Cytochrome P450BM-3 (D168L/E435T) variant enzyme as well as coding genes and application thereof

A cytochrome and variant technology, applied in the field of variant enzymes, can solve the problems of low efficiency and low affinity of indigo

Inactive Publication Date: 2012-10-31
NINGBO INST OF TECH ZHEJIANG UNIV ZHEJIANG
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0008] The invention provides a cytochrome P450BM-3 variant enzyme, which solves the problem of low affinity between the traditional variant enzyme and the substrate and low efficiency of catalyzing indole to produce indigo

Method used

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  • Cytochrome P450BM-3 (D168L/E435T) variant enzyme as well as coding genes and application thereof
  • Cytochrome P450BM-3 (D168L/E435T) variant enzyme as well as coding genes and application thereof
  • Cytochrome P450BM-3 (D168L/E435T) variant enzyme as well as coding genes and application thereof

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Embodiment Construction

[0020] 1. Plasmid pET-28a(+)-P450BM-3(F87V / A74G / L 188Q)

[0021] The plasmid pET-28a(+)-P450BM-3(F87V / A74G / L188Q) used in the present invention was donated by Professor R.D.Schmid of the University of Stuttgart, Germany. It is a variant of cytochrome P450 BM-3(F87V / A74G / L188Q) The enzyme gene was inserted downstream of the promoter of the original vector pET-28a (+), and the sequence of the mutant gene and the structure of the recombinant plasmid had been recorded in Chinese Patent 00810943.5, Chinese Patent 200610052588.0 and literature ("Directed Evolution of the Fatty-Acid Hydroxylase P450 BM-3 into an Indole-Hydroxylating Catalyst. Chemistry-A European Journal. 2000, 6(9):1531-1536"). The construction method of the recombinant plasmid can refer to the existing general plasmid construction method. The purpose of using the plasmid is to obtain the cytochrome P450BM-3 (F87V / A74G / L188Q) variant enzyme gene, and there is no specific requirement for the carrier.

[0022] 2. Des...

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Abstract

The invention discloses a cytochrome P450BM-3 (D168L/E435T) variant enzyme as well as coding genes and application thereof. The amino acid sequence of the variant enzyme is shown in SEQ ID No.1: two mutational sites are increased on basis of a cytochrome P450BM-3 (F87V/A74G/L188Q) variant enzyme, namely aspartic acid (GAT) at the 168th site is mutated to be leucine (CTT) and glutamic acid at the 435th site (GAA) is mutated to be threonine (ACG). Compared with a male parent, the variant enzyme disclosed by the invention has higher appetency with the substrate, and higher efficiency in catalyzing indole to be indigo.

Description

technical field [0001] The invention relates to a variant enzyme, in particular to a cytochrome P450BM-3 (D168L / E435T) variant enzyme, its encoding gene and its application. Background technique [0002] Indigo is a natural dye with a long history. Whether it is some clothing worn by ancient Egyptian mummies or the blue linen fabric unearthed from the Mawangdui Han Tomb in my country, their color comes from indigo. The most important reason why indigo has been favored by people for a long time and widely is that it has dyeing fastness and light fastness unmatched by other dyes. Therefore, indigo is also known as the "king of dyes". [0003] In ancient times, indigo was mainly obtained from indigo-containing plants (such as woad, indigo, indigo, etc.). In the middle of the 19th century, because the production of natural indigo could not meet the needs of the rapid development of the textile industry, people began to try to synthesize indigo by chemical methods. In 1897, the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/63C12N1/21C12P17/16C12R1/19
Inventor 梅乐和张澎湃胡升胡桂香雷引林
Owner NINGBO INST OF TECH ZHEJIANG UNIV ZHEJIANG
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