyces of gene order-checking Obtain lipase gene l-1 in (Streptomyces sp.) SCSIO 13580, overall length for 1005bp (from initiation codon to Terminator codon), encode 334 amino acid；The zymoprotein is a completely new lipase, with other fatty enzyme amino acid sequences Maximum similarity be 51%.It is converted afterwards greatly by cloning lipase gene l-1 and connecting expression vector pET-28a (+) Enterobacteria BL21 (DE3) is cultivated and after induced expression, the lipase L-1 recombinantly expressed.Lipase L-1 is as catalysis Agent is catalyzed cinnamyl alcohol and acry radical donor is reacted, and cinnamyl acetate is prepared, the cinnamyl acetate yield of acquisition is reachable 48.3%.Lipase L-1 has the advantages that stability height, high catalytic efficiency, available for biological medicine, cosmetics and fine chemistry industry The fields of grade.

Description of the drawings

Fig. 1 is influence of the p-nitrophenyl phenolic ester of different acyl length to lipase L-1 enzyme activity.

Fig. 2 is the optimal pH of lipase L-1 and pH stability, and A is optimal pH curve, and B is pH stability curves.

Fig. 3 is the optimal reactive temperature and temperature stability of lipase L-1, and A is optimal reactive temperature curve, and B is temperature Stability curve.

Fig. 4 is the gas chromatogram of lipase L-1 synthesis of acetic acid cinnamic esters, is successively followed successively by according to retention time：1st, it is interior Mark dodecane, 2, cinnamyl alcohol and 3, cinnamyl acetate.

Fig. 5 is the PAGE gel of lipase L-1 purifying, desalination as a result, wherein, 1 is protein markers, and 2 are IPTG induction before total protein control, 3 for IPTG induction after total protein control, 4 for IPTG induction after cell supernatant, 5 Liquid is penetrated for nickel column, 6 be ni-sepharose purification albumen, and 7 be desalination albumen (Lipase protein i.e. in figure, molecular mass：38kD).

Specific embodiment

Following embodiment is the further explanation to the present invention rather than limitation of the present invention.

The lipase gene l-1 of the present invention comes from streptomycete (Streptomyces sp.) SCSIO 13580, is stored in Chinese Academy of Science Nanhai Ocean Research Institute, the gene can also be obtained by the means such as artificial synthesized.

Embodiment 1：Lipase gene l-1 design of primers and open reading frame boundary determine

The genomic DNA of actinomyces (Streptomyces sp.) SCSIO 13580 is extracted, is verified through 16S rRNA errorless Afterwards, it hands over to Shanghai Mei Ji bio tech ltd and is sequenced.Genome is annotated using bioinformatics means, analyzes it In lipase gene, it is determined that the wherein open reading frame of lipase gene l-1, nucleotide sequence such as SEQ ID NO.1 Shown, overall length is 1005bp (from initiation codon to terminator codon), and the amino acid sequence of the lipase L-1 of coding is such as Shown in SEQ ID NO.2, totally 334 amino acid；The zymoprotein is a completely new lipase, with other lipase amino acid sequences The maximum similarity of row is 51%.According to the lipase gene l-1 sequences that analysis obtains, design overall length amplimer is as follows：Just To primer：5′-CACGGATCCATGAGGATCGATCCGCCCG-3 ', underscore part are BamH I restriction enzyme sites；Reversely draw Object：5′-CATCTCGAGTTAGGCGGGCTGTGGGGTC-3 ', underscore part are Xho I restriction enzyme sites.

Embodiment 2：The clone of lipase gene l-1 and vector construction

2.1 PCR amplification

Primer (5 '-the CAC of forward primer that embodiment 1 is designedGGATCCATGAGGATCGATCCGCCCG-3 ' reversely draws 5 '-CAT of objectCTCGAGTTAGGCGGGCTGTGGGGTC-3 ') supreme marine growth Engineering Co., Ltd synthetic primer is sent, synthesis Primer is diluted to 10 μM using TE, extracts the total DNA of actinomyces (Streptomyces sp.) SCSIO 13580 as DNA moulds Plate establishes reaction system as shown in table 1：

1 PCR reaction systems of table

Use following PCR amplification program amplification lipase gene l-1：A.95 DEG C denaturation 5min；DEG C b.95 denaturation 1min, 55 ~65 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 1000bp or so.PCR product is recycled according to the method for plastic recovery kit, uses 20 μ L sterile waters Elution obtains the PCR product of purifying recycling.

