Polymer modified beta-glucosidase, preparation thereof and application of polymer modified beta-glucosidase in lignocellulose enzymolysis

A technology of glucosidase and polymer, applied in the direction of glycosylase, biochemical equipment and methods, enzymes, etc., can solve the problems of unfavorable cost, high energy consumption, inactivation of cellulase, etc., and achieve the change of surface properties , the effect of increasing affinity

Active Publication Date: 2021-07-23
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, high-temperature recovery of enzymes is easy to inactivate cellulase, and the energy consumption is high, which is not conducive to cost reduction

Method used

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  • Polymer modified beta-glucosidase, preparation thereof and application of polymer modified beta-glucosidase in lignocellulose enzymolysis
  • Polymer modified beta-glucosidase, preparation thereof and application of polymer modified beta-glucosidase in lignocellulose enzymolysis
  • Polymer modified beta-glucosidase, preparation thereof and application of polymer modified beta-glucosidase in lignocellulose enzymolysis

Examples

Experimental program
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Effect test

Embodiment 1

[0051] (1) The synthetic method of polymer modified β-glucosidase bgl-P (AM-b-AA-b-AN):

[0052] Dissolve 5g of acrylamide (AM) and 0.04g of azobisisobutyronitrile with 30ml of ethanol, transfer to a three-necked flask, deoxygenate with nitrogen, add 0.15g of chain transfer agent bis(carboxymethyl)trithiocarbonate, The reaction was stirred at 80°C for 8h, and 1.28g of acrylic acid (AA) and 1g of acrylonitrile (AN) were added dropwise to continue the reaction for 6h. Cool and centrifuge, take the precipitate and add methanol to heat up and resuspend, repeat three times, and vacuum dry to obtain a gel-like product. Add 45 mg of the above product into 20 ml of 10 mg / ml β-glucosidase phosphate buffer, adjust the pH to 8.0, add 0.45 g of EDC and 0.3 g of NHS in sequence, and react with magnetic stirring at 50 ° C for 2 h. The product was purified and concentrated by 100kDa membrane ultrafiltration, adjusted to pH 4.0, stored at 4°C, and tested for its enzyme activity and phase tra...

Embodiment 2

[0056] (1) The synthetic method of modified β-glucosidase bgl-P (AM-b-AA-b-DMAEMA):

[0057] Dissolve 4.95g of acrylamide and 0.022g of ammonium persulfate in 30ml of pure water, transfer to a three-necked flask, deoxygenate with nitrogen, add 0.08g of chain transfer agent mercaptopropionic acid, stir and react at 80°C for 8h, add dropwise 1.28g of acrylic acid (AA ) and 7.8g dimethylaminoethyl methacrylate (DMAEMA) continued to react for 5h. Cool and centrifuge, take the precipitate and add pure water to raise the temperature and resuspend, repeat three times, and vacuum dry to obtain a gelatinous product. Take 30 mg of the above product and add it into 10 ml of 10 mg / ml β-glucosidase phosphate buffer solution, adjust the pH to 8.0, followed by 0.45 g of EDC and 0.3 g of NHS, and react with magnetic stirring at 40 ° C for 2 h. The product was purified and concentrated by 100kDa membrane ultrafiltration, adjusted to pH 4.0, stored at 4°C, and tested for its enzyme activity an...

Embodiment 3

[0061] (1) The synthetic method of polymer modified β-glucosidase bgl-P (AM-b-AA-b-st):

[0062] Dissolve 4.95g of acrylamide (AM) and 0.02g of azobisisobutyronitrile in 10ml of dimethyl sulfoxide, transfer to a three-necked flask, deoxygenate with nitrogen, add 0.15g of chain transfer agent 2-(dodecyltri Thiocarbonate group)-2-methylpropionic acid, stirred at 70°C for 8h, added dropwise 1.28g of acrylic acid and 1.05g of styrene (st) to continue the reaction for 20h. Cool and centrifuge, take the precipitate and add methanol to heat up and resuspend, repeat three times, and vacuum dry to obtain a gel-like product. Add 45 mg of the above product into 10 ml of 10 mg / ml β-glucosidase phosphate buffer, adjust the pH to 8.0, add 0.45 g of EDC and 0.3 g of NHS in sequence, and react with magnetic stirring at 50 ° C for 2 h. The product was purified and concentrated by 100kDa membrane ultrafiltration, adjusted to pH 4.0, stored at 4°C, and tested for its enzyme activity and phase t...

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Abstract

The invention discloses polymer modified beta-glucosidase, preparation thereof and application of the polymer modified beta-glucosidase in lignocellulose enzymolysis. The preparation method comprises the following steps: grafting a block polymer containing acrylamide, acrylic acid and cationized and/or hydrophobized monomers on the surface of beta-glucosidase, compounding with a cellulase preparation, and adding into an enzymolysis system taking lignocellulose as a substrate, so that polymer modified enzyme is preferentially adsorbed on lignin, ineffective adsorption of endonuclease and exonuclease on lignin is reduced, enzymolysis strengthening of lignocellulose is achieved, and meanwhile electrostatic adsorption between polymer modified enzymes is adjusted through acrylic acid blocks. After the enzymolysis is finished, when the temperature of the system is reduced to 0-45 DEG C, the polymer modified beta-glucosidase with temperature response is precipitated and separated out, and the modified beta-glucosidase is recovered through solid-liquid separation.

Description

technical field [0001] The invention belongs to the technical field of lignocellulose enzymatic hydrolysis, and in particular relates to a polymer-modified β-glucosidase and its preparation and application in strengthening lignocellulose enzymatic hydrolysis and enzyme recovery. Background technique [0002] At present, the energy crisis and environmental problems are prominent. In order to solve this series of problems, the development of technologies related to the production of cellulosic ethanol by using renewable lignocellulosic resources has attracted much attention. In the production process of cellulosic ethanol, the enzymatic hydrolysis of lignocellulose has the advantages of safety, cleanness and strong reaction specificity, and is considered to be a method with great development potential for degrading cellulose. However, because lignin in lignocellulose is attached to the surface of cellulose, the accessibility of cellulose is low, which limits the improvement of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12P7/10C12P19/14
CPCC12N9/2445C12P7/10C12P19/14C12Y302/01021Y02E50/10
Inventor 楼宏铭覃飞扬李飞云焦芮庞煜霞邱学青
Owner SOUTH CHINA UNIV OF TECH
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