Cytochrome P450 BM-3 (L148S/Q 229R) variant enzyme and coding gene and use thereof

A cytochrome and variant technology, applied in the field of mutant enzymes, can solve problems such as poor thermal stability, low catalytic indole efficiency, and low affinity

Inactive Publication Date: 2013-10-30
NINGBO INST OF TECH ZHEJIANG UNIV ZHEJIANG
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0008] The invention provides a cytochrome P450BM-3 (L148S / Q229R) variant enzyme, which solves the problems of low affinity between the traditional variant enzyme and the substrate, poor thermal stability, and low efficiency of catalyzing indole to produce indigo

Method used

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  • Cytochrome P450 BM-3 (L148S/Q 229R) variant enzyme and coding gene and use thereof
  • Cytochrome P450 BM-3 (L148S/Q 229R) variant enzyme and coding gene and use thereof
  • Cytochrome P450 BM-3 (L148S/Q 229R) variant enzyme and coding gene and use thereof

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Embodiment Construction

[0018] 1. Plasmid pET-28a(+)-P450BM-3(F87V / A74G / L 188Q)

[0019] 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.

[0020] 2. Err...

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Abstract

The invention discloses a cytochrome P450 BM-3 (L148S / Q 229R) variant enzyme and a coding gene and use of the cytochrome P450 BM-3 (L148S / Q 229R) variant enzyme. The amino acid sequence of the variant enzyme is shown as the SEQ ID NO. 1. Two mutational sites are added on the basis of the cytochrome P450 BM-3 (F87V / A74G / L188Q) variant enzyme, i.e., the No. 148 leucine (L) is mutated into serine (S), and the No. 229 glutamine (Q) is mutated into arginine (R). Compared with a male parent, the variant enzyme disclosed by the invention is higher in affinity to substances and thermal stability.

Description

technical field [0001] The invention relates to a mutant enzyme, in particular to a cytochrome P450BM-3 (L148S / Q229R) variant enzyme, its coding gene and its use. 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/63C12N1/21C12P17/16C12R1/19
Inventor 梅乐和张澎湃胡升金志华胡桂香
Owner NINGBO INST OF TECH ZHEJIANG UNIV ZHEJIANG
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