P450BM-3Asp168His variant gene capable of catalyzing indole to generate indigo blue and its use

A technology of p450bm-3asp168his and indole catalysis, applied in the field of genetic engineering, can solve the problems of large-scale industrialization of indigo and limited improvement, and achieve the effects of improved catalytic efficiency, high catalytic activity and broad application prospects

Inactive Publication Date: 2007-01-24
ZHEJIANG UNIV
View PDF2 Cites 11 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0010] Design of Saturation Mutation Primers and Acquisition of PCR Amplified Products

[0011] Upstream primer 5'-CAGCTTTTACCGA NNN CAGCCTCATCC-3'

[0012] Downstream primer 5'-GGATGAGGCTG NNN TCGGTAAAAGCTG-3'

[0013] To a 500 μL eppendorf tube, add 100 pmol of dNTPs, 15 pmol each of upstream and downstream primers, approximately 2 ng pET28α(+)P450BM-3 as template DNA, 2.5 U of PfuDNA polymerase, 5 μL of MgCl containing 25 mmol / L 2 Pfu PCR buffer, then add sterile distilled water to a total volume of 50 μL. The reaction parameters are denaturation at 95°C for 1min, followed by 30s at 95°C, primer 39: 48°C-56°C, 2min; primer 168: 50°C-56°C, 2min, 16min at 72°C, after 18 cycles, then 72°C Extend for 2 min to obtain the PCR amplification product.

[0014] Mutation library construction

[0015] The above-mentioned PCR amplified PCR product was digested with DpnI for 2 hours, then transformed into E.coliBL21, spread on LB agar plate containing 30 μg / ml kanamycin, and cultur...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The present invention discloses one kind of P450BM-3Asp168His 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-3Asp168His 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 histidine 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-3Asp168His 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...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12P17/10
Inventor 梅乐和李红梅
Owner ZHEJIANG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap