Unimolecule embedding method for enzyme

A single-molecule, molecular technology, applied in the field of single-molecule encapsulation of enzymes, can solve the problems of variable enzyme conformation, thick shell, poor stability, etc., and achieve the effect of low mass transfer resistance, mild conditions and strong stability

Inactive Publication Date: 2012-05-02
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the immobilized enzyme prepared by this method often has a thicker shell, and the enzyme is not easy to contact with the substrate; the encapsulation material is not cross-linked with the enzyme, resulting in variable conformation and poor stability of the enzyme, and the single-molecule embedding method can solve these problems

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] First, take horseradish peroxidase powder (or any of the above enzyme powders, the same below) and dissolve it in phosphate buffer solution to prepare a 2g / L enzyme solution for later use. N-acryloyloxysuccinyl Imine (NAS) was dissolved in dimethyl sulfoxide to prepare 100 g / L NAS liquid for use. Then, mix 10ml of horseradish peroxidase solution with 0.1ml of NAS solution, place in a constant temperature water bath at 30°C and shake for 3 hours to make the enzyme molecules attach double bonds, and then dialyze with buffer solution for 12 hours to remove unreacted small molecules . Add 3ml of polyethylene glycol 400 dimethacrylate and 50mg of acrylamide into the modified enzyme solution above, pass N 2 After 10 minutes, after the gas flow was stabilized, 0.3ml of ammonium persulfate with an initiator mass concentration of 5%, and 0.015ml of N,N,N',N'-tetramethyldiethylamine were added to initiate the reaction, and the reaction was carried out at 35°C for 4h. Then grind...

Embodiment 2

[0023] First, horseradish peroxidase powder was weighed and dissolved in phosphate buffer solution to prepare a 10 g / L enzyme solution for later use, and acryloyl chloride was dissolved in chloroform to prepare a 500 g / L acryloyl chloride solution for later use. Mix 10ml of horseradish peroxidase solution with 1ml of acryloyl chloride solution, shake and react at 20°C for 5 hours to attach double bonds to the enzyme molecules, and then dialyze with buffer solution for 24 hours to remove unreacted small molecules. Add 4ml tetraethylene glycol dimethacrylate into the modified enzyme solution, pass N 2 After 10 minutes, after the gas flow is stable, add 0.1ml of ammonium persulfate with an initiator mass concentration of 5%, and 0.05ml of N,N,N',N'-tetramethyldiethylamine to initiate the reaction, and react at 35°C for 3h. Then grind, wash, filter and freeze-dry to make enzyme single-molecule embedded particles. The measured enzyme activity yield was 85%, and the polymerization ...

Embodiment 3

[0025] First, weigh chymotrypsin powder and dissolve it in boric acid buffer solution to prepare a 20g / L enzyme solution for later use; dissolve N-acryloyloxysuccinimide in dimethyl sulfoxide to prepare a 300g / L enzyme solution NAS solution is reserved. Mix 10ml of chymotrypsin solution with 10ml of NAS solution, shake and react at 30°C for 3h to attach double bonds to the enzyme molecules, and then dialyze with buffer solution for 48h to remove unreacted small molecules. Add 6ml of polyethylene glycol 200 diacrylate and 200mg of trimethylolpropane trimethacrylate into the modified enzyme solution above, pass N 2 After 10 minutes, after the gas flow stabilized, 1 ml of potassium persulfate with an initiator mass concentration of 4% and 0.1 g of sodium bisulfite were added to initiate the reaction, and the reaction was carried out at 20°C for 8 hours. Then grind, wash, filter and freeze-dry to make enzyme single-molecule embedded particles. The activity yield of the enzyme wa...

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Abstract

The invention discloses a unimolecule embedding method for enzyme. The method comprises the following steps of: reacting an enzyme modifying agent of N-acryloxysuccinimide, acryloyl chloride or itaconic anhydride with amino groups on enzyme molecules so as to make each enzyme molecule connect a double bond; and reacting the double bond with a crosslinking agent to embed the individual enzyme molecule. By adopting the method, the center of each embedded particle only contains the individual enzyme molecule, and the thickness of the grid structure on the outer layer of the enzyme molecule is often prepared into the nanometer class, so that the enzyme in which the unimolecules are embedded has the characteristics of large specific surface area, low diffusion mass transfer resistance, high catalytic activity, high stability and the like. The method has the advantages of simple process, mild condition, high yield of enzyme activity, wide environmental application range and the like.

Description

technical field [0001] The invention relates to an enzyme single-molecule embedding method. Background technique [0002] Enzyme is a vital biocatalyst in organisms. Its high efficiency, specificity, mild reaction conditions, and environmental protection have attracted people's attention since the birth of human civilization. However, due to the protein nature of the enzyme, its biological structure is easily changed or destroyed by the influence of the environment, so the enzyme has poor stability and is easy to inactivate. The application range is often limited to aqueous solutions and cannot adapt to most industrial applications. Enzymes are often immobilized. [0003] Since the 1970s, enzyme immobilization has become an important research field in enzyme engineering and has been widely used in practice. Immobilization is to use a reliable method to stably link the enzyme to a soluble or insoluble carrier, thus solving the shortcomings of poor stability of the enzyme, n...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/04
Inventor 宋锡瑾徐佳音厉瑾王杰
Owner ZHEJIANG UNIV
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