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Plasmid pET28a(+)-P450BM3-gdh0310 capable of catalytic preparing indigo from indole, preparation process and use thereof

A technology of -p450bm3-gdh0310, P450BM3, applied in the field of indigo production by genetically engineered bacteria in biochemical industry, can solve the problems of limited practicality, achieve the effects of improved catalytic activity, increased production, and broad application prospects

Inactive Publication Date: 2008-10-29
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, P450 BM3 catalyzes indole to indigo and requires the participation of the reduced coenzyme NADPH, which limits the practicality of P450 BM3 for the synthesis of indigo

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0032]Using pQE30-gdh0310 as a template, the gdh0310 gene fragment was amplified by PCR, and the resulting gdh0310 gene fragment was detected by electrophoresis, and the glucose dehydrogenase gene fragment was recovered from the agarose gel, and connected to pMD18- On the T plasmid, pMD18-gdh0310 was obtained; the obtained pMD18-gdh0310 was transformed into E.coli JM109, spread on LB agar plates with a final concentration of 50 μg / ml ampicillin, and cultured overnight at 37°C; about 8 clones were selected Inoculate into 3ml of LB liquid medium containing 50μg / ml ampicillin, culture overnight at 37°C to amplify the plasmid; extract the amplified plasmid by alkaline lysis, and digest it with EcoRI and XhoI at 37°C, and digest the obtained fragment Directionally ligated to pET28a(+)-P450BM3 treated with the same double restriction enzymes to construct pET28a(+)-P450BM3-gdh0310.

[0033] Transform the recombinant plasmid pET28a(+)-P450 BM3-gdh0310 into E.coli DH5α, spread it on th...

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Abstract

The invention discloses a together-expressing P450BM3 and glucose dehydrogenase and plasmid pET28a (+)-P450BM3-gdh0310, making method and utility for catalyzing indole to synthesize indigo, which is characterized by the following: connecting P450BM3 gene and glucose dehydrogenase gene on the same expressing carrier pET28a(+); transferring the plasmid into E.coliBL21 to obtain bacterial strain to express cytochrome P450BM3 monooxygenase and glucose dehydrogenase simultaneously; catalyzing indole to synthesize indigo through synergic action; improving catalytic activity by 22-27 times; providing wide appliance prospect for biological transferring manufacturing indigo dye.

Description

technical field [0001] The invention relates to the technical field of indigo production by genetically engineered bacteria in biochemical industry, in particular to a plasmid pET28a(+)-P450BM3 capable of catalyzing indole to generate indigo and co-expressing cytochrome P450 BM3 monooxygenase and glucose dehydrogenase -gdh0310, preparation method and use thereof. Background technique [0002] Indigo, a blue pigment, was one of the first natural dyes discovered. According to statistics, as of 1998, the world's total annual output of dyes was 800,000 tons, of which indigo accounted for 80,000 tons. The dye indigo was originally extracted from plants until its chemical structure was elucidated in 1883, and its chemical synthesis preparation method was widely used. It quickly became popular due to its advantages of simple production, sufficient raw materials, high purity, and convenient use, and soon replaced it. plant indigo. However, chemically synthesized indigo has seriou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/53C12N15/64C12N1/21C12P17/10
Inventor 梅乐和陆燕
Owner ZHEJIANG UNIV