CN104388404A - Microbial fermentation and extraction separation method of lipase - Google Patents

Abstract

The invention discloses a microbial fermentation and extraction separation method of lipase, relating to the technical field of bioengineering. The invention overcomes the defects of low enzyme yield, low enzyme-producing activity, short enzyme life and the like in the traditional fermentation technology, and realizes efficient fermentation production and extraction separation of lipase. The method disclosed by the invention is simple to operate, low in cost and easy to amplify; and the enzyme is high in yield, high in activity, high in purity, favorable in stability and long in service life. The invention has important application value, and can be used in the fields of grease and cheese processing, bread baking, catalytic synthesis of medicine, feed processing, biodiesel synthesis, sewage treatment, textile degreasing and the like.

Description

The fermentable of lipase and extraction and separation method

Technical field

The present invention relates to technical field of bioengineering, be specifically related to a kind of fermentable and extraction and separation method of lipase.

Background technology

Lipase (EC3.1.1.3, Lipase), also known as GEH, refers to the enzyme of the tri-glyceride ester bond decomposing or synthesize higher fatty acid and glycerol formation.Lipase is the special ester linkage hydrolyzing enzyme of a class, only acts on outphasing system, namely only acts on water-oil interface, and only have when substrate with particulate, little polymerization dispersion state or in emulsified particle time, lipase just has significant katalysis to substrate hydrolysis.Extensively there is microbial lipase most species in animal, plant and microorganism in lipase, extensively be present in bacterium, yeast and mould, the most easily obtaining and scale operation, and have pH, the thermal adaptability wider than andvegetable fats enzyme, is therefore the important sources of industrial lipase.

The production of lipase and use cost are one of key factors of its industrial applications of restriction.Because the ability of wild mushroom yielding lipase is low, enzyme system is complicated, and more difficult separation and purification, generally cannot be directly used in scale operation.Build by genetic engineering technique the expression amount that suitable expression system can not only improve lipase, and the separation purifying technique of subsequent protein can be simplified, thus reduce production cost.In addition, the stereoselectivity enzyme preparing purifying is very important for Stereoselective reaction, can avoid the impact of other non-cubic selectivity enzymes.Pichia pastoris phaff (Pichia pastoris) be grew up in recent years comparatively perfect, be widely used for expressing the methanotrophic yeast expression system of foreign protein.Major advantage has: (1) has strong alcohol oxidase (Alochol Oxidase, AOX1) gene promoter, strictly can regulate and control the expression of foreign protein; (2) as eukaryotic expression system, the processing after can translating the albumen of expressing and modification, thus make the albumen given expression to have biological activity; (3) nutritional requirement is low, growth is fast, substratum is cheap, is convenient to suitability for industrialized production; (4) can cultivate by high density fermentation, in fermentor tank, dry cell weight even can reach more than 120g/L; (5) expression amount is high, and much albumen can reach more than g/L level; (6) albumen of expressing both can be present in cell, can be secreted into again outside born of the same parents, and the albumen of self secretion is considerably less, is extremely conducive to purifying; (7) degree of glycosylation is low, and the immunogenicity of secreted glycoprotein is lower, is more conducive to clinical application.

For a long time, the lipase market of China all monopolize by offshore company, and expensive, therefore develop the lipase of China's autonomous innovation and preparation thereof for promotion China's scientific-technical progress with realize that national prosperity is rich and powerful has important progressive meaning.The domestic fermentable about lipase and extractive technique still immature, lipase preparation does not also have a manufacturing enterprise on a large scale in existing zymin manufacturing enterprise of China, the problem such as its product also also exists that catalytic activity is low, poor stability, work-ing life are short.Therefore, active research exploitation lipase and preparation thereof, be conducive to breaking foreign technology monopolization, realizes lipase and change to industrialization and drive the development of related industries, realize the prosperous and powerful of China.

