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A kind of multi-enzyme system of polyethylenimine metal coordination immobilization and preparation method thereof

A polyethylenimine and multi-enzyme system technology, applied in multi-enzyme systems, immobilized on/in organic carriers, oxidoreductases, etc., can solve problems such as decreased enzyme activity, achieve synergistic effects, immobilization Quickly, the effect of improving catalytic efficiency

Active Publication Date: 2018-08-10
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the covalent bond formed by glutaraldehyde cross-linking, it is easy to cause a significant decrease in enzyme activity for oxidoreductases that are easily inactivated

Method used

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  • A kind of multi-enzyme system of polyethylenimine metal coordination immobilization and preparation method thereof
  • A kind of multi-enzyme system of polyethylenimine metal coordination immobilization and preparation method thereof
  • A kind of multi-enzyme system of polyethylenimine metal coordination immobilization and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0033] 1) Construction of engineering bacteria: construct the coenzyme oxidase gene with a histidine tag as described in SEQ ID NO.2 by PCR method, double digest the above gene and pET32a plasmid with BamHI and xholI respectively, and connect them with T4DNA ligase Transform Escherichia coli (DH5α), then extract the plasmid and transform Escherichia coli BL21 (DE3) to prepare the engineering bacteria E.coli BL21 (DE3) / pET32a that can express the coenzyme oxidase NOX; the original sequence of the coenzyme oxidase gene is derived from Lactobacillusbrevis;

[0034] 2) Preparation of crude enzyme solution: Inoculate engineering bacteria E.coli BL21(DE3) / pET32a into 200mL LB medium containing ampicillin with 1% inoculation amount; the composition of LB medium is 10.0g / L tryptone , 5.0g / L yeast powder, 10g / LNaCl, ampicillin was added before inoculation to make the final concentration 50-150μg / mL, the culture conditions were: initial pH 7.0, the volume fraction of the filling volume w...

Embodiment 2

[0041] 1) Construction of engineering bacteria: construct the glycerol dehydrogenase gene with histidine tag as described in SEQ ID NO.1 by PCR method, use BamHI and xholI to double-enzyme digest the above gene and pET32a plasmid respectively, and use T4DNA ligase Connect and transform Escherichia coli (DH5α), then extract the plasmid and transform Escherichia coli BL21 (DE3) to prepare engineering bacteria E.coli BL21 (DE3) / pET32a that can express glycerol dehydrogenase GDH; the original sequence source of the glycerol dehydrogenase gene in Klebsiellapneumonia;

[0042] 2) Preparation of crude enzyme solution: Inoculate engineering bacteria E.coli BL21(DE3) / pET32a into 200mL LB medium containing ampicillin with 1% inoculation amount; the composition of LB medium is 10.0g / L tryptone , 5.0g / L yeast powder, 10g / LNaCl, ampicillin was added before inoculation to make the final concentration 50-150μg / mL, the culture conditions were: initial pH 7.0, the volume fraction of the fillin...

Embodiment 3

[0048] 1) Construction of engineering bacteria: Utilize the coenzyme oxidase gene with histidine tag in embodiment 1, the glycerol dehydrogenase gene with histidine tag in embodiment 2, adopt overlapping PCR method to construct such as SEQ For the glycerol dehydrogenase-coenzyme oxidase fusion enzyme gene with a histidine tag described in ID NO.3, the above gene and pET32a plasmid were double-digested with BamHI and xholI respectively, and transformed into Escherichia coli (DH5α ), then extract the plasmid and transform it into Escherichia coli BL21(DE3) to prepare engineering bacteria E.coli BL21(DE3) / pET32a that can express glycerol dehydrogenase-coenzyme oxidase fusion enzyme;

[0049] 2) Preparation of crude enzyme solution: Inoculate engineering bacteria E.coli BL21(DE3) / pET32a into 200mL LB medium containing ampicillin with 1% inoculation amount; the composition of LB medium is 10.0g / L tryptone , 5.0g / L yeast powder, 10g / LNaCl, ampicillin was added before inoculation to ...

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Abstract

The invention discloses a multi-enzyme system with immobilized polyethylenimine and metal coordination and a method for preparing the multi-enzyme system, and belongs to the technical field of immobilized enzymes. The method includes immobilizing glycerol dehydrogenase, coenzyme oxidase and glycerol dehydrogenase-coenzyme oxidase fusion enzymes by the aid of coordination of polyethylenimine and metal ions; forming netted polyethylenimine frameworks by polyethylenimine molecules under coordination effects of imine and the metal ions in immobilizing procedures; forming coordination bonds by the metal ions coordinated on the polyethylenimine, C-end histidine tags of the glycerol dehydrogenase and the coenzyme oxidase and C-end histidine tags of the glycerol dehydrogenase-coenzyme oxidase fusion enzymes so as to acquire the immobilized multi-enzyme system. Coordination crosslinking effects can be realized by the metal ions in the immobilizing procedures, and the catalysis efficiency of the multi-enzyme system can be improved. The multi-enzyme system and the method have the advantages that the multi-enzyme system is high in multi-enzyme coupling efficiency, preparation conditions are mild, and processes are simple and feasible; the immobilized enzymes are high in immobilizing rate and activity recovery rate and good in reuse stability, the temperature stability can be obviously improved, and the like.

Description

technical field [0001] The invention relates to a multi-enzyme system with polyethylenimine metal coordination immobilization and a preparation method thereof. Background technique [0002] Dihydroxyacetone (Dihydroxyacetone) is widely used in the preparation of pharmaceutical and pesticide intermediates, and can be used to synthesize heterocyclic compounds, triglycerides, and ketone-substituted compounds. Dihydroxyacetone can be used as a component of sunscreen cosmetics. In the oxidation pathway of microbial metabolic conversion of glycerol to dihydroxyacetone, glycerol is dependent on NAD + Dihydroxyacetone is formed under the action of glycerol dehydrogenase (Glycerol dehydrogenase, GDH for short, EC 1.1.1.6). Glycerol dehydrogenase is also widely used in medical diagnostic analysis, for example, for enzymatic analysis of blood lipid content. [0003] In the process of oxidoreductase catalysis, the regeneration of coenzyme is very important. In the enzymatic productio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/18C12N11/08C12N9/04C12N9/02
Inventor 王世珍方柏山张永辉林鹏
Owner XIAMEN UNIV
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