CN101701211A - A method for efficiently extracting and purifying rapeseed lipase - Google Patents
A method for efficiently extracting and purifying rapeseed lipase Download PDFInfo
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- CN101701211A CN101701211A CN200910235070A CN200910235070A CN101701211A CN 101701211 A CN101701211 A CN 101701211A CN 200910235070 A CN200910235070 A CN 200910235070A CN 200910235070 A CN200910235070 A CN 200910235070A CN 101701211 A CN101701211 A CN 101701211A
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- 235000019421 lipase Nutrition 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title claims abstract description 19
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- 244000188595 Brassica sinapistrum Species 0.000 title 1
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- YNGNVZFHHJEZKD-UHFFFAOYSA-N (4-nitrophenyl) dodecanoate Chemical compound CCCCCCCCCCCC(=O)OC1=CC=C([N+]([O-])=O)C=C1 YNGNVZFHHJEZKD-UHFFFAOYSA-N 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
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Abstract
The invention relates to a high-efficient extraction and purification method of rape lipase, which comprises the following steps: (1) determining the optimal time for extracting the lipase from rape; (2) adding solid ammonium sulfate in the extracted crude lipase liquid under stirring, precipitating proteins, and using distilled water for dissolving, thereby obtaining salting-out solution, wherein the recovery rate of the lipase is 93.49% and the purification fold achieves 1.06; (3) then selecting an ultrafiltration membrane with the intercepted molecular weight of 100000Da for concentrating the salting-out solution of the lipase; and (4) adopting the ion exchange chromatography for purifying the lipase in ultra-filtrate, thereby obtaining rape lipase proteins, wherein the purity is 3.13u/mg and 9.14u/mg respectively, and the overall recovery rate is 43.94%. The purification process of the rape lipase provided by the invention is different from the separation and purification technology of microbial lipase, and liquid nitrogen is adopted for grinding and breaking plant cell walls, thereby dissolving the lipase in plant cytoplasms and the lipase in plant plasma membranes. Thus, the invention provides the method for extracting and purifying the lipase secreted in plant cells, which has the advantages of high purity, high recovery rate and enzyme purification fold of 4.83.
Description
Technical field
The present invention relates to a kind of method of extracting purifying vegetation fat enzyme, particularly extract the method for purifying rape lipase.
Background technology
Lipase belongs to the hydrolase that decomposes triglyceride, and on water-oil interface, the ester linkage hydrolyzing of lipase-catalyzed triacylglycerol discharges and contains still less glyceryl ester or the glycerine and the lipid acid of ester bond.Lipase all is widely used in the industries such as improvement, medicine, the degreasing of leather silk spinning, washing composition and makeup of fat hydrolysis and refining, flavour of food products and fragrance.
Lipase is present in (Rohit S Y, Uttam C B 2001) in plant and the microorganism widely.The vegetation fat enzyme is because the starting material growth cycle is long, extract than factors such as difficulties, many (Wu Dan waits 2004 for Wu Qiuming, Ye Xingqian) that its researchdevelopment is slower than microbial lipase, and present, the research patent for preparing lipase with vegetable material is not appeared in the newspapers.The patent of inventions such as conventional method for preparing lipase such as middle tail are just grand is given and is used microorganism in method-this method of describing among the open NO.WO2007/066779, also has Shu Zhengyu (biotechnology journal, 2007) He Hubo investigators such as (microorganism circular, 2007) prepares lipase from microorganism.But the safety coefficient of vegetation fat enzyme is higher than microbial lipase, and is with low cost, has more wide application value and prospect.
It is material that the present invention selects the rape of one of topmost oil crops of China for use.When Semen Brassicae campestris was sprouted, lipase can generate carbohydrate with the collaborative catalytic decomposition oil substances that plays a role of other enzyme, and seed root necessary nutriment and energy (Teissere M, Borel M, Caillol B, et al 1995) are provided.Therefore the rape seedling of germination period can be used as the vegetable material that lipase extracts.The present invention is with reference to the method for extracting purifying lipase from microorganism, as " saltouing → dialyse → anion-exchange chromatography " operational path (Shu Zhengyu that separating and purifying lipase adopted from aspergillus niger F044, Yang Jiangke, monarch's Yan Yun biotechnology journal, 2007), and from having a liking for " the hydrophobic chromatography anion-exchange chromatography of saltouing " operational path (Hu Bo that cold subtilis purifying low-temperature lipase is adopted, Wu Sheng, microorganisms such as willow circular, 2007), from extracting lipase and carry out purifying the rape seedling in high period, obtain the higher lipase of purity, thereby set up a kind of advantages of simplicity and high efficiency rape lipase purification process than vigor.
