Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Carbonyl reductase mutant with improved thermal stability

A mutant and reductase technology, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of poor thermal stability of carbonyl reductase ChKRED12, and achieve the effects of flexibility, improved thermal stability, and good industrial application prospects

Active Publication Date: 2019-06-04
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
View PDF13 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved in the present invention is: the problem of poor thermal stability of carbonyl reductase ChKRED12

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

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Carbonyl reductase mutant with improved thermal stability
  • Carbonyl reductase mutant with improved thermal stability
  • Carbonyl reductase mutant with improved thermal stability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 14

[0035] Example 1 Construction of 4 single point mutants

[0036] The results of the prediction of potential heat-resistant sites by the rational design method are shown in the appendix of the manual. figure 1. The single point mutants S79P, L128M, V162I and G163A were all constructed using the codon-optimized gene SEQ ID NO.1 of carbonyl reductase ChKRED12 as a template, and the primers used were:

[0037] S79P-F:5′–GGATGCGAAT CCG AGCGCCGTGG–3′

[0038] S79P-R:5′–CCACGGCGCT C G ATTCGCATCC–3′

[0039] L128M-F: 5′–CTTTATTCCAGAAA ATG CTGCGTAA C–3′

[0040] L128M-R:5′–GTTACGCAG CAT TTTCTGGATAAA G–3′

[0041] V162I-F:5′–GGCGCGCTG ATT GGGGCGACC–3′

[0042] V162I-R:5′–GGTCGCCCC AAT CAGCGCGCC–3′

[0043] G163A-F: 5′–GGCGCGCTGGTG GCA GCGACCAAAGC–3′

[0044] G163A-R:5′–GCTTTGGTCGC TGC CACCAGCGCGCC–3′

[0045] The PCR conditions are: 5×HF Buffer 10μL, MgCl 2 (1mM) 1μL, primer (50ng / μL) 1.5μL each, dNTP (2.5mM) 4μL, Phu (1U) 1μL, plasmid 50ng, ultrapure water to m...

Embodiment 2

[0046] Embodiment 2 crude enzyme liquid preparation and the mensuration of enzyme activity

[0047] 2.1 Preparation of crude enzyme solution

[0048] Each mutant plasmid in Example 1 was chemically transformed into E. coli expression strain BL21 (DE3), coated with an LB plate containing kanamycin (50 μg / mL), cultured overnight at 37 ° C, and single clones were picked in 10 mL In LB liquid medium containing kanamycin (50 μg / mL), culture overnight at 37° C., 180 rpm. Inoculate 1% inoculum in 200 mL of TB medium containing kanamycin (50 μg / mL), culture at 37 °C for 3 h, add final concentration of 0.5 mM IPTG, induce at 30 °C for 18 h, centrifuge at 5000 rpm and 4 °C for 10 min , discard the supernatant, wash the cells twice with normal saline, then add 15 mL of 0.1M potassium phosphate buffer (pH 8.0) to resuspend the cells, and disrupt the cells with ultrasonic waves (working conditions: working time 3s, intermittent time 3s , working times 99, power 200W), cell disruption sol...

Embodiment 3

[0060] Example 3 Construction of multi-site combination mutants

[0061] 3.1 Construction of mutant M722

[0062] The site-directed mutagenesis method mutated the 128th leucine of the mutant S79P to methionine (the DNA sequence changed from TTA to ATG), and the 162nd valine to isoleucine (the DNA sequence changed from GTG to ATG). For ATT), construct mutant M722, the primers used are as follows:

[0063] L128M-F: 5′–CTTTATTCCAGAAA ATG CTGCGTAA C–3′

[0064] L128M-R:5′–GTTACGCAG CAT TTTCTGGATAAA G–3′

[0065] V162I-F:5′–GGCGCGCTG ATT GGGGCGACC–3′

[0066] V162I-R:5′–GGTCGCCCC AAT CAGCGCGCC–3′

[0067] The PCR conditions and operations were the same as in Example 1, and a new mutant M722 was obtained.

[0068] 3.2 Construction of mutant M723

[0069] The site-directed mutagenesis method mutated the 128th leucine of the mutant S79P to methionine (the DNA sequence changed from TTA to ATG), and the 163rd glycine to alanine (the DNA sequence changed from GGG to GCA) ,...

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 invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a carbonyl reductase ChKRED12 heat-resistant mutant. The carbonyl reductase ChKRED12 can be used for efficiently catalytically preparing duloxetine chiral alcohol and other intermediates, but has relatively poor heat stability. By utilizing the rationally designed scheme, the enzyme can be subjected to molecular evolution, heat resistance of the mutant can be greatly improved, and industrial application can be favored.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and enzyme engineering, and specifically relates to a carbonyl reductase mutant with improved heat resistance. Background technique [0002] Carbonyl reductase (Carbonyl reductase, EC 1.1.1.x) is a member of the oxidoreductase (Oxidoreductases) family, which refers to a class of oxidoreductases that can catalyze the reduction of carbonyl groups to generate corresponding alcohols or their reverse reactions. The essence is the transfer of hydrogen or electrons between the substrate and the hydrogen donor. Chiral alcohols are an important class of organic synthesis building blocks, and have high application value in medicine, agriculture, food and chemical industries, and carbonyl reductase can catalyze the asymmetric reduction of carbonyl groups to generate corresponding chiral alcohols. [0003] With the development of industrial biotechnology and protein engineering technology, carbo...

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
IPC IPC(8): C12N9/04C12N15/70
Inventor 吴中柳李孜一刘艳
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com