P450 BM-3Asp168Leu variant gene capable of catalyzing indole to generate indigo blue and its use

Abstract

The present invention discloses a kind of P450BM-3Asp168Leu variant gene capable of catalyzing indole to generate indigo blue, and the gene is obtained by means of saturated mutation and directional evolution technology mediated random mutation and screening. The P450BM-3Asp168Leu variant gene has base sequence as shown in SEQ ID No. 1 of the sequence list, and the aspartic acid codon in the 168-th site of the original P450BM-3(F87V / A74G / L188Q) gene mutated into leucine codon. The enzymic kinetic curve measurement shows that the variant enzyme the obtained variant gene expresses has catalysis efficiency obviously higher than that in available technology. The present invention provides new practical enzyme for the biological synthesis of dye indigo blue and wide application foreground for the biological production of dye indigo blue.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering in biochemical industry, and in particular relates to a P450BM-3Asp168Leu variant gene capable of catalyzing indole to generate indigo and its application. Background technique [0002] As one of the first discovered natural dyes, the use of indigo dye has quite a long history. From some clothes worn by ancient Egyptian mummies to the blue linen fabrics unearthed in Mawangdui in my country, they are all dyed with indigo. Indigo was also dyed on the flags of the French Revolution and the American Revolution more than 200 years ago. The reason why this kind of dye has been loved by people for a long time and widely is that it is unmatched by other dyes in terms of dyeing fastness and light fastness, so it is often called "the king of dyes". In ancient times, it was mainly obtained from indigo plants such as woad, indigo, and indigo. In the middle of the 19th century, the industrial re...

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Problems solved by technology

In the previous patent applications CN200410089167.6 and CN200410089174.6 of the inventors of this patent, it was also disclosed that on the basis of P450BM-3 (F87V / A74G / L188Q), error-prone PCR directed evolution technology was used to obtain a higher catalytic indole activity. Variant enzymes, but the degree of improvement is limited, and there are still some difficulties in the large-scale industrialization of biotransformation and synthesis of indigo