Summary of the invention
The present invention for overcome microbial lipase be applied to the food service industry safety coefficient low wait not enough, provide a kind of from rape the method for efficiently extracting and purifying lipase.Realize that technology of the present invention is as follows:
(1) determines the rape optimum extraction lipase time: cultivate the peaceful oil of cabbage type rape variety No. 12 at incubator, the Semen Brassicae campestris duration of germination only provides not additional carbon of water, take a sample for the first time when the seed imbibition is complete, got one time sample later on every 1 day, totally 10 times, the sample seedling with liquid nitrogen grinding to powder, then with the sodium phosphate buffer (0.025M that contains 1.2% triton x-100 (Triston X-100), pH 7) mix at 3: 10 with quality and volume ratio, get supernatant liquor behind the centrifugal 20min of 12000rpm, get crude enzyme liquid, measure crude enzyme liquid fat specific activity of enzyme respectively; The fatty specific activity of enzyme of finding 6 days rape seedling of cultivation is 1.89U/mg, and it is active in the highest;
(2) with the crude enzyme liquid that extracts while stirring to wherein adding solid ammonium sulfate, making ammonium sulfate saturation ratio wherein is 10%, leave standstill 30min after shaking up, centrifugal 10min under rotating speed 4000r/min then, abandoning supernatant, precipitation is used the ammonium sulfate precipitation of 40% saturation ratio again, leave standstill 30min after shaking up, centrifugal 10min under rotating speed 4000r/min then, abandoning supernatant, protein precipitation is obtained the liquid of saltouing with dissolved in distilled water, and the rate of recovery of lipase is 93.49% under this condition, and the purifying multiple reaches 1.06;
(3) selecting molecular weight cut-off then for use is that the ultra-filtration membrane of 100000Da concentrates the lipase liquid of saltouing, and the ratio vigor of lipase is 2.46u/mg, and the rate of recovery is 75.84%;
(4) adopt lipase in the M-Bondapak-C18 ion-exchange chromatography purification ultrafiltration liquid, behind the dress post, use the distilled water balance; With the treatment solution that previous step obtains, upper prop behind the centrifugal 10min of 12000r/min behind 3 times of the distilled water dilutings, successively with ammoniacal liquor with contain the 0.05mol/L sodium phosphate buffer wash-out of the pH8 of 1mol/L NaCl, flow velocity is 5mL/min, 2 elution peaks occur; Collect active peak, dialysis, lyophilize obtains rape lipase albumen, and purity is respectively 3.13u/mg and 9.14u/mg, and total yield is 43.94%.
Advantage of the present invention:
1. the present invention adopts and to saltout and method that ultrafiltration combines, can comparatively make things convenient for and obtain lipase efficiently, further passes through the M-Bondapak-C18 column chromatography, can reach and the close level of market pig steapsase vigor.
2. in the rape lipase purge process provided by the invention, be different from the microbial lipase separation purifying technique, adopt the broken plant cell wall of liquid nitrogen grinding, lipase in lipase and the plant cell membrane in the dissolving plant cytoplasm, a kind of method of extracting purifying vegetable cell internal secretion lipase is provided, the purity height, rate of recovery height, enzyme purification multiple are 4.83.
Description of drawings
The variation of the fatty specific activity of enzyme of the Brassica campestris L seedling of the different sprouting of Fig. 1 fate
Fig. 2 ammonium sulfate saturation ratio is to the influence of the lipase rate of recovery
Fig. 3 ammonium sulfate saturation ratio is to the influence of lipase purifying multiple
Fig. 4 M-Bondapak-C18 ion exchange chromatography elution curve
Embodiment
Test reagent: triton x-100 (Triston X-100) is available from Amresco company.The p-nitrophenyl laurate (ρ-nitrophenol laurate, ρ-NPL) available from Sigma-Aldrich company.Bovine serum albumin (BSA) is available from Beijing Baeyer enlightening company.Virahol, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC are analytical pure, and ammoniacal liquor is chemical pure.The ultrafiltration pipe is available from MILLIPORE company.Chromatography column 2.0cm*50cm is available from the special Analytical Instrument Co., Ltd of Shanghai fine jade.
