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Method for regulating activity of glucose oxidase by utilizing polyisopropylacrylamide

A technology of glucose oxidase and propylacrylamide, applied in the field of regulating the activity of glucose oxidase by polyisopropylacrylamide

Inactive Publication Date: 2014-04-23
QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the separation and reassembly process of FAD and glucose oxidase is cumbersome, and most of the reagents in the fixation or modification process are toxic, causing great harm to the environment, it is difficult to be used in large-scale production and application.

Method used

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  • Method for regulating activity of glucose oxidase by utilizing polyisopropylacrylamide
  • Method for regulating activity of glucose oxidase by utilizing polyisopropylacrylamide
  • Method for regulating activity of glucose oxidase by utilizing polyisopropylacrylamide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1. Take 0.527 g of RAFT reagent and 0.238 g of 2-mercaptothiazoline and dissolve them in 10 ml of dichloromethane. Then add 0.497 g of the dehydrating agent dicyclohexylcarbodiimide (DCC) and 24.5 mg of the catalyst 4-dimethylaminopyridine (DMAP), and stir at room temperature for 7 hours. After the reaction, the solid particles were removed by suction filtration, and then the product was purified through a silica gel column. Dissolve 3.5 mg of RAFT reagent with mercaptothiazoline end groups in 0.1 ml of tetrahydrofuran. 20 mg of glucose oxidase was dissolved in 5 ml of phosphate buffer with pH=7.4, stirred slowly in an ice bath, and then slowly dropped into the RAFT reagent solution, and reacted in an ice bath for 5 hours. After the reaction, 3.5 mg azobisisobutyronitrile salt initiator (VA-044) and 0.45 g isopropylacrylamide (NIPAAm) were added. Among them, the molar ratio of RAFT reagent to NIPAAm is 1:400. Deoxidize with nitrogen for 40 minutes, then seal t...

Embodiment 2

[0016] Example 2. Change the molar ratio in 1 to RAFT reagent: NIPAAm=1:100, and the others are as in Example 1.

Embodiment 3

[0017] Example 3. Change the molar ratio in 1 to RAFT reagent: NIPAAm=1:200, and the others are as in Example 1.

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Abstract

The invention aims to provide a method for preparing novel glucose oxidase which is prepared by adopting an in-situ polymerization method, and has high thermal stability, wherein the activity of glucose oxidase can be regulated along with the temperature. The method comprises the following steps: modifying glucose oxidase through amino groups on the surface of glucose oxidase by utilizing an RAFT (Reversible Addition Fragmentation Chain) reagent with a mercaptothiazoline terminal group, and polymerizing isopropylacrylamide (NIPAAm) high polymer on the surface of enzyme through an RAFT in-situ polymerization method. According to the modified glucose oxidase, a macromolecular chain on the surface has high temperature sensitivity, polymer at the temperature higher than 32 DEG C is twisted and curled on the surface of the enzyme, and a contact area between the enzyme and the outside is increased, so that the activity of the enzyme is improved. Moreover, because the temperature is higher than the optimum temperature, the activity of standard enzyme is reduced. Therefore, the modified glucose oxidase has controllable activity and high thermal stability.

Description

technical field [0001] The method involves a new method for regulating the activity of glucose oxidase. We connect a RAFT reagent with a mercaptothiazoline end group to the surface of the glucose oxidase through the amino group on the surface of the glucose oxidase to make it modified. The modified glucose oxidase polymerizes a temperature-sensitive polymer ---polyisopropylacrylamide (PNIPAAm) on the surface of the enzyme through the "in situ" polymerization method. After the "in situ" polymerized glucose oxidase, when When the temperature is 40°C (the temperature-sensitive minimum critical solution temperature is 32°C), its enzyme activity becomes 1.6 times that of 30°C, while the standard enzyme activity is reduced, which belongs to the field of biological enzyme research. Background technique [0002] High-purity glucose oxidase is light yellow powder, easily soluble in water, completely insoluble in ether, chloroform, butanol, etc. The molecular weight is about 160KDa, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/96C12N9/04
CPCC12N9/0006C12Y101/03004
Inventor 不公告发明人
Owner QINGDAO UNIV