Summary of the invention

For the above-mentioned deficiency of China's prior art, the invention provides a kind of fermentable and extraction and separation method of lipase, technical scheme of the present invention is as follows:

The fermentable of lipase and an extraction and separation method, its method is as follows:

(1) fermented bacterium

Using genetic engineering bacterium Pichia pastoris GS115/pPIC9K-proRCL as fermentative production bacterial classification;

(2) nutrient solution

A, shake flask culture

Yeast extract 10 g/L, peptone 20 g/L, K ₂hPO ₄3g/L, H ₂pO ₄11.8g/L, add water 890mL/L, 121 DEG C of sterilizing 20 min, then treats that temperature is down to less than 60 DEG C and on super clean bench, is added 10 × YNB 100mL/L (13.4 g/L), 500 × vitamin H 1mL/L (4 × 10 ^-4g/L), glycerine 10mL/L;

B, fermentation culture

85% H ₃pO ₄26.7mL/L, CaSO ₄2H ₂o 0.93g/L, K ₂sO4 18.2g/L, MgSO ₄2H ₂o 14.9g/L, KOH 4.13 g/L, glycerine 40g/L, PTM ₁4.0mL/L(is glycerine, PTM wherein ₁add again after the real end that disappears);

c、PTM ₁

CuSO ₄5H ₂o 6.0g/L, KI 0.088g/L, MnSO ₄h ₂o 3.0g/L, Na ₂moO ₄2H ₂o 0.2g/L, H ₃bO ₃0.02g/L, CoC1 ₂6H ₂o 0.5g/L, ZnCl ₂20.0g/L, FeSO ₄7H ₂o 65.0g/L, vitamin H 0.2 g/L, dense H ₂sO ₄5 mL/L;

D, feed-batch culture liquid

(W/V, containing 12mL/L PTM for 50% glycerine ₁) feed supplement liquid;

E, BMMY nutrient solution

Yeast extract 10 g/L, peptone 20 g/L, K ₂hPO ₄3g/L, KH ₂pO ₄11.8g/L, add water 895mL/L, 121 DEG C of sterilizing 20 min, then treats that temperature is down to less than 60 DEG C and on super clean bench, is added 100 × YNB 100mL/L (13.4 g/L), 500 × vitamin H 1mL/L (4 × 10 ^-4g/L), methyl alcohol 5m/L;

F, methanol induction nutrient solution

100% methyl alcohol is (containing 12mL/L PTM ₁);

(3) strain fermentation is produced

A, select a single bacterium colony, be placed in the 250ml shaking flask that 25ml shake flask culture is housed, cultivate 16-18 h in 28-30 DEG C/250-300 rpm;

B, preparation fermentation culture (glycerine, PTM ₁wouldn't add) and add in fermentor tank, demarcate pH electrode and DO electrode and insert tank body, sealing and fermenting tank, connect steam, to air system and the tank body enforcement sterilizing of fermentor tank, treat that tank pressure rises to 0.12MPa, during temperature 121 DEG C, pressurize, insulated sterilizing 20min, treat the real end that disappears, and connects freeze cycle water and temperature is cooled to 28-30 DEG C;

C, inoculation mouth around put a circle cotton ball soaked in alcohol, light, aseptically open inoculation mouth, shake flask culture is inoculated in fermentor tank rapidly by 5-10% inoculum size, and add the glycerine 40g/L of filtration sterilization, PTM ₁4.0mL/L, set the parameters such as temperature, mixing speed, air flow, ferment, ferment about 20 h, glycerine in fermentation culture is consumed totally, now DO rises to 100% rapidly, start flow feeding nutrient solution, it is 6-24 h that the stream of feed-batch culture liquid adds the time, during the fermentation, keeps temperature 28-30 DEG C in tank, dissolved oxygen DO>20% in tank, pH value 5-7 in tank, by the height regulating air flow and mixing speed to control dissolved oxygen, the aseptic ammoniacal liquor of auto-feeding mass concentration 25% controls the pH value size of fermentation culture;

D, treat that feed-batch culture liquid stream adds end, stop feed supplement, maintain the hungry 0.5-1 h of cell, then stream adds BMMY nutrient solution 0.5-1 h and allows yeast adapt to methyl alcohol environment, start stream when DO rises to 100% again and add methanol induction nutrient solution, in the hierarchy of control, the mass concentration of methyl alcohol is 1.0-2.0% in 0-1h, be 3.0-5.0% in 2-4h, be 2.0-3.0% in 4-8h, be 0.5-1.0% after 8h, between inductive phase, temperature controls at 20-28 DEG C, pH value controls at 5.5-6.5, dissolved oxygen DO>20%;

E, methanol induction start to put tank, are transferred to by fermented liquid in the centrifugal 5-10min of 4 DEG C/1500-3000g in refrigerated centrifuge after cultivating 36-72h, collect centrifuged supernatant;

