A carbonyl reductase mutant mut-accr(g152l/y189n) and its application and coding gene

A mut-accr, reductase technology, applied in oxidoreductase, application, genetic engineering and other directions, can solve the problems of poor substrate tolerance and low enzyme activity, and achieve high substrate tolerance, high activity, good selective effect

Active Publication Date: 2021-05-14
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 ( G152L / Y189N)

Method used

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  • A carbonyl reductase mutant mut-accr(g152l/y189n) and its application and coding gene
  • A carbonyl reductase mutant mut-accr(g152l/y189n) and its application and coding gene
  • A carbonyl reductase mutant mut-accr(g152l/y189n) and its application and coding gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 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 (sequence In the name of the database) 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 152G and 189Y sites were selected as mutation sites.

Embodiment 2

[0027] The changes between carbonyl reductase and ethyl 2-oxo-4-phenylbutyrate were analyzed by molecular docking; carbonyl reductase AcCR, mutant mut-AcCR (mut-G152L / Y189N) and 2- Molecular docking of oxo-4-phenylbutyric acid ethyl ester to analyze the distance between the enzyme active site Ser142, Tyr155 and the coenzyme NADH nicotinamide ring C4 located between the substrate 2-oxo-4-phenyl-butyric acid ethyl ester and force changes. The results are shown in Figure 1. The docking results of AcCR and mut-G152L / Y189N with ethyl 2-oxo-4-phenylbutyrate Figure 1a , Figure 1b shown. It can be seen from the figure that the distances between OPBE and the enzyme active site Ser142, Tyr155 and the C4 hydrogen atom on the nicotinamide ring of NADH are shortened respectively (24.5%), (14.8%) and (10.5%). On the one hand, the mutation of Gly to Leu enhances the hydrophobic interaction near the active center of the enzyme; on the other hand, the Trp with a large R group near th...

Embodiment 3

[0029]Using PrimeSTAR Max DNA Polymerase, the plasmid pGEX-mut-G152L / Y189N containing the mutant mut-AcCR (G152L / Y189N) gene was obtained by amplifying the whole pGEX-acr plasmid. Primers used for site-directed mutagenesis: the mutation primer at site G152L is Primer 1: 5'-ACCCAATGTTGGCCGCCTATAAC-3', Primer2: 5'-GTTATAGGCGGCCAACATTGGGT-3'; the mutation primer at site Y189N is: Primer 3: 5'-CGGCAATATCTGGACACCTATGGTGG-3 ', Primer 4: 5'-CCACCATAGGTGTCCAGATATTGCCG-3'.

[0030] The PCR amplification system and reaction conditions used for site-directed mutagenesis are as follows:

[0031] Polymerase Chain Reaction (PCR) Amplification System

[0032]

[0033] PCR reaction conditions:

[0034]

[0035] 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:

[0036]

[0037] Place the prepared digestion reaction system at 37°C and incubate for ...

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Abstract

The invention discloses a carbonyl reductase mutant mut-AcCR (G152L / Y189N) and its application and coding 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 , to obtain the mutant mut-AcCR (G152L / Y189N), the specific enzyme activity of the mutant for 2-oxo-4-phenylbutyrate ethyl ester can reach 88.9U / mg, which is higher than that of the carbonyl reductase before the mutation The specific vitality is increased by 61.3 times. The substrate tolerance concentration was increased from 50mmol / L to 200mmol / L. The mutant catalyzed ethyl 2‑oxo‑4‑phenyl‑butyrate with absolute selectivity, and the enantiomeric excess value of the product increased from 82.9% to >99%. The carbonyl reductase mutant of the present invention plays an important role in catalyzing the asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate.

Description

technical field [0001] The invention belongs to the field of enzyme molecular modification, and in particular relates to a carbonyl reductase mutant mut-AcCR (G152L / Y189N) 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. Compa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/55C12P7/62
CPCC12N9/0006C12P7/62C12Y101/01184
Inventor 娄文勇魏萍宗敏华
Owner SOUTH CHINA UNIV OF TECH
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