Variant enzyme of cytochrome P450BM-3D168S and method for preparing indirubin using the same

A P450BM-3D168S, cytochrome technology, applied in oxidoreductase, fermentation and other directions, can solve the problems of cost and economic value without economic value, difficult to use indirubin for large-scale biological preparation, increase separation and purification costs, etc. Achieve the effect of reducing the burden of separation and purification, avoiding the destruction of natural resources and the environment, and improving the utilization rate of raw materials

Inactive Publication Date: 2008-12-31
ZHEJIANG UNIV
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Problems solved by technology

Therefore, the product composition of the P450 BM-3 enzymes that can catalyze the hydroxylation of indole are all indigo-based. If these enzymes are used for the bio-production of indirubin, not only the yield of indirubin is low, but also To separate a small amount of indirubin from a large amount of indigo will increase the cost of separation and purification, and will also cause greater waste
It can be said that using the P450 BM-3 enzyme disclosed in the existing patent to carry out the biological preparation of indirubin has no economic value in terms of cost and economic value, and it is difficult to use the P450 BM-3 enzyme disclosed in the existing patent to produce Performing Large-Scale Bioproduction of Indirubin

Method used

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preparation example Construction

[0033] The preparation method of the cytochrome P450 BM-3 D168S variant enzyme disclosed in the present invention specifically includes the following steps:

[0034] 1. Design random primers for the 168th codon of P450 BM-3 and perform PCR amplification

[0035] According to the principle of Stratagene's Quik-Change Kit, the following upstream and downstream primers for codon 168 are designed:

[0036] Upstream primer: 5’-CAGCTTTTACCGANNNCAGCCTCATCC-3’

[0037] Downstream primer: 5’-GGATGAGGCTGNNNTTGGTAAAAGCTG-3’

[0038] PCR reaction system composition: 5μL of 25mmol / L MgCl 2 Pfu PCR buffer, 10nmol of dNTPs, 15pmol of upstream and downstream primers, pET28α(+)P450BM-3 (F87V / A74G / L188Q / E435T) template DNA and PfuDNA polymerase, add sterile water to a total volume of 50μL.

[0039] PCR reaction parameters: after denaturation at 95°C for 1 min, 18 cycles at 95°C for 30 s, 53°C for 2 min, and 72°C for 16 min, and finally extension at 72°C for 2 min to obtain PCR amplification product...

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Abstract

The invention discloses a cytochrome P450 BM-3 D168Smutant enzyme as well as a method applying the enzyme to prepare indirubin. The enzyme adopts the directed evolution technique, and takes P450 BM-3 (F87V / A74G / L188Q / E435T) as a male parent, and is obtained by implementing the fixed-point saturation and mutation at the codon for the aspartic acid residue in position 168 of the sequence in the male parent gene through screening. The base sequence is shown as SEQ ID NO.1 in the sequence table, the codon at the original point is mutated to TGG from GAT, and the corresponding amino acid residue is mutated to aspartic acid from serine. The P450 BM-3 D168Smutant enzyme of the invention takes indirubin as main acquired product when catalyzing indole, and the product shows obvious red color.

Description

Technical field [0001] The present invention relates to the field of genetic engineering and biopharmaceuticals in bioengineering, in particular to the cytochrome P450 BM-3D 168S mutant enzyme with new catalytic properties, its genes and the use of this enzyme in biocatalysis, especially It is used in the preparation of indirubin. Background technique [0002] Indirubin is a new type of leukemia treatment drug discovered by Chinese scientists after years of research in the traditional prescription Angelica Longhui Pills in the last century. In the mid-1960s, the Institute of Hematology of the Chinese Academy of Medical Sciences found that Danggui Longhui Pills had better curative effects in the treatment of chronic myelogenous leukemia. After more than 10 years of pharmacological analysis of the 11 components in the pill, the organization found that Qingdai was finally determined to be an effective component against leukemia. After further research, the indirubin isolated from In...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12P17/16
Inventor 梅乐和胡升虞青黄俊
Owner ZHEJIANG UNIV
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