(4) lipase extraction and isolation

A, ammonium sulfate precipitation

Ammonium sulfate powder is joined in centrifuged supernatant while stirring, ammonium sulfate final concentration is made to be 2%, be put in 4 DEG C of refrigerators and leave standstill 0.5-1 h, then in the centrifugal 5-10min of 4 DEG C/3000-5000g, collect centrifuged supernatant, again ammonium sulfate powder is joined in centrifuged supernatant in the same way, ammonium sulfate final concentration is made to be 60%, be put in 4 DEG C of refrigerators and leave standstill 0.5-1 h, then in the centrifugal 5-10min of 4 DEG C/3000-5000g, collect centrifugation, centrifugation neutral phosphate buffer liquid is dissolved, obtains crude enzyme liquid;

B, gel permeation chromatography

Chromatography column is filled with the gel particle of isolated molecule amount near 16-200KD scope, with neutral phosphate buffer liquid balance chromatography column, the crude enzyme liquid obtained is loaded on chromatography column, wash-out is carried out with neutral phosphate buffer liquid, collect the elutriant corresponding to Peak Activity, the elutriant of gained is carried out ultrafiltration and concentration, obtains gel-filtration crude enzyme liquid;

C, ion exchange chromatography

Ion exchange column is filled with anionite-exchange resin, with neutral phosphate buffer liquid counterion exchange column, gel-filtration crude enzyme liquid is loaded on ion exchange column, with deionized water rinsing ion exchange column, wash-out is carried out again with neutral phosphate buffer liquid, collect the elutriant corresponding to Peak Activity, the elutriant of gained is carried out ultrafiltration and concentration, obtains ion-exchange crude enzyme liquid;

D, affinity chromatography

Affinity column is filled with Sepharose4B, lipase inhibitor is coupled on Sepharose4B as part by covalent linkage, with neutral phosphate buffer liquid balance chromatography column, ion-exchange crude enzyme liquid is loaded on chromatography column, with deionized water rinsing chromatography column, then carries out wash-out with 0.1M pH3-5 citrate buffer solution, collect elutriant, the elutriant of gained is neutralized to neutrality with weakly alkaline phosphate buffered saline buffer immediately, carries out ultrafiltration and concentration and desalination, obtain pure enzyme liquid;

E, polyethylene glycol precipitation

In the pure enzyme liquid obtained, add polyoxyethylene glycol, lipase is precipitated, then in the centrifugal 5-10min of 4 DEG C/3000-5000g, collect centrifugation, centrifugation is put into freeze drier inner drying, obtain lipase lyophilized powder.

Beneficial effect of the present invention:

1, instant invention overcomes the shortcomings such as traditional zymotic technology yield of enzyme is few, inulinase-producing activity is low, the enzyme life-span is short, the high-efficiency fermenting achieving lipase is produced and extraction and isolation, the present invention is simple to operate, with low cost, be easy to amplify, and production of enzyme is high, activity is high, purity is high, good stability, life-span are long.

2, the present invention has important using value, may be used for grease cheese processing, the field such as bread baking, medicine catalyze and synthesize, feed manufacturing, biodiesel synthesis, sewage disposal, weaving degreasing.

Embodiment

Below in conjunction with the embodiment of the present invention, be clearly and completely described the technical scheme in the present invention, every simple distortion that other are made when not departing from core of the present invention or amendment all fall into protection scope of the present invention.

Embodiment:

(1) fermented bacterium

Using genetic engineering bacterium Pichia pastoris GS115/pPIC9K-proRCL as fermentative production bacterial classification;

(2) nutrient solution

A, shake flask culture

Yeast extract 10 g/L, peptone 20 g/L, K ₂hPO ₄3g/L, H ₂pO ₄11.8g/L, add water 890mL/L, 121 DEG C of sterilizing 20 min, then treats that temperature is down to less than 60 DEG C and on super clean bench, is added 10 × YNB 100mL/L (13.4 g/L), 500 × vitamin H 1mL/L (4 × 10 ^-4g/L), glycerine 10mL/L;

B, fermentation culture

85% H ₃pO ₄26.7mL/L, CaSO ₄2H ₂o 0.93g/L, K ₂sO4 18.2g/L, MgSO ₄2H ₂o 14.9g/L, KOH 4.13 g/L, glycerine 40g/L, PTM ₁4.0mL/L(is glycerine, PTM wherein ₁add again after the real end that disappears);

c、PTM ₁

CuSO ₄5H ₂o 6.0g/L, KI 0.088g/L, MnSO ₄h ₂o 3.0g/L, Na ₂moO ₄2H ₂o 0.2g/L, H ₃bO ₃0.02g/L, CoC1 ₂6H ₂o 0.5g/L, ZnCl ₂20.0g/L, FeSO ₄7H ₂o 65.0g/L, vitamin H 0.2 g/L, dense H ₂sO ₄5 mL/L;

