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Lipase gel particle and preparation thereof

A gel particle, lipase technology, applied in the direction of enzyme stabilization, etc., can solve the problems of limited effect of stability improvement, complex genetic engineering methods, unable to solve the problem of cheap production, etc., achieve controllable particle size scale, improve activity and stability High performance, easy to industrially implement the effect of magnification

Active Publication Date: 2010-12-22
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The stability of natural lipase, including thermal stability, organic phase activity and organic phase stability, is the main limiting factor of lipase, which is difficult to meet the needs of practical applications
At 50°C, the half-life of most lipase activities is only about 30 minutes, and its stability is even worse in a mixed system composed of some strong polar organic solvents and aqueous solutions
At present, chemical additive methods, immobilization methods, genetic engineering and other methods can improve the stability of enzyme molecules. However, additive methods need to add a large amount of additives, which will bring new impurities and interference to the reaction system; traditional immobilization methods High mass transfer resistance will be introduced, resulting in a significant decrease in enzyme catalytic activity; while genetic engineering methods are more complex, costly, have limited effect on improving stability, and cannot solve the problem of mass and cheap production
Therefore, it is of great application value to develop a lipase nano-polymer biocatalytic gel particle with high stability, high biocatalytic activity, easy implementation, and controllable particle size, which can solve the problem of poor thermal stability of lipase, resistance to The problem of low organic solvent capacity

Method used

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  • Lipase gel particle and preparation thereof
  • Lipase gel particle and preparation thereof
  • Lipase gel particle and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1, preparation, activity detection and stability detection of lipase gel particles

[0025] 1. Preparation of lipase gel particles

[0026] 1) Add 10 g of lipase (purchased from Sigma, U.S., Candida rugosa lipase) and 2 g of succinimide acrylate (enzyme modifier, purchased from Sigma, U.S.) to 200 mL of 100 mM In acetic acid buffer solution (24.5 mg sodium acetate trihydrate and 49 mg acetic acid dissolved in 200 mL water), react at 20 °C for 6 hours, put the reaction solution into a dialysis bag with a molecular weight cut-off of 10,000, and dialyze in water for 24 hours. Change the water every 6 hours to remove succinimide acrylate;

[0027] 2) Add the reaction solution of the above step 1) to remove the succinimide acrylate ester into 200mL of 2% (mass percentage) dimethyl sulfoxide in an organic solvent aqueous solution, and then add 20g of acrylamide to the reaction solution (vinyl monomer), and an initiator composed of 4g ammonium persulfate and 6g N,...

Embodiment 2

[0036] Embodiment 2, preparation, activity detection and stability detection of lipase gel particles

[0037] 1. Preparation of lipase gel particles

[0038] 1) Add 10 g of lipase (purchased from Sigma, U.S., Aspergillus niger lipase) and 40 g of succinimide acrylate (enzyme modifier, purchased from Sigma, U.S.) to 200 mL of 100 mM acetic acid buffer with a pH of 9.4 (127.5mg of sodium acetate trihydrate and 3.6mg of acetic acid dissolved in 200mL of water) were reacted at 30°C for 4 hours, the reaction solution was put into a dialysis bag with a molecular weight cut-off of 10,000, and dialyzed in water for 24 hours. Change the water every hour to remove succinimide acrylate;

[0039] 2) Add the reaction solution of removing the succinimide acrylate ester in the above step 1) to 200mL of 5% (mass percentage) dimethyl sulfoxide in an organic solvent aqueous solution, and then add 200g of acrylamide ( Vinyl monomer), and an initiator composed of 4g ammonium persulfate and 6g N...

Embodiment 3

[0048] Example 3, preparation, activity detection and stability detection of lipase gel particles

[0049] 1. Preparation of lipase gel particles

[0050] 1) Add 10 g of lipase (purchased from Sigma, U.S., Aspergillus oryzae lipase) and 2 g of succinimide acrylate (enzyme modifier, purchased from Sigma, U.S.) to 200 mL of 100 mM acetic acid buffer at pH 4 (24.5mg of sodium acetate trihydrate and 49mg of acetic acid dissolved in 200mL of water), reacted at 30°C for 5 hours, put the reaction solution into a dialysis bag with a molecular weight cut-off of 10,000, and dialyzed in water for 24 hours, every 6 hours Change the water once to remove succinimide acrylate;

[0051] 2) The reaction solution of the above step 1) for removing succinimide acrylate ester is added to 200mL of 2% (mass percentage) dimethyl sulfoxide in an aqueous organic solvent solution, and then 20g of propylene is added to the reaction solution. Vinyl monomer composed of amide and 0.2g N,N'-methylene bisac...

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Abstract

The invention discloses a lipase gel granule and a preparation method thereof; the lipase gel granule provided by the invention consists of a nucleus and a nucleocapsid; wherein, the nucleus is lipase, the nucleocapsid is polymer material which is formed by the polymerization of vinyl monomers, and the nucleus and the nucleocapsid are connected by chemical bonds. The lipase gel granule of the invention has high activity, strong heat stability, strong organic solvent resistance, small granular size, high specific surface area and no mass transfer and diffusion resistance, etc.; as a nano-enzyme preparation with high performance, the lipase gel granule has wide application prospect in the field of nanoscience and biotechnology; the preparation method of the lipase gel granule of the invention is characterized in that the reaction condition is simple and moderate and the prepared gel granule can be controlled and is easy to be implemented industrially and expanded.

Description

technical field [0001] The invention relates to a lipase gel particle and a preparation method thereof. Background technique [0002] Lipase is currently the most important and widely used hydrolytic enzyme, which can hydrolyze substances containing ester bonds, and can also catalyze the synthesis of ester substances in the organic phase. Lipase has mild reaction conditions, high activity, easy source, and low cost. It has a wide range of applications in organic synthesis, food industry, detergent industry, energy industry, biotransformation, biomedicine, and biosensors. The stability of natural lipase, including thermal stability, organic phase activity and organic phase stability, is the main limiting factor of lipase, which is difficult to meet the needs of practical applications. At 50°C, the half-life of most lipase activities is only about 30 minutes, and its stability is even worse in a mixed system composed of some strong polar organic solvents and aqueous solutions...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/96
Inventor 戈钧刘铮卢滇楠
Owner TSINGHUA UNIV
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