Embodiment: the purifying of rape lipase
1. the measuring method of fatty specific activity of enzyme adopts People's Republic of China's industry standard (Ministry of Light Industry of the People's Republic of China (PRC), 1993).
1) mensuration of lipase activity
The sodium phosphate buffer that contains 50mmol/L in the reaction system of 1mL, 0.6% triton x-100 (Triston X-100) solution, the p-NP laurate of 100 μ mol/L (substrate solution of ρ-NPL) and enzyme solution (0mg/mL～2.5mg/mL).Substrate is dissolved in the 10mL Virahol, slowly adds 100mL 50mmol/L, and the Triston X-100 sodium phosphate buffer of pH7.0 is made into reaction solution.Per 900 μ L reaction solutions add 100 μ L enzyme liquid, behind 25 ℃ of insulation 15min, and the centrifugal 30s stopped reaction of 12000rpm.Under experiment condition, it is a unit of activity that per minute generates 1 μ moL p-NP, and the mensuration of p-NP adopts spectrophotometry.
2) mensuration of total protein
The coomassie brilliant blue staining method is measured, and is standard protein with BSA.
3) than the mensuration of vigor
Than vigor is the unit of activity number of lipase in the 1mg protein.
4) mensuration of the rate of recovery
The rate of recovery of lipase is defined as the ratio of the vigor of lipase in the vigor of lipase in the treatment solution and the extracting solution.The rate of recovery H calculation formula of lipase is as follows:
H(％)＝C1V1/C2C2
The vigor of lipase in the C1-extracting solution, U;
The cumulative volume of V1-extracting solution, mL;
The vigor of lipase in the treatment solution that C2-lipase purifying obtains, U;
The cumulative volume of the treatment solution that V2-lipase purifying obtains, mL;
5) mensuration of purifying multiple
The ratio of the ratio vigor of lipase in the ratio vigor that the purifying multiple of lipase is defined as lipase in the treatment solution and the extracting solution.
2. the preparation of crude enzyme liquid
No. 12 rape strains of the peaceful oil of experimental raw are provided by the Jiangsu Province Agriculture Science Institute.Cultivate No. 12 Semen Brassicae campestriss of peaceful oil in incubator, cultivate sampling in 6 days, sample retention is in-70 ℃ of refrigerators.
Get the sample that-70 ℃ of refrigerators are preserved, in liquid nitrogen, grind, the powder that obtains is with the ratio and the sodium phosphate buffer (0.025M that contains 1.2% triton x-100 (Triston X-100) of 3: 10 (W/V), pH 7) mix, the centrifugal 20min of concussion back 12000rpm, supernatant liquor is crude enzyme liquid, and its fatty specific activity of enzyme is 1.89U/mg
3. saltout
Make ammonium sulfate saturation ratio wherein reach 10%～80% respectively, leave standstill 30min after shaking up, centrifugal 10min under rotating speed 4000r/min then, abandoning supernatant obtains the liquid of saltouing with protein precipitation with dissolved in distilled water.The test-results of saltouing is shown in Fig. 2,3, and the ammonium sulfate saturation ratio is 10% o'clock, a spot of albumen precipitation only occurs.When saturation ratio is 20%, apparent in view protein precipitation is arranged, at this moment the purifying multiple maximum of lipase.When saturation ratio continuation increase, the purifying multiple reduces a lot.When saturation ratio was 40%, the rate of recovery of lipase had reached the highest.When the saturation ratio of solution surpassed 60%, because solution density is bigger, the lipase throw out was suspended in the surface of supernatant liquor, is not easy separated.Take all factors into consideration, earlier with the ammonium sulfate precipitation of 10% saturation ratio, except that foreigh protein removing, adopting saturation ratio then is that 40% ammonium sulfate is saltoutd.Adopt this method that lipase is carried out preliminary purification, measure fatty specific activity of enzyme according to the method for embodiment 1, find that the rate of recovery of lipase is 93.49%, the purifying multiple reaches 1.06.