D, feed-batch culture liquid

(W/V, containing 12mL/L PTM for 50% glycerine ₁) feed supplement liquid;

E, BMMY nutrient solution

Yeast extract 10 g/L, peptone 20 g/L, K ₂hPO ₄3g/L, KH ₂pO ₄11.8g/L, add water 895mL/L, 121 DEG C of sterilizing 20 min, then treats that temperature is down to less than 60 DEG C and on super clean bench, is added 100 × YNB 100mL/L (13.4 g/L), 500 × vitamin H 1mL/L (4 × 10 ^-4g/L), methyl alcohol 5m/L;

F, methanol induction nutrient solution

100% methyl alcohol is (containing 12mL/L PTM ₁);

(3) strain fermentation is produced

A, select a single bacterium colony, be placed in the 250ml shaking flask that 25ml shake flask culture is housed, cultivate 18 h in 28 DEG C/280rpm;

B, preparation fermentation culture (glycerine, PTM ₁wouldn't add) and add in fermentor tank, demarcate pH electrode and DO electrode and insert tank body, sealing and fermenting tank, connect steam, to air system and the tank body enforcement sterilizing of fermentor tank, treat that tank pressure rises to 0.12MPa, during temperature 121 DEG C, pressurize, insulated sterilizing 20min, treat the real end that disappears, and connects freeze cycle water and temperature is cooled to 30 DEG C;

C, inoculation mouth around put a circle cotton ball soaked in alcohol, light, aseptically open inoculation mouth, shake flask culture is inoculated in fermentor tank rapidly by 10% inoculum size, and add the glycerine 40g/L of filtration sterilization, PTM ₁4.0mL/L, set the parameters such as temperature, mixing speed, air flow, ferment, ferment about 20 h, glycerine in fermentation culture is consumed totally, now DO rises to 100% rapidly, start flow feeding nutrient solution, it is 12 h that the stream of feed-batch culture liquid adds the time, during the fermentation, keeps temperature 28 DEG C in tank, dissolved oxygen DO>20% in tank, pH value 5 in tank, by the height regulating air flow and mixing speed to control dissolved oxygen, the aseptic ammoniacal liquor of auto-feeding mass concentration 25% controls the pH value size of fermentation culture;

D, treat that feed-batch culture liquid stream adds end, stop feed supplement, maintain the hungry 0.5h of cell, then stream adds BMMY nutrient solution 0.5h and allows yeast adapt to methyl alcohol environment, start stream when DO rises to 100% again and add methanol induction nutrient solution, in the hierarchy of control, the mass concentration of methyl alcohol is 1.0% in 0-1h, is 4.0% in 2-4h, be 2.5% in 4-8h, be 0.6% after 8h, between inductive phase, temperature controls at 25 DEG C, pH value controls 6, dissolved oxygen DO>20%;

E, methanol induction start to put tank, are transferred to by fermented liquid in the centrifugal 5min of 4 DEG C/3000g in refrigerated centrifuge after cultivating 60h, collect centrifuged supernatant;

(4) lipase extraction and isolation

A, ammonium sulfate precipitation

Ammonium sulfate powder is joined in centrifuged supernatant while stirring, make ammonium sulfate final concentration be 2%, be put in 4 DEG C of refrigerators and leave standstill 1 h, then in the centrifugal 5min of 4 DEG C/5000g, collect centrifuged supernatant, again ammonium sulfate powder is joined in centrifuged supernatant in the same way, make ammonium sulfate final concentration be 60%, be put in 4 DEG C of refrigerators and leave standstill 1 h, then in the centrifugal 5min of 4 DEG C/5000g, collect centrifugation, centrifugation neutral phosphate buffer liquid is dissolved, obtains crude enzyme liquid;

B, gel permeation chromatography

Chromatography column is filled with the gel particle of isolated molecule amount near 16-200KD scope, with neutral phosphate buffer liquid balance chromatography column, the crude enzyme liquid obtained is loaded on chromatography column, wash-out is carried out with neutral phosphate buffer liquid, collect the elutriant corresponding to Peak Activity, the elutriant of gained is carried out ultrafiltration and concentration, obtains gel-filtration crude enzyme liquid;

