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
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[0032] Example 1
[0033] 1) Construction of engineering bacteria: PCR method is used to construct the histidine tag coenzyme oxidase gene as described in SEQ ID NO.2, the above gene and pET32a plasmid are double-cut with BamHI and xhol I, and the large intestine is ligated and transformed with T4 DNA ligase E.coli BL21(DE3) / pET32a which can express the coenzyme oxidase NOX by extracting the plasmid and transforming E.coli BL21(DE3); the original sequence of the coenzyme oxidase gene is from Lactobacillusbrevis;
[0034] 2) Preparation of crude enzyme solution: Inoculate engineering bacteria E.coli BL21(DE3) / pET32a into 200 mL of LB medium containing ampicillin at 1% inoculum; the composition of LB medium is 10.0g / L tryptone, 5.0g / L yeast powder, 10g / L NaCl, add ampicillin before inoculation to make the final concentration 50~150μg / mL, the culture conditions are: initial pH 7.0, filling volume fraction of 10%, culture temperature 37 ℃, shaker rotation speed 200rpm, after 6 hours o...
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[0040] Example 2
[0041] 1) Construction of engineering bacteria: PCR method is used to construct the glycerol dehydrogenase gene with histidine tag as described in SEQIDNO.1, double enzyme digestion of the above gene and pET32a plasmid with BamHI and xholI, and ligation and transformation with T4DNA ligase Escherichia coli (DH5α), then the plasmid was extracted and transformed into Escherichia coli BL21(DE3) to prepare an engineered strain E.coli BL21(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 engineering bacteria E.coli BL21(DE3) / pET32a into 200 mL of LB medium containing ampicillin at 1% inoculum; the composition of LB medium is 10.0g / L tryptone, 5.0g / L yeast powder, 10g / L NaCl, add ampicillin before inoculation to make the final concentration 50~150μg / mL, the culture conditions are: initial pH 7.0, filling volum...
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[0047] Example 3
[0048] 1) Construction of engineering bacteria: using the coenzyme oxidase gene with histidine tag in Example 1 and the glycerol dehydrogenase gene with histidine tag in Example 2, using overlapping PCR to construct as SEQIDNO .3 The glycerol dehydrogenase-coenzyme oxidase fusion enzyme gene with histidine tag, the above-mentioned gene and pET32a plasmid were double-cut with BamHI and xholI, respectively, and E. coli (DH5α) was ligated and transformed with T4 DNA ligase. Then the plasmid was extracted and transformed into E. coli BL21(DE3) to prepare the engineered strain E.coli BL21(DE3) / pET32a that can express the glycerol dehydrogenase-coenzyme oxidase fusion enzyme;
[0049] 2) Preparation of crude enzyme solution: Inoculate engineering bacteria E.coli BL21(DE3) / pET32a into 200 mL of LB medium containing ampicillin at 1% inoculum; the composition of LB medium is 10.0g / L tryptone, 5.0g / L yeast powder, 10g / L NaCl, add ampicillin before inoculation to make the ...
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