4. ultrafiltration
The ultrafiltration pipe that adopts is a MILLIPORE company, and molecular weight cut-off is 10000Da.
Get the treatment solution that previous step obtains and put into the ultrafiltration pipe, in 4 ℃ of centrifugal 8min of following 4000g, full distilled water is mended in centrifugal back in the ultrafiltration pipe centrifugal again, repeats 3 times.Be the ultrafiltration terminal point when detect seeing through in the liquid no white precipitate with BaCl2 solution.Then the ultrafiltration pipe is upside down in the whizzer,, in pipe, adds the distilled water vibration dissolving ultrafiltration product of 1mL again in 4 ℃ of centrifugal 20min of following 4000g.
Measure fatty specific activity of enzyme according to the method for embodiment 1, the discovery molecular weight cut-off is that the rate of recovery of the ultra-filtration membrane purifying lipase of 10000Da is 94.62%.
5.M-Bondapak-C18 ion exchange chromatography
M-Bondapak-C18 handles by the product description requirement.Behind the dress post (2.0cm*50cm post), use the distilled water balance.With the treatment solution that previous step obtains, upper prop behind the centrifugal 10min of 12000r/min behind 3 times of the distilled water dilutings, successively with ammoniacal liquor with contain the pH80.05mol/L sodium phosphate buffer wash-out of 1mol/L NaCl, flow velocity is 5mL/min, and the peak is collected, and elution volume is 400mL; Collect active peak, dialysis, lyophilize is standby.
As shown in Figure 4, separate to obtain two active peaks, be respectively ammoniacal liquor and contain the elution peak that the pH8 0.05mol/L sodium phosphate buffer wash-out of 1mol/L NaCl obtains.Collect active peak, measure total protein, the gross activity at each peak, calculate then than vigor, purifying multiple, the rate of recovery.The result of the lipase separation and purification that obtains at last is as shown in table 1.The purification effect of finding chromatographic peak 2 is higher than chromatographic peak 1 far away.Measure fatty specific activity of enzyme according to the method for embodiment 1, find that the last rate of recovery of chromatographic peak 2 has reached 34.13%, the purifying multiple is about 5 times.
Claims (1)
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Cited By (6)
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| CN103013951A (en) * | 2012-12-27 | 2013-04-03 | 江南大学 | Method for extracting and purifying wheat germ lipase |
| CN105219695A (en) * | 2015-10-11 | 2016-01-06 | 华中农业大学 | A kind of method of rape root cytoplasmic membrane Purification and Characterization and application thereof |
| CN109652386A (en) * | 2019-01-03 | 2019-04-19 | 重庆工商大学 | A method of enzyme is recycled in catalysis reaction |
| CN109735517A (en) * | 2019-03-12 | 2019-05-10 | 内蒙古博格农牧业开发有限责任公司 | Using the method for immobilization horse antibody extraction purification lipase |
| CN110523491A (en) * | 2019-09-30 | 2019-12-03 | 中南大学湘雅医院 | a fat cutter |
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| CN1854292A (en) * | 2005-04-18 | 2006-11-01 | 广州市开恒生物科技有限公司 | Method for separating and purifying lipase efficiently |
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2009
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| CN103013951A (en) * | 2012-12-27 | 2013-04-03 | 江南大学 | Method for extracting and purifying wheat germ lipase |
| CN103013951B (en) * | 2012-12-27 | 2014-03-26 | 江南大学 | Method for extracting and purifying wheat germ lipase |
| CN105219695A (en) * | 2015-10-11 | 2016-01-06 | 华中农业大学 | A kind of method of rape root cytoplasmic membrane Purification and Characterization and application thereof |
| CN109652386A (en) * | 2019-01-03 | 2019-04-19 | 重庆工商大学 | A method of enzyme is recycled in catalysis reaction |
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| CN109735517A (en) * | 2019-03-12 | 2019-05-10 | 内蒙古博格农牧业开发有限责任公司 | Using the method for immobilization horse antibody extraction purification lipase |
| CN110523491A (en) * | 2019-09-30 | 2019-12-03 | 中南大学湘雅医院 | a fat cutter |
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