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Preparation method of immobilized beta-glucosidase

A technology of glucosidase and fructose, which is applied in directions such as being fixed on/in an organic carrier, can solve the problems of high production and use costs, inactivation of β-glucosidase, etc., and achieves a simple removal method without pollution, load The effect of high amount and reducing the cost of enzymatic hydrolysis

Active Publication Date: 2017-06-20
PETROCHINA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to prepare immobilized β-glucosidase by sol-gel embedding method in view of the high cost of production and use of β-glucosidase, and the problem that it is easily inactivated by lignin in the lignocellulose substrate. Glucosidase

Method used

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  • Preparation method of immobilized beta-glucosidase
  • Preparation method of immobilized beta-glucosidase
  • Preparation method of immobilized beta-glucosidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Add 32g of tetraethyl orthosilicate and 7.5mL of 0.034mol hydrochloric acid into a 500mL three-neck round bottom flask, under nitrogen atmosphere, magnetically stir (1500rpm) for 15min, then raise the temperature of the water bath, and reflux for 1h when it rises to 60°C , then cooled to room temperature and degassed with nitrogen.

[0048] Vacuum was applied under magnetic stirring until the weight loss was about 50%. Add 50% fructose aqueous solution, stir evenly, and then sonicate for 5 minutes to remove air bubbles. The obtained liquid was evenly divided into three 100mL small beakers, and 1.155mL of enzyme solution (a mixture of β-glucosidase and 50mM, pH5.0 acetate buffer solution) was added, stirred for 2min, sealed with plastic wrap, and Prick 30-40 holes on the membrane and put it in a refrigerator at 4°C for 48 hours to make it gel.

[0049] After 48 hours, put the gelled immobilized β-glucosidase sample into a vacuum drying oven to dry in vacuum until the wei...

Embodiment 2

[0052] Add 32g of tetraethyl orthosilicate and 7.5mL of 0.034mol hydrochloric acid into a 500mL three-neck round bottom flask, under nitrogen atmosphere, magnetically stir (1500rpm) for 15min, then raise the temperature of the water bath, and reflux for 2h when it rises to 55°C , then cooled to room temperature and degassed with nitrogen.

[0053] Vacuum was applied under magnetic stirring until the weight loss was about 50%. Add 50% fructose aqueous solution, stir evenly, and then sonicate for 5 minutes to remove air bubbles. The obtained liquid was evenly divided into three 100mL small beakers, and 2.31mL of enzyme solution (a mixture of β-glucosidase and 50mM, pH5.0 acetate buffer solution) was added, stirred for 2min, sealed with plastic wrap, and Prick 30-40 holes on the membrane and put it in a refrigerator at 4°C for 96 hours to make it gel.

[0054] After 96 hours, put the gelatinized immobilized β-glucosidase sample into a vacuum drying oven for vacuum drying until ...

Embodiment 3

[0056] Add 32g of tetraethyl orthosilicate and 7.5mL of 0.034mol hydrochloric acid into a 500mL three-neck round bottom flask, under nitrogen atmosphere, magnetically stir (1500rpm) for 15min, then increase the temperature of the water bath, and reflux for 1h when it rises to 65°C , then cooled to room temperature and degassed with nitrogen.

[0057] Vacuum was applied under magnetic stirring until the weight loss was about 50%. Add 60% fructose aqueous solution, stir evenly, and then sonicate for 5 minutes to remove air bubbles. The obtained liquid was evenly divided into three 100mL small beakers, and 1.155mL of enzyme solution (a mixture of β-glucosidase and 50mM, pH5.0 acetate buffer solution) was added, stirred for 2min, sealed with plastic wrap, and Prick 30-40 holes on the membrane and put it in a refrigerator at 4°C for 48 hours to make it gel.

[0058] After 48 hours, put the gelled immobilized β-glucosidase sample into a vacuum drying oven to dry in vacuum until th...

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Abstract

The invention discloses a preparation method of immobilized beta-glucosidase in the field of enzyme preparations. According to the method, fructose is adopted as a pore-foaming agent, and the immobilized beta-glucosidase is prepared through the tetraethoxysilane hydrolysis reaction sol-gel method. Different immobilized beta-glucosidase samples are prepared by adding fructose water solutions with different concentrations and beta-glucosidase of different dosages. For the obtained immobilized beta-glucosidase, the invalid adsorption in the enzymolysis process of cellulose can be avoided, so that the enzymolysis conversion efficiency of cellulose is obviously improved, the immobilized enzyme has the advantages of good mechanical strength and multiple times of recycling, and the hydrolysis and saccharifying costs of cellulose are reduced.

Description

technical field [0001] The invention belongs to the field of enzyme preparations, in particular to a preparation method of immobilized β-glucosidase. Background technique [0002] Cellulose is the most abundant high-molecular carbohydrate on earth, providing an abundant raw material for the renewable energy and materials fields. The biotransformation of cellulose is an important transformation technology of cellulose raw materials, and the separation and purification of cellulase is the key technical link. Cellulase is a multi-component compound enzyme system of glycoside hydrolase, which is divided into three types of components according to the catalytic function: endoglucanase; exoglucanase, namely cellobiohydrolase and β-glucose Glucosidase [12,19~21]. [0003] β-glucosidase mainly acts on β-(1,4) glycosidic bonds, and also acts on β-(1,1), (1,2), (1,3), (1,6) glycosidic bonds. It can hydrolyze the non-reducing β-D-glucosidic bond bound to the terminal, and simultaneo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/08C12N11/04
Inventor 齐泮仑李十中何皓杜然胡徐腾范桂芳孙洪磊李顶杰雪晶张佳张家仁侯丹孙立明李萌萌
Owner PETROCHINA CO LTD
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