2.2 digestion

The PCR product for purifying recycling is subjected to double digestion, digestion time 1h using following system.Digestion system is：BamH I1 μ L, XhoI1 μ L, DNA<0.3 μ g, the distilled water of sterilizing add to 30 μ L.Purifying recycling obtains the PCR by double digestion after digestion Product.

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, with BamHI and XhoI by following system double digestion digestion, digestion time 1h.Digestion body It is to be：11 μ L of μ L, XhoI of BamH I, Plasmid DNA<1 μ g, the distilled water of sterilizing add to 20 μ L.Purifying recycling obtains after digestion By pET-28a (+) carrier of double digestion.

Quick restriction endonuclease of the restriction enzyme that above-mentioned double digestion uses for the production of Thermo companies, 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 will connect by the PCR product of double digestion and pET-28a (+) carrier of double digestion according to 5: 1 molar ratio It connects.The T4 ligases used are connected purchased from Beijing Quan Shijin biotech companies, it is 5U/5 μ L connectors to connect the enzyme amount used System, connection temperature are 22 DEG C, Connection Time 20min.

2.4 conversion and screening

It takes in 10 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 50s, 500 μ L LB fluid nutrient mediums are added in after ice bath 2min, 37 DEG C of 200rpm cultivate 1h.Culture 4000rpm is centrifuged After 1min, abandon supernatant 400 μ L, remaining 100 μ L and be coated on the LB tablets containing 50 μ L/mL kanamycins, list is selected after cultivating 20h Bacterium colony.Single bacterium is fallen within be incubated overnight in 5mL LB culture mediums after extract plasmid, carry out double digestion verification, endonuclease bamhi is big with gene Small identical as 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 Fat enzyme gene l-1 nucleotide sequences are compared, and confirmation is by lipase gene l-1 (its nucleotide sequence such as SEQ ID NO.1 It is shown) it is inserted into pET-28a (+) plasmid, as a result completely correct rear confirmation obtains the pET-28a with lipase gene l-1 (+) plasmid (is named as pET-28a (+)-l-1), available for carrying out next step experiment.

Embodiment 3：High efficient expressions of the lipase L-1 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 fluid nutrient 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 in ice water is rapidly cooled to 0 DEG C, stock culture is dispensed to the centrifuge tube (50mL) being pre-chilled to ice, ice It puts several minutes；

D, 4 DEG C, 4000rpm centrifugation 10min recycling cells do residual liquid air (rapid)；

E, the CaCl of the 10mL 0.1M of ice precooling₂Cell is resuspended, 4 DEG C, 4000rpm centrifugations 10min recycles cell；

F, the CaCl of 10mL 0.1M₂Cell, more than ice bath 1h is resuspended；

G, 4 DEG C, 4000rpm centrifugation 10min recycling cells；

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 (+)-l-1 plasmids and 50 μ L BL21 (DE3) competent cells obtained in Example 2 Mixing, ice bath 30min add in 500 μ L LB fluid nutrient mediums after 42 DEG C of water-baths heat shock 90s, ice bath 2min, 37 DEG C 200rpm cultivates 1h.The kanamycins LB tablets of 50 μ L/mL are coated with after culture centrifugation, single bacterium is selected after cultivating 20h.Thus To the e. coli bl21 (DE3) containing pET-28a (+)-l-1.

Embodiment 4：The expression and purifying of lipase L-1

4.1 protein induced expression

E. coli bl21 (DE3) containing pET-28a (+)-l-1 is cultivated left for 0.5 to OD600 in LB culture mediums The right side adds IPTG to concentration 0.2mM, 22 DEG C of 16 hours of culture.300mL bacterium solutions 4000rpm, 4 DEG C of centrifugation 10min collect thalline, Thalline is resuspended with 20mL (50mM, pH 7.2) PBS buffer solution, ultrasonic 400w, super 4s stop 6s, crush 10min minutes, centrifuge, and receive Collect supernatant.Supernatant is prepared into the thick enzyme powders of lipase L-1 in -80 DEG C of freeze overnights, freeze-drying.

