Process method of oxidized graphene directional immobilization glucose oxidase

A technology of glucose oxidase and process method, which is applied in the field of directional immobilization of glucose oxidase on graphene oxide, to achieve the effects of directional immobilization, small loss of enzyme activity, and improvement of immobilization efficiency

Active Publication Date: 2013-01-16
HEBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is no relevant report on the directional immobilization of glucose oxidase using graphene oxide as a carrier in the literature.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0018] Graphene oxide was dissolved in distilled water and ultrasonically mixed for 60 minutes to obtain a uniformly dispersed graphene oxide suspension with a concentration of 1 mg / mL. Take 50 μL of 1 mg / mL graphene oxide suspension, add 100 μg N-hydroxysulfosuccinimide and 44.1 μg 1-ethyl-3-(3-dimethylaminopropyl) carbodiamine to it, and use 2 -(N-Morpholino)ethanesulfonic acid buffer (0.1M, pH 6.0) was fixed to make the final concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiamine 20mM, shake vigorously After shaking for 30min, centrifuge at 13000r / min for 20min, discard the supernatant, add 0.05M 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.0) to the precipitate for washing, centrifuge, discard the supernatant , repeating the steps of washing and centrifugation three times, and finally the precipitate was vacuum-dried at 40°C for 24 hours to obtain esterified graphene oxide;

[0019] Resuspend the above-obtained esterified graphene oxide in distilled water, son...

example 2

[0022] The esterification method of graphene oxide is with example 1;

[0023] Resuspend the esterified graphene oxide in distilled water, sonicate for 10 minutes to obtain 50 μL of a uniformly dispersed suspension of esterified graphene oxide with a concentration of 1 mg / mL, and add 700 μL of activated 0.1 mg / mL concanavalin A to it (pH 7.0) reacted for 60min, centrifuged at 13000r / min 4°C for 20min, washed 3 times with 0.05M 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.0) to obtain a precipitate; add 700μL pH React with 0.5 mg / mL glucose oxidase solution of 5.5 for 60 minutes, centrifuge at 13000 r / min at 4°C for 20 minutes, wash with PBS three times, and vacuum freeze-dry the precipitate for 24 hours to obtain directional immobilized glucose oxidase. The activity of the immobilized enzyme was measured by a colorimetric method, and the activity of the immobilized enzyme was 195 U / mg.

[0024] Enzyme assay method is the same as example 1.

example 3

[0026] The esterification method of graphene oxide is with example 1;

[0027] Resuspend the esterified graphene oxide in distilled water, sonicate for 10 min to obtain 50 μL of a uniformly dispersed suspension of esterified graphene oxide with a concentration of 1 mg / mL, and add 1000 μL of activated 0.1 mg / mL concanavalin to it A (pH7.0) was reacted for 60min, centrifuged at 13000r / min at 4°C for 20min, washed 3 times with 0.05M 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.0) to obtain a precipitate; add React 1000 μL of 0.5 mg / mL glucose oxidase solution with pH 7.0 for 120 minutes, centrifuge at 13000 r / min at 4°C for 20 minutes, wash with PBS three times, and vacuum freeze-dry the precipitate for 24 hours to obtain directional immobilized glucose oxidase. The activity of the immobilized enzyme was measured by a colorimetric method, and the activity of the immobilized enzyme was 180 U / mg.

[0028] Enzyme assay method is the same as example 1.

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Abstract

The invention relates to a process method of oxidized graphene directional immobilization glucose oxidase. The process method comprises the following steps that 1) oxidized graphene is dissolved in distilled water, and oxidized graphene turbid liquid with the concentration being 1mg/mL is obtained through ultrasonic mixing; N-hydroxy sulfo-succinimide and 1-ethide-3-(3-dimethyl aminopropyl) carbodiimide are added into the turbid liquid, the washing and the separation are carried out after shaking, and esterified oxidized graphene is obtained; and 2) activated concanavalin A is added into the uniform esterified oxidized graphene, the centrifugation is carried out after reaction, and sediments are fully washed; and then, glucose oxidase solution is added, the full washing is carried out after reaction, and the directional immobilization glucose oxidase is obtained after the vacuum freeze-drying for 24 hours. The method for preparing the immobilized enzyme has the advantages that the immobilization efficiency is high, and the defects that the orientation of enzyme activity sites is inconsistent, or the enzyme activity sites are covered, and the like are avoided, so the activity of the immobilized enzyme is improved, the enzyme activity loss is low, and the enzyme activity can reach 150 to 195U/mg.

Description

technical field [0001] The invention belongs to the field of immobilized enzymes, in particular to a process method for directional immobilized glucose oxidase on graphene oxide. Background technique [0002] Compared with traditional chemical catalysis, enzymatic catalysis has the advantages of mild conversion conditions, high reaction rate, and excellent selectivity. Under the new situation of vigorously advocating the greening of the process industry, energy saving and emission reduction, enzyme catalysis will play an increasingly important role in the process replacement of bulk chemicals and fine chemicals and environmental chemistry. Due to the advantages of good stability, reusability, and easy separation, immobilized enzymes have become a research hotspot and frontier in the field of enzyme catalysis. However, due to the constraints of the following aspects, the wider application of enzyme catalysis is limited: (1) out of the intracellular microenvironment, the enzy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14
Inventor 高静周丽亚姜艳军
Owner HEBEI UNIV OF TECH
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