Variant enzyme of cytochrome P450BM-3D168R and method for preparing indirubin using the same

A technology of P450BM-3D168R and cytochrome, applied in oxidoreductase, fermentation, etc., can solve problems such as cost, economic value without economic value, difficulty in using indirubin for large-scale biological preparation, increased separation and purification costs, etc. Achieve the effects of reducing the burden of separation and purification, avoiding the damage 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 D168R variant enzyme disclosed in the present invention specifically comprises the following steps:

[0034] 1. Design random primers for P450 BM-3168 codon and carry out PCR amplification

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

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

[0037] Downstream primer: 5'-GGATGAGGCTGNNNTCGGTAAAAGCTG-3'

[0038] Composition of PCR reaction system: 5 μL of 25 mmol / 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 ...

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Abstract

The invention discloses a cytochrome P450 BM-3 D168R mutant 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 CGG from GAT, and the corresponding amino acid residue is mutated to arginine from aspartic acid. The P450 BM-3 D168R mutant 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 in bioengineering and the field of biopharmaceuticals, in particular to a cytochrome P450 BM-3 D168R variant enzyme with new catalytic properties, its gene and the use of this enzyme in biocatalysis, Especially in the application of indirubin preparation. Background technique [0002] Indirubin is a new type of leukemia treatment drug discovered by Chinese scientists in the last century after years of research from the traditional prescription Danggui Longhui Pills. In the mid-1960s, the Institute of Hematology of the Chinese Academy of Medical Sciences found that Danggui Longhui Pills had a good curative effect in the treatment of chronic myelogenous leukemia. The agency has conducted pharmacological analysis of 11 ingredients in the pill for more than 10 years, among which Qingdai was finally identified as an effective anti-leukemia ingredient. After further research, indirubin isola...

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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