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Carbonyl reductase AcCR and encoding gene and application thereof

A carbonyl reductase and gene technology, applied in the field of genetic engineering, can solve the problems of many side reactions, low stereoselectivity, and low space-time yield, and achieve the effects of reducing production costs, efficient coenzyme regeneration cycle system, and mild conditions

Inactive Publication Date: 2016-02-24
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It currently has the problems of low space-time yield of reaction, many side reactions and low stereoselectivity of reaction in some cases

Method used

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  • Carbonyl reductase AcCR and encoding gene and application thereof
  • Carbonyl reductase AcCR and encoding gene and application thereof
  • Carbonyl reductase AcCR and encoding gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Embodiment 1 The acquisition of carbonyl reductase AcCR gene accr

[0049] Acetobacter XZY003 was shaken and cultivated at a temperature of 30°C and a rotational speed of 100rpm for 24h; then the bacteria were centrifuged (4°C, 9000rpm) for 5min to collect the bacteria and washed three times with 0.85% normal saline to remove Remove the remaining medium, and then use the bacterial genome extraction kit of Shanghai Jierui Bioengineering Co., Ltd. to extract the genomic DNA of Acetobacter XZY003. For specific methods, refer to the method provided in the kit. Using the extracted genomic DNA of Acetobacter XZY003 as a template, primer 1 (5'-TCC CCCGGG AATGGCACGTGTAGCAGGCAAGGTT-3') and primer 2 (5'-CCG GAATTC CTCATTGTGCTGTTGACCTCCCATCCAT-3') are amplification upstream and downstream primers, wherein the restriction sites are AvaI and EcoRI (underlined); PCR amplification obtains the target fragment carbonyl reductase AcCR gene accr. Among them, the enzyme kit used for PCR...

Embodiment 2

[0050] The acquisition of embodiment 2 glucose dehydrogenase gene gdh

[0051] The gene of glucose dehydrogenase is derived from Bacillus subtilis Bacillus subtilis 168 (purchased from Guangdong Microorganism Culture Collection Center), the medium formula and culture conditions are provided by the method provided by the Culture Collection Center, and the extraction of Bacillus subtilis genomic DNA is carried out according to the Shanghai Jierui Biotechnology Co., Ltd. Bacterial Genome Extraction Kit from Engineering Ltd. Using the genomic DNA of Bacillus subtilis168 as a template, the primer 5 (5'-GA AGATCT CATGTATCCGGATTTAAAAGGAAAAGTCGTCG-3') and primer 6 (5'-CCG CTCGAG TTAACCGCGGCCTGCCTGGAAT-3') are upstream and downstream primers respectively, wherein the restriction sites are respectively BglII and XhoI (underlined); the target fragment glucose dehydrogenase GDH gene gdh is obtained by PCR amplification. The PCR reaction system (25 μL) is: KODFX buffer 12.5 μL; 2mMdNTP...

Embodiment 3

[0052] The construction of embodiment 3 recombinant vector pGEX-accr

[0053] (1) The carbonyl reductase AcCR gene accr fragment (recovered product) obtained in Example 1 and the plasmid pGEX-2T (purchased from Guangzhou Qiyun Biotechnology Co., Ltd.) were subjected to double enzyme digestion; wherein, the gene fragment enzyme digestion system System (30μL) is: ddH 2 O15 μL; buffer 3 μL; gene fragment 10 μL; AvaI+EcoRI 1 μL each; plasmid pGEX-2T enzyme digestion system (20 μL) is: ddH 2 O6-14 μL; buffer 2 μL; carrier 2-10 μL; AvaI+EcoRI 1 μL each; digestion conditions: 37°C for 30 min;

[0054] (2) Recover the products after enzyme digestion in step (1) respectively. For the recovery method and steps, refer to the PCR Product Recovery Kit of Shanghai Jierui Bioengineering Co., Ltd. The recovered product was ligated, and the ligation system (20 μL) was: vector 5 μL; gene fragment 10 μL; 10×T4buffer 2 μL; T4DNAligase 1 μL; ddH 2 O2 μL; connection conditions: 22°C for 30 minut...

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Abstract

The invention belongs to the technical field of gene engineering and particularly relates to carbonyl reductase AcCR and an encoding gene and application thereof. The amino acid sequence of the carbonyl reductase AcCR is shown as SEQ ID NO.1. The nucleotide sequence of the gene for encoding the carbonyl reductase AcCR is shown as SEQ ID NO.2. The carbonyl reductase AcCR comes from bacillus aceticus XZY003 and can catalyze 13 carbonyl compounds to generate corresponding chiral alcohol of single enantiomers. The carbonyl reductase AcCR and glucose dehydrogenase GDH are co-expressed in a prokaryotic expression system or a eukaryotic expression system, the biological reaction process of biological catalytic conversion is conducted through the carbonyl reductase, in-situ regeneration of coenzyme is achieved, production cost is greatly reduced, the optical purity of an obtained product is high, the reaction process is efficient, and conditions are moderate.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a carbonyl reductase AcCR and its coding gene and application. Background technique [0002] Optically pure chiral alcohols and their derivatives are important building blocks for the synthesis of chiral drugs, functional materials and chiral pesticides. It is estimated that the global sales of chiral alcohol building blocks are as high as tens of billions of dollars. Due to the importance and huge commercial value of these chiral alcohol building blocks in the synthesis of chiral drugs, the development of their synthesis technology has become the commanding height of the development strategy of major pharmaceutical companies around the world. Currently, there are mainly two methods for the synthesis of chiral alcohol building blocks: chemical and biological. Traditional chemical methods have problems such as harsh reaction conditions (high temperature a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N15/65C12P7/22C12P7/62C12P9/00C12P13/00C12N1/21C12R1/19
CPCC12N9/0006C12N15/65C12N15/70C12P7/22C12P7/62C12P9/00C12P13/008C12Y101/01184
Inventor 娄文勇魏萍宗敏华
Owner SOUTH CHINA UNIV OF TECH
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