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High-efficient extraction and purification method of rape lipase

A technology of lipase and rapeseed, applied in the direction of hydrolase, can solve the problems of slow research and development, difficult extraction, long growth cycle of raw materials, etc., and achieve the effect of high recovery rate and high purity

Inactive Publication Date: 2012-05-23
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to factors such as long growth cycle and difficult extraction of raw materials, the research and development of plant lipase is much slower than that of microbial lipase (Wu Qiuming, Ye Xingqian, Wu Dan, etc. 2004). At present, there is no patent for the research of lipase prepared from plant materials. see the report

Method used

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  • High-efficient extraction and purification method of rape lipase
  • High-efficient extraction and purification method of rape lipase
  • High-efficient extraction and purification method of rape lipase

Examples

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Embodiment

[0018] Example: Purification of Rapeseed Lipase

[0019] 1. The assay method of lipase specific activity adopts the industry standard of the People's Republic of China (Ministry of Light Industry of the People's Republic of China, 1993).

[0020] 1) Determination of lipase activity

[0021] 1mL reaction system contains 50mmol / L sodium phosphate buffer, 0.6% Triton X-100 (TristonX-100) solution, 100μmol / L p-nitrophenol laurate (ρ-NPL) substrate solution And enzyme solution (0mg / mL~2.5mg / mL). The substrate was dissolved in 10 mL of isopropanol, and 100 mL of 50 mmol / L, pH 7.0 Triston X-100 sodium phosphate buffer solution was slowly added to prepare a reaction solution. Add 100 μL of enzyme solution to every 900 μL of reaction solution, incubate at 25°C for 15 minutes, and centrifuge at 12,000 rpm for 30 seconds to stop the reaction. Under the experimental conditions, 1 μmoL p-nitrophenol was generated per minute as an activity unit, and the determination of p-nitrophenol was...

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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, theinvention 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 [0001] The invention relates to a method for extracting and purifying plant lipase, in particular to a method for extracting and purifying rapeseed lipase. Background technique [0002] Lipase belongs to the hydrolytic enzymes that decompose triacylglycerol. At the oil-water interface, lipase catalyzes the hydrolysis of the ester bond of triacylglycerol, releasing glyceride or glycerol and fatty acid with fewer ester bonds. Lipase is widely used in oil hydrolysis and refining, improvement of food flavor and fragrance, medicine, leather silk spinning degreasing, detergent and cosmetics and other industries. [0003] Lipases are widely found in plants and microorganisms (Rohit S Y, Uttam C B 2001). Due to factors such as long growth cycle and difficult extraction of raw materials, the research and development of plant lipase is much slower than that of microbial lipase (Wu Qiuming, Ye Xingqian, Wu Dan, etc. 2004). At present, there is no patent for the researc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/20
Inventor 谭小力魏明玉袁伟伟王政张志燕
Owner JIANGSU UNIV