The purifying of 4.2 lipase

Lipase L-1 (the figures for being purified to purify to the supernatant collected in step 4.1 with nickel ion affinity chromatograph column 5), the albumen size about 35kD of purifying, meets theory expectation.Specific embodiment is as follows：5 columns are eluted using the imidazoles of 5mM Volume, 20~100mM imidazoles elute 30 column volumes, finally 5 column volumes are eluted using 100~1000mM imidazoles, in collection Between 3.5mL.Desalination is carried out to above-mentioned lipase with desalting column SephadexG25, concrete operation method is with reference to the operation of GE companies Handbook carries out.

4.3 lipase L-1 enzyme activity determinations

Lipase L-1 vitality tests use p-nitrophenyl phenolic ester, and specific method is as follows：1. prepare the p-nitrophenol of 10mM Ester；2. 220 μ L Tris-HCl buffer (50mM, pH 8.0) are added in 1mL reaction systems, 10 μ L ethyl alcohol, 20 μ L fat Enzyme L-1 enzyme solutions (3.26mg/mL)；3. at 35 DEG C, after 3~5min, 410nm measures absorbance.

Enzyme-activity unit defines：Hydrolysis p-nitrophenyl phenolic ester 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 L-1

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 L-1 effect different acyl length, as a result such as Fig. 1, it is seen that lipase L-1 cannot act on C16, illustrate to long-chain p-nitrophenyl phenolic ester poor specificity；And for moderate-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 the buffer solution (Tris-HCl described in determination condition in 4.3 (using p-nitrophenol capronate as substrate) Buffer it) is replaced according to the buffer solution in table 2, measures the influence of the buffer solution of different PH to the enzyme activity of lipase L-1, As a result illustrate lipase L-1 enzyme activity highest in the range of neutrality to alkalescent, optimal pH 8.0 is preferred with Tris-HCl；PH high It all can drastically decline (Fig. 2A) in 8.0, less than 7.0 enzymatic activitys.

Lipase L-1 is first placed in the buffer solution of different PH and handles 4h, by determination condition in 4.3 (with p-nitrophenol oneself Acid esters is as substrate) lipase L-1 enzyme activity is measured, lipase L-1 enzyme activity is the (figure of stability highest in 8.5 buffer solutions in pH 2B).5.3 optimum temperatures and temperature stability

Using pH8.0, Tris-HCl is as buffer solution, by the reaction mixture in 4.3 (with p-nitrophenol capronate As substrate) it is placed at different temperature and handles 30min, lipase L-1 is then added in, measures enzyme activity, lipase L-1 is most suitable anti- Temperature is answered at 40 DEG C or so, higher than 45 DEG C enzyme activity slowly reduce, and 60 DEG C of enzyme activity are essentially 40 DEG C of 60% (Fig. 3 A).

Enzyme is pre-processed into 1h for (25~70 DEG C) in different temperatures, at 40 DEG C, in the buffer solution of pH8.0, Tris-HCl, The enzyme activity of lipase L-1 is measured by 4.3 assay methods (using p-nitrophenol capronate as substrate), as a result illustrates lipase L- 1 is best in 20 DEG C of stability, and as temperature increases, stability drastically reduces, and enzyme activity is essentially 0 (figure after 40 DEG C of processing 1h 3B)。

5.4 metal ions inhibit

Different metal ions solution is prepared by solvent of the Tris-HCl of 50mM pH7.0, each concentration of metal ions is Lipase L-1 enzyme solutions are handled 1h by 2mM in various metal ion solutions at 37 DEG C；Be not added with the 50mM of metal ion, The Tris-HCl solution of pH7.0 is control (control).Enzyme activity is measured, the results are shown in Table 3, it is seen that only Li²⁺、Mg²⁺To enzyme activity There is facilitation, to Ca²⁺Tolerance also relatively preferably, other metal ions show inhibiting effect to lipase L-1.

