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Multi-enzyme system with immobilized polyethylenimine and metal coordination and method for preparing multi-enzyme system

A technology of polyethylenimine and multi-enzyme system, applied in multi-enzyme system, fixed on/in organic carrier, oxidoreductase, etc., can solve the problem of enzyme activity decline, achieve synergistic effect and operation method Simple, fast-fixing effect

Active Publication Date: 2015-11-18
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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  • Multi-enzyme system with immobilized polyethylenimine and metal coordination and method for preparing multi-enzyme system
  • Multi-enzyme system with immobilized polyethylenimine and metal coordination and method for preparing multi-enzyme system
  • Multi-enzyme system with immobilized polyethylenimine and metal coordination and method for preparing multi-enzyme system

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Experimental program
Comparison scheme
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, use BamHI and xholI to double-enzyme digest the above gene and pET32a plasmid respectively, and use T4DNA ligase to ligate and transform the large intestine bacillus (DH5α), then extract the plasmid and transform Escherichia coli BL21 (DE3) to prepare the engineering bacteria E.coliBL21 (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 the engineered bacteria E.coliBL21(DE3) / pET32a into 200 mL of LB medium containing ampicillin at an inoculum size of 1%; the composition of the LB medium is 10.0 g / L tryptone, 5.0g / L yeast powder, 10g / LNaCl, add ampicillin before inoculation to make the final concentration 50-150μg / mL, the culture conditions are: initial pH 7.0, the volume fraction o...

Embodiment 2

[0041] 1) Construction of engineering bacteria: construct the glycerol dehydrogenase gene with a histidine tag as described in SEQ ID NO.1 by PCR method, use BamHI and xholI to double-enzyme cut the above gene and pET32a plasmid respectively, and use T4DNA ligase to ligate and transform Escherichia coli (DH5α), then extract the plasmid transformation Escherichia coli BL21 (DE3) to prepare the engineering bacteria E.coliBL21 (DE3) / pET32a that can express glycerol dehydrogenase GDH; the original sequence of the glycerol dehydrogenase gene is derived from Klebsiellapneumonia;

[0042] 2) Preparation of crude enzyme solution: Inoculate the engineered bacteria E.coliBL21(DE3) / pET32a into 200 mL of LB medium containing ampicillin at an inoculum size of 1%; the composition of the LB medium is 10.0 g / L tryptone, 5.0g / L yeast powder, 10g / LNaCl, add ampicillin before inoculation to make the final concentration 50-150μg / mL, the culture conditions are: initial pH 7.0, the volume fraction o...

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 SEQIDNO .3 said glycerol dehydrogenase-coenzyme oxidase fusion enzyme gene with histidine tag, use BamHI and xholI to double-digest the above-mentioned gene and pET32a plasmid respectively, connect and transform Escherichia coli (DH5α) with T4DNA ligase, Then extract the plasmid and transform Escherichia coli BL21 (DE3) to prepare engineering bacteria E.coliBL21 (DE3) / pET32a that can express glycerol dehydrogenase-coenzyme oxidase fusion enzyme;

[0049] 2) Preparation of crude enzyme solution: Inoculate the engineered bacteria E.coliBL21(DE3) / pET32a into 200 mL of LB medium containing ampicillin at an inoculum size of 1%; the composition of the LB medium is 10.0 g / L tryptone, 5.0g / L yeast powder, 10g / LNaCl, add ampicillin 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 (Glyceroldehydrogenase, GDH for short, EC1.1.1.6). 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 production ...

Claims

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Application Information

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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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