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A method for immobilizing bifunctional acidic urease through graphene oxide and chitosan microspheres

An acid urease, graphene technology, applied in biochemical equipment and methods, immobilized on or in an inorganic carrier, immobilized on/in an organic carrier, etc., can solve the problem of weak pH stability, low enzyme amount, Problems such as poor temperature stability, to achieve the effect of improving mechanical strength, improving the utilization rate of enzymes, and facilitating column packing

Active Publication Date: 2018-03-23
JIANGNAN UNIV RUGAO FOOD BIOTECH RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The first object of the present invention is to provide a method for immobilizing bifunctional acid urease with graphene oxide and chitosan microspheres, which overcomes the less enzyme amount, poor temperature stability, and stable pH of the enzyme immobilized by the previous immobilization materials. Weakness and other shortcomings

Method used

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  • A method for immobilizing bifunctional acidic urease through graphene oxide and chitosan microspheres
  • A method for immobilizing bifunctional acidic urease through graphene oxide and chitosan microspheres
  • A method for immobilizing bifunctional acidic urease through graphene oxide and chitosan microspheres

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Embodiment 1

[0038] Step 1. Firstly activate the graphene oxide. Weigh 0.2g of GO and sonicate it in a buffer (20mL 50mM pH7.0) for 2h to make it uniformly dispersed, then add 5mL 0.5mM EDC and 5mL 0.5mM NHS in the above dispersion In the medium, shaking for 2 hours for activation, weighing 0.2 g of chitosan and using 20 ml of 2.0% acetic acid solution to activate, shaking for 2 hours for activation. According to the mass ratio of chitosan and graphene oxide of 1: (1 / 3~3), the solution of chitosan and graphene oxide carrier is mixed and reacted for 4-12 hours.

[0039] Step 2. Obtain the precipitates of graphene oxide and chitosan by centrifugation. Add 4 mL of 1% to 5% (v / v) glutaraldehyde for cross-linking per 0.03g of precipitate, cross-linking time 4 to 12 hours, deionized water Wash and remove uncrosslinked substances, and freeze-dry the precipitate at -65°C.

[0040] Step 3. Add 4mL of recombinant bifunctional acid urease with a specific enzyme activity of 2U / mg to 0.03g graphene oxide / c...

Embodiment 2

[0043] Step 1. Firstly activate the graphene oxide. Weigh 0.2g of GO and sonicate it in a buffer (20mL 50mM pH7.0) for 2h to make it uniformly dispersed, then add 5mL 0.5mM EDC and 5mL 0.5mM NHS in the above dispersion In the medium, shaking for 2 hours for activation, weighing 0.2 g of chitosan and using 20 ml of 2.0% acetic acid solution to activate, shaking for 2 hours for activation. According to the weight ratio of graphene oxide and chitosan 3 / 2, the solution of chitosan and graphene oxide carrier is mixed and reacted for 4-12 hours.

[0044] Step 2. Obtain the precipitates of graphene oxide and chitosan by centrifugation. Add 4 mL of 1% to 5% (v / v) glutaraldehyde for cross-linking per 0.03g of precipitate, cross-linking time 4 to 12 hours, deionized water Wash and remove uncrosslinked substances, and freeze-dry the precipitate at -65°C.

[0045] Step 3. Add 4mL of bifunctional acid urease with a specific enzyme activity of 2U / mg to 0.03g graphene oxide / chitosan cross-linked...

Embodiment 3

[0050] Step 1. Firstly activate the graphene oxide. Weigh 0.2g of GO and sonicate it in a buffer (20mL 50mM pH7.0) for 2h to make it uniformly dispersed, then add 5mL 0.5mM EDC and 5mL 0.5mM NHS in the above dispersion In the medium, shaking for 2 hours for activation, weighing 0.2 g of chitosan and using 20 ml of 2.0% acetic acid solution to activate, shaking for 2 hours for activation. According to the mass ratio of chitosan and graphene oxide of 3 / 2, the solution of chitosan and graphene oxide carrier was mixed and reacted for 8 hours.

[0051] Step 2. Obtain the precipitates of graphene oxide and chitosan by centrifugation, add 4mL 2.5% (v / v) glutaraldehyde for cross-linking per 0.03g of precipitates, cross-linking time 8h, deionized water cleaning and removal Uncrosslinked material, the precipitate is freeze-dried at -65°C.

[0052] Step 3. Add 4mL of bifunctional acid urease with a specific enzyme activity of 2U / mg to 0.03g graphene oxide / chitosan cross-linked carrier, put i...

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Abstract

A method for immobilizing bifunctional acidic urease through graphene oxide and chitosan microspheres is disclosed, and belongs to the technical field of enzyme preparations. The enzyme immobilizationamount, pH stability and temperature stability are obviously improved by utilizing a graphene oxide-chitosan crosslinked carrier. Complex application conditions of the bifunctional acidic urease areovercome, and the enzyme application rate is increased. The immobilized enzyme can be used for column packing conveniently so that a yellow wine treatment manner is facilitated. The immobilized enzymehas extremely low influences on volatile compounds in yellow wine, and has good urea and ethyl carbamate removing effects.

Description

Technical field [0001] The invention relates to a method for immobilizing bifunctional acid urease with graphene oxide and chitosan microspheres, and belongs to the technical field of enzyme preparations. Background technique [0002] Ethyl urethane (EC) has been identified as a carcinogen. It has multi-site carcinogenicity and genotoxicity and has been confirmed to be widely present in fermented foods and alcoholic beverages. In fermented wine, EC mainly produces ethanol and urea, so the removal of EC and urea in food and wine is very important. [0003] The inventor obtained the recombinant bifunctional acid urease "Expression ofan AcidUrease with Urethanase Activity in E.coli and Analysis of Urease Gene.[J].Molecular Biotechnology,2017,59(2-3):84- 97.", can degrade urea and EC, and maintain high activity in the acidic environment of alcoholic beverages, but the acquisition rate of this enzyme is less, and the application environment is more complicated. Therefore, it is necessa...

Claims

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Application Information

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IPC IPC(8): C12N11/14C12N11/10C12H1/056C12H1/044
CPCC12H1/0408C12H1/0424C12N9/80C12N11/10C12N11/14C12Y305/01005
Inventor 田亚平刘小锋杨柳周楠迪
Owner JIANGNAN UNIV RUGAO FOOD BIOTECH RES INST
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