C, ion exchange chromatography

Ion exchange column is filled with anionite-exchange resin, with neutral phosphate buffer liquid counterion exchange column, gel-filtration crude enzyme liquid is loaded on ion exchange column, with deionized water rinsing ion exchange column, wash-out is carried out again with neutral phosphate buffer liquid, collect the elutriant corresponding to Peak Activity, the elutriant of gained is carried out ultrafiltration and concentration, obtains ion-exchange crude enzyme liquid;

D, affinity chromatography

Affinity column is filled with Sepharose4B, lipase inhibitor is coupled on Sepharose4B as part by covalent linkage, with neutral phosphate buffer liquid balance chromatography column, ion-exchange crude enzyme liquid is loaded on chromatography column, with deionized water rinsing chromatography column, then carries out wash-out with 0.1M pH4.5 citrate buffer solution, collect elutriant, the elutriant of gained is neutralized to neutrality with weakly alkaline phosphate buffered saline buffer immediately, carries out ultrafiltration and concentration and desalination, obtain pure enzyme liquid;

E, polyethylene glycol precipitation

In the pure enzyme liquid obtained, add polyoxyethylene glycol, lipase is precipitated, then in the centrifugal 5min of 4 DEG C/5000g, collect centrifugation, centrifugation is put into freeze drier inner drying, obtain lipase lyophilized powder.

Claims (1)

1. the fermentable of lipase and an extraction and separation method, it is characterized in that, its method is as follows:

(1) fermented bacterium

Using genetic engineering bacterium Pichia pastoris GS115/pPIC9K-proRCL as fermentative production bacterial classification;

(2) nutrient solution

A, shake flask culture

Yeast extract 10 g/L, peptone 20 g/L, K ₂hPO ₄3g/L, H ₂pO ₄11.8g/L, add water 890mL/L, 121 DEG C of sterilizing 20 min, then treats that temperature is down to less than 60 DEG C and on super clean bench, is added 10 × YNB 100mL/L (13.4 g/L), 500 × vitamin H 1mL/L (4 × 10 ^-4g/L), glycerine 10mL/L;

B, fermentation culture

85% H ₃pO ₄26.7mL/L, CaSO ₄2H ₂o 0.93g/L, K ₂sO4 18.2g/L, MgSO ₄2H ₂o 14.9g/L, KOH 4.13 g/L, glycerine 40g/L, PTM ₁4.0mL/L(is glycerine, PTM wherein ₁add again after the real end that disappears);

c、PTM ₁

CuSO ₄5H ₂o 6.0g/L, KI 0.088g/L, MnSO ₄h ₂o 3.0g/L, Na ₂moO ₄2H ₂o 0.2g/L, H ₃bO ₃0.02g/L, CoC1 ₂6H ₂o 0.5g/L, ZnCl ₂20.0g/L, FeSO ₄7H ₂o 65.0g/L, vitamin H 0.2 g/L, dense H ₂sO ₄5 mL/L;

D, feed-batch culture liquid

(W/V, containing 12mL/L PTM for 50% glycerine ₁) feed supplement liquid;

E, BMMY nutrient solution

Yeast extract 10 g/L, peptone 20 g/L, K ₂hPO ₄3g/L, KH ₂pO ₄11.8g/L, add water 895mL/L, 121 DEG C of sterilizing 20 min, then treats that temperature is down to less than 60 DEG C and on super clean bench, is added 100 × YNB 100mL/L (13.4 g/L), 500 × vitamin H 1mL/L (4 × 10 ^-4g/L), methyl alcohol 5m/L;

F, methanol induction nutrient solution

100% methyl alcohol is (containing 12mL/L PTM ₁);

(3) strain fermentation is produced

A, select a single bacterium colony, be placed in the 250ml shaking flask that 25ml shake flask culture is housed, cultivate 16-18 h in 28-30 DEG C/250-300 rpm;

B, preparation fermentation culture (glycerine, PTM ₁wouldn't add) and add in fermentor tank, demarcate pH electrode and DO electrode and insert tank body, sealing and fermenting tank, connect steam, to air system and the tank body enforcement sterilizing of fermentor tank, treat that tank pressure rises to 0.12MPa, during temperature 121 DEG C, pressurize, insulated sterilizing 20min, treat the real end that disappears, and connects freeze cycle water and temperature is cooled to 28-30 DEG C;