Influence of 3 metal ion of table to lipase L-1 enzyme activities

The influence of 5.5 organic solvents and chelating agent to lipase active

To obtain after purification lipase L-1 be added in several organic solvents in table 4 handle 1h (control for distilled water, A concentration of volume fraction of other solution), it is measured according to 4.3 assay method (using p-nitrophenol capronate as substrate) surplus Remaining enzyme activity.The results are shown in Table 4, illustrates that lipase L-1 has short chain alcohol, hydro carbons and Tween-20 a preferable tolerance, and 10% Triton X-100 can improve enzyme activity to 119.48 ± 25.39%, and not high to SDS tolerances, and specially lipase L-1 is to first Alcohol, ethyl alcohol, isopropanol, the tert-butyl alcohol, isobutanol, n-amyl alcohol, n-hexyl alcohol, TritonX-100, toluene, 1,4- dioxane, hexamethylene Alkane, normal heptane, normal octane, n-decane, Tween-20 and acetone have preferable tolerance, and it can improve lipase L-1 enzymes It is living.

The influence of 4 organic solvent of table and chelating agent to lipase L-1 activity

Embodiment 6：Applications of the lipase L-1 in synthesis of acetic acid cinnamic ester

This law is using the transesterification reaction in organic phase come synthesis of acetic acid cinnamic ester.

Cinnamyl acetate is a kind of important fragrance, and enzymatic clarification cinnamyl acetate is organic molten by acry radical donor, reaction The influence of all many conditions of agent type, substrate ratios, reaction temperature etc..Present invention optimizes above-mentioned parameters, realize acetic acid meat The synthesis of osmanthus ester.Under optimal conditions, in the reaction medium of 5mL Isosorbide-5-Nitraes-dioxane, add in final concentration of 300mM's Vinyl acetate adds the thick enzyme powders of 50mg lipase L-1 as acry radical donor and 50mM cinnamyl alcohols, in 25 DEG C, 500rpm items Under part, cinnamyl alcohol is made to be converted to cinnamyl acetate after reacting 96h, conversion ratio reaches 48.3% (Fig. 4).

Concrete analysis condition is：Liquid after reaction takes out 3mL, and 12000rpm centrifugations 1min removes the thick enzymes of lipase L-1 Powder adds in the dodecane of 0.1mM as internal standard compound, after mixing, adds in anhydrous sodium sulfate and remove moisture, take out 100-1000 μ L detect for GCMS.Chromatographic column RXI-5MS, 220 DEG C of injector temperature, input mode：Shunting, column flow：1.20mL/min 200 DEG C of ion source temperature, 280 DEG C of interface temperature.50 DEG C of post case temperature, constant temperature keep 1min, are risen with the rate of 12 DEG C/min 300 DEG C are risen to 150 DEG C, then with the rate of 20 DEG C/min, constant temperature keeps 1min.3 peaks are respectively internal standard compound 12 in Fig. 4 Alkane, substrate cinnamyl alcohol and acetic acid product cinnamic ester, appearance time are respectively 8.385min, 9.799min, 11.022min.

Claims

1. a kind of lipase L-1, which is characterized in that its amino acid sequence is as shown in SEQ ID NO.2.

2. a kind of lipase gene for encoding lipase L-1 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 L-1 described in claim 1 in cinnamyl acetate is prepared.

5. application according to claim 4, which is characterized in that the application is：Lipase L-1 is taken in reaction medium In, cinnamyl alcohol and acry radical donor are added, is reacted, cinnamyl acetate is prepared.

6. application according to claim 5, which is characterized in that the reaction medium is Isosorbide-5-Nitrae-dioxane, tetrahydrochysene furan It mutters, one kind in the tert-butyl alcohol, isopropanol, dichloromethane, toluene, hexamethylene, n-hexane, normal heptane and isooctane.

7. application according to claim 5, which is characterized in that the acry radical donor is vinyl acetate, isopropyl acetate One kind in enester, acetic anhydride, acetic acid, sec-butyl acetate and ethyl acetate.

8. lipase L-1 described in claim 1 is in tolerance short chain alcohol, hydro carbons, acetone, Tween-20 or TritonX-100 rings The application of catalyzing hydrolysis p-nitrophenol capronate under border.

9. application according to claim 8, which is characterized in that the short chain alcohol is methanol, ethyl alcohol, isopropanol, tertiary fourth Alcohol, isobutanol, n-amyl alcohol or n-hexyl alcohol；The hydro carbons for toluene, 1,4- dioxane, hexamethylene, normal heptane, normal octane or N-decane.