Enzyme directed immobilization method based on protein surface screening

A technology of immobilizing enzymes and immobilizing chambers, applied in immobilizing enzymes, biochemical equipment and methods, immobilizing on or in inorganic carriers, etc., can solve the problem of low sensitivity of enzyme test paper and enzyme biosensor, and uneven biofilm activity. , low remaining enzyme activity, etc., to achieve the effect of good enzyme activity maintenance, wide application range and large enzyme immobilization capacity

Inactive Publication Date: 2014-09-17
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the traditional enzyme immobilization methods only stay in the adsorption and immobilization of the random orientation of the enzyme protein, which often leads to the ever-changing orientation of the enzyme on the carrier, and those poor spatial orientations hinder the proximity orientation effect between the enzyme and the substrate. The remaining activity of the enzyme is low, the sensitivity of the enzyme test paper and the enzyme biosensor is low, and the activity of the biofilm is not uniform and the interchangeability is poor.
These problems cannot be effectively solved by relying on the existing immobilized enzyme technology, which has become the current technical bottleneck

Method used

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  • Enzyme directed immobilization method based on protein surface screening
  • Enzyme directed immobilization method based on protein surface screening

Examples

Experimental program
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Effect test

Embodiment 1

[0053] Embodiment 1 - human erythrocyte catalase

[0054] Human erythrocyte catalase (HUMAN ERYTHROCYTE CATALASE) was selected, and the three-dimensional crystal structure of human erythrocyte catalase molecular number 1DGB was used in the Protein Data Bank (Protein Data Bank, PDB) (the three-dimensional crystal structure was obtained from Protein Data Bank http: / / www .rcsb.org / pdb), analyzed the surface of human erythrocyte catalase by Sybyl software, and obtained that its catalytic center was located in the enzyme center, with the 147th asparagine (Asn147) and the 74th histidine (His74 ) midpoint as the center within range. Centered on the 176th leucine (Leu176) There are obvious depressions on the surface of human erythrocyte catalase protein in the range, which is far away from the catalytic center of human erythrocyte catalase, with a moderate volume, which can accommodate molecules with a molecular weight of 100-2000, and the interior of the depression is relatively ...

Embodiment 2

[0061] Example 2 - Catalase

[0062] Human erythrocyte catalase (HUMAN ERYTHROCYTE CATALASE) was selected, and the three-dimensional crystal structure of human erythrocyte catalase molecular number 1DGB was used in the Protein Data Bank (Protein Data Bank, PDB) (the three-dimensional crystal structure was obtained from Protein Data Bank http: / / www .rcsb.org / pdb), analyzed the surface of human erythrocyte catalase by Sybyl software, and obtained that its catalytic center was located in the enzyme center, with the 147th asparagine (Asn147) and the 74th histidine (His74 ) near the midpoint as the center within range. Centered around the 176th leucine (Leu176) There is a significant depression on the surface of human red blood cell catalase protein near the range, which is far away from the catalytic center of human red blood cell catalase, and has a moderate volume, which can accommodate molecules with a molecular weight of 100-2000, and the interior of the depression is re...

Embodiment 3

[0068] Example 3 - Acetylcholinesterase

[0069] Select acetylcholinesterase (Acetylcholine Esterase, AChE), using the three-dimensional crystal structure of human acetylcholinesterase molecule with PDB number 1F8U (the three-dimensional crystal structure comes from Protein Data Bank http: / / www.rcsb.org / pdb), through Sybyl software The surface analysis of acetylcholinesterase shows that its catalytic center is located in the enzyme center, with the 203rd serine (Ser203) as the center within range. Centered on the 400th aspartic acid (Asp400) There are obvious depressions on the surface of the acetylcholinesterase enzyme protein in the range, which is far away from the catalytic center of acetylcholinesterase, and has a moderate volume, which can accommodate molecules with a molecular weight of 100-2500, and the interior of the depression is relatively hydrophobic. Because the depression near the 400th aspartic acid (Asp400) has the above characteristics, it is selected as t...

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Abstract

The invention provides a novel enzyme directed immobilization method. The method comprises the following steps: (1) conducting enzyme surface analysis, and selecting a depression away from catalytic center on the enzyme as a fixed cavity; (2) screening docking molecules, and selecting small molecules with strong binding with the fixed cavity and weak binding with the catalytic center; and (3) preparing an oriented immobilized enzyme: connecting the small molecules to carrier surface and incubating with the enzyme molecule, so as to obtain the composite oriented immobilized enzyme. The immobilized enzyme catalyst obtained by the method has the advantages of high efficiency, good stability and superiority to the traditional immobilized enzyme.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the field of enzyme immobilization. Background technique [0002] Enzyme is a protein-based (very small amount of non-RNA, DNA-based) biocatalyst. There is a site in the enzyme molecule that can directly bind to the substrate molecule and catalyze the chemical reaction of the substrate, and this site becomes the catalytic center of the enzyme. Compared with chemical catalysts, enzymes have the advantages of mild reaction conditions, high catalytic efficiency, strong specificity, and low pollution. They are widely used in various fields such as industry, agriculture, medicine, and food. [0003] Enzyme immobilization technology (Enzyme immobilization) refers to a type of technology that binds or restricts enzymes to a certain area, but still retains its catalytic properties and can be recycled and reused. Compared with free enzymes, immobilized enzymes mainly have the following adva...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12N11/08C12N11/00
Inventor 孙英刁剑雄
Owner CHINA AGRI UNIV
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