C, inoculation mouth around put a circle cotton ball soaked in alcohol, light, aseptically open inoculation mouth, shake flask culture is inoculated in fermentor tank rapidly by 5-10% inoculum size, and add the glycerine 40g/L of filtration sterilization, PTM ₁4.0mL/L, set the parameters such as temperature, mixing speed, air flow, ferment, ferment about 20 h, glycerine in fermentation culture is consumed totally, now DO rises to 100% rapidly, start flow feeding nutrient solution, it is 6-24 h that the stream of feed-batch culture liquid adds the time, during the fermentation, keeps temperature 28-30 DEG C in tank, dissolved oxygen DO>20% in tank, pH value 5-7 in tank, by the height regulating air flow and mixing speed to control dissolved oxygen, the aseptic ammoniacal liquor of auto-feeding mass concentration 25% controls the pH value size of fermentation culture;

D, treat that feed-batch culture liquid stream adds end, stop feed supplement, maintain the hungry 0.5-1 h of cell, then stream adds BMMY nutrient solution 0.5-1 h and allows yeast adapt to methyl alcohol environment, start stream when DO rises to 100% again and add methanol induction nutrient solution, in the hierarchy of control, the mass concentration of methyl alcohol is 1.0-2.0% in 0-1h, be 3.0-5.0% in 2-4h, be 2.0-3.0% in 4-8h, be 0.5-1.0% after 8h, between inductive phase, temperature controls at 20-28 DEG C, pH value controls at 5.5-6.5, dissolved oxygen DO>20%;

E, methanol induction start to put tank, are transferred to by fermented liquid in the centrifugal 5-10min of 4 DEG C/1500-3000g in refrigerated centrifuge after cultivating 36-72h, collect centrifuged supernatant;

(4) lipase extraction and isolation

A, ammonium sulfate precipitation

Ammonium sulfate powder is joined in centrifuged supernatant while stirring, ammonium sulfate final concentration is made to be 2%, be put in 4 DEG C of refrigerators and leave standstill 0.5-1 h, then in the centrifugal 5-10min of 4 DEG C/3000-5000g, collect centrifuged supernatant, again ammonium sulfate powder is joined in centrifuged supernatant in the same way, ammonium sulfate final concentration is made to be 60%, be put in 4 DEG C of refrigerators and leave standstill 0.5-1 h, then in the centrifugal 5-10min of 4 DEG C/3000-5000g, collect centrifugation, centrifugation neutral phosphate buffer liquid is dissolved, obtains crude enzyme liquid;

B, gel permeation chromatography

Chromatography column is filled with the gel particle of isolated molecule amount near 16-200KD scope, with neutral phosphate buffer liquid balance chromatography column, the crude enzyme liquid obtained is loaded on chromatography column, wash-out is carried out with neutral phosphate buffer liquid, collect the elutriant corresponding to Peak Activity, the elutriant of gained is carried out ultrafiltration and concentration, obtains gel-filtration crude enzyme liquid;

C, ion exchange chromatography

Ion exchange column is filled with anionite-exchange resin, with neutral phosphate buffer liquid counterion exchange column, gel-filtration crude enzyme liquid is loaded on ion exchange column, with deionized water rinsing ion exchange column, wash-out is carried out again with neutral phosphate buffer liquid, collect the elutriant corresponding to Peak Activity, the elutriant of gained is carried out ultrafiltration and concentration, obtains ion-exchange crude enzyme liquid;

D, affinity chromatography

Affinity column is filled with Sepharose4B, lipase inhibitor is coupled on Sepharose4B as part by covalent linkage, with neutral phosphate buffer liquid balance chromatography column, ion-exchange crude enzyme liquid is loaded on chromatography column, with deionized water rinsing chromatography column, then carries out wash-out with 0.1M pH3-5 citrate buffer solution, collect elutriant, the elutriant of gained is neutralized to neutrality with weakly alkaline phosphate buffered saline buffer immediately, carries out ultrafiltration and concentration and desalination, obtain pure enzyme liquid;

E, polyethylene glycol precipitation

In the pure enzyme liquid obtained, add polyoxyethylene glycol, lipase is precipitated, then in the centrifugal 5-10min of 4 DEG C/3000-5000g, collect centrifugation, centrifugation is put into freeze drier inner drying, obtain lipase lyophilized powder.