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Cytochrome P450 BM-3 variant enzyme, coding gene and application thereof

A cytochrome and variant technology, applied in the field of variant enzymes, can solve the problems of poor thermal stability, low efficiency, low affinity between variant enzymes and substrates, etc.

Inactive Publication Date: 2012-10-24
NINGBO INST OF TECH ZHEJIANG UNIV ZHEJIANG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The invention provides a cytochrome P450BM-3 variant enzyme, which solves the problems of low affinity between the existing variant enzyme and the substrate, poor thermal stability, and low efficiency of catalyzing indole to generate indigo

Method used

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  • Cytochrome P450 BM-3 variant enzyme, coding gene and application thereof
  • Cytochrome P450 BM-3 variant enzyme, coding gene and application thereof
  • Cytochrome P450 BM-3 variant enzyme, coding gene and application thereof

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Embodiment Construction

[0022] 1. Plasmid pET-28a(+)-P450BM-3(F87V / A74G / L 188Q)

[0023] 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 enzyme of cytochrome P450BM-3(F87V / A74G / L188Q) The gene is 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 have 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.

[0024] 2. Des...

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Abstract

The invention discloses a cytochrome P450 BM-3 variant enzyme, a coding gene and application of the cytochrome P450 BM-3 variant enzyme. An amino acid sequence of the variant enzyme is shown in SEQ ID NO.1; three mutational sites are added on the basis of the cytochrome P450 BM-3 (F87 / A74G / L188Q) variant enzyme, namely aspartic acid (GAT) at the 168th site mutates into aminocaproic acid (CTT), glutamic acid (GAA) at the 435th site mutates into threonine (ACG), and valine (GTG) at the 445th site mutates into aminopropionic acid (GCG). Compared with male parent enzyme and protoenzyme, the variant enzyme disclosed by the invention has high affinity with substrate and the efficiency of catalyzing benzazole to generate indigo is higher.

Description

technical field [0001] The invention relates to a variant enzyme, in particular to a cytochrome P450 BM-3 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 synthetic in...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/63C12N1/21C12P17/16C12R1/19
Inventor 梅乐和张澎湃胡升雷引林金志华胡桂香
Owner NINGBO INST OF TECH ZHEJIANG UNIV ZHEJIANG
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