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A carbonyl reductase mutant mut-accr (e144a/g152l) and its application and coding gene

A mut-accr, reductase technology, applied in oxidoreductase, application, genetic engineering, etc., can solve the problems of poor substrate tolerance and low enzyme activity

Active Publication Date: 2021-06-08
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the shortcomings of low enzyme activity and poor substrate tolerance, the primary purpose of the present invention is to provide a carbonyl reductase mutant mut-AcCR ( E144A / G152L)

Method used

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  • A carbonyl reductase mutant mut-accr (e144a/g152l) and its application and coding gene
  • A carbonyl reductase mutant mut-accr (e144a/g152l) and its application and coding gene
  • A carbonyl reductase mutant mut-accr (e144a/g152l) and its application and coding gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The gene sequence of AcCR was translated into its amino acid sequence by standard methods, searched in the PDB database with this sequence, and selected 4RF2, 1ZJY, 1NXQ and 1ZK3 (database The name of the protein sequence) tertiary structure as a template, homology modeling, and energy minimization, the tertiary structure model of carbonyl reductase AcCR was obtained. Further use Ramachandran Plot and Profile-3D to evaluate the structural rationality of each amino acid residue in the homology modeling results and the matching degree between the protein model and the amino acid sequence of the protein. It is determined that the model built is reasonable and can be used for subsequent experimental analysis. The tertiary structure of carbonyl reductase AcCR was docked with the coenzyme NADH, and the mutation hotspots of carbonyl reductase were predicted by HotSpot 2.0, and the 144E and 152G sites were selected as mutation sites.

Embodiment 2

[0026] The changes between carbonyl reductase and 4'-chloroacetophenone before and after mutation were analyzed by molecular docking; carbonyl reductase AcCR, mutant mut-AcCR (E144A / G152L) were docked with 4'-chloroacetophenone, respectively, The changes in the distance and force between the enzyme active site Ser142, Tyr155 and the coenzyme NADH nicotinamide ring C4 located on the substrate 4'-acetophenone were analyzed. see results Figure 1a , Figure 1b The docking results of AcCR and mut-E144A / G152L with 4'-chloroacetophenone are shown. from Figure 1a , Figure 1b It can be seen that the distance between the catalytic site S142, Y155 and the hydrogen atom at the 4-position of the NADH nicotinamide ring and the carbonyl oxygen atom of 4’-chloroacetophenone is significantly reduced after the mutation, shortening respectively (22.4%), with (5.2%). After the mutation, the steric hindrance between the substrate and the active center of the enzyme is reduced, which is...

Embodiment 3

[0028] The plasmid pGEX-mut-E144A / G152L containing the mutant mut-AcCR (E144A / G152L) gene was obtained by amplifying the whole pGEX-acr plasmid using PrimeSTAR Max DNA Polymerase.

[0029] Primers used for site-directed mutagenesis: the mutation primer at site E144A is Primer1: 5'-ATCTGTCTTCCATTGCCGGACTGAT-3';

[0030]Primer2: 5'-ATCAGTCCGGCAATGGAAGACAGAT-3'; the mutant primer at position G152L is

[0031] Primer3: 5'-ACCCAATGTTGGCCGCCTATAAC-3';

[0032] Primer 4: 5'-GTTATAGGCGGCCAACATTGGGT-3'.

[0033] The PCR amplification system and reaction conditions used for site-directed mutagenesis are as follows:

[0034] Polymerase Chain Reaction (PCR) Amplification System

[0035]

[0036] PCR reaction conditions:

[0037]

[0038] After the reaction, the reaction product was treated with restriction endonuclease DpnI to act on the Gm6A^TC site to digest the template plasmid in the system. The reaction system is:

[0039]

[0040]

[0041] Place the prepared digest...

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Abstract

The invention discloses a carbonyl reductase mutant mut-AcCR (E144A / G152L) and its application and encoding gene. Carbonyl reductase AcCR can catalyze the asymmetric reduction of various latent chiral carbonyl compounds, but its activity and substrate tolerance to aromatic compounds are low. The present invention adopts enzyme molecular transformation method to mutate glycosylation reductase AcCR , the mutant mut‑AcCR (E144A / G152L) was obtained, the specific activity of the mutant for 4'‑chloroacetophenone could reach 92.7 U / mg, which was 17.93 times higher than that of the unmutated carbonyl reductase. The substrate tolerance concentration was increased from 50 mmol / L to 200 mmol / L. The carbonyl reductase mutants of the present invention are widely used in the asymmetric reduction of carbonyl compounds.

Description

technical field [0001] The invention belongs to the field of enzyme molecular transformation, and in particular relates to a carbonyl reductase mutant mut-AcCR (E144A / G152L) and its application and a gene encoding the mutant. Background technique [0002] Optically pure chiral alcohols and their derivatives are important chiral intermediates for the synthesis of chiral drugs, liquid crystal materials, flavors and fragrances, and pesticides, and occupy an important position in the fields of medicine and other chemical industries. Chiral alcohols can be synthesized chemically and biologically. Chemical synthesis generally requires harsh conditions such as high temperature and high pressure; a large amount of organic reagents are used, causing serious environmental pollution; the preparation process is complicated, and there are often multiple protection and deprotection steps; more importantly, the enantiomers of the products obtained by chemical methods Low selectivity. Com...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12P7/02
CPCC12N9/0006C12P7/02C12Y101/01184
Inventor 娄文勇魏萍宗敏华郭泽望区晓阳
Owner SOUTH CHINA UNIV OF TECH
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