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Carbonyl reductase mutant as well as coding gene and application thereof

A reductase and mutant technology, which is applied to carbonyl reductase mutants and their coding genes and application fields, can solve the problems of difficult post-processing and low catalytic efficiency, and achieve the effect of easy post-processing and high catalytic efficiency

Inactive Publication Date: 2018-10-09
宿迁阿尔法科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The invention provides a solution to the problems of low enzyme catalytic efficiency and difficult post-reaction treatment, and provides a recombinant carbonyl reductase with high catalytic efficiency and easy post-treatment, as well as a recombinant expression vector and genetic engineering containing the carbonyl reductase gene. Bacteria, the preparation method of its recombinant enzyme, and the use of the recombinant enzyme to asymmetrically catalyze the preparation of optically active chiral alcohols, especially to catalyze the synthesis of important chiral intermediates ((3R,5R)-6-cyano Method for tert-butyl-3,5-dihydroxyhexanoate

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] The establishment of embodiment 1 genetically engineered bacteria

[0016] According to the Candida magnoliae ADH3 (GenBank: DQ288128.1) published by Genebank, the gene fragment was artificially synthesized, and the gene fragment was used as a template to amplify the target gene fragment by polymerase chain reaction (PCR) (NdeI was added on both sides of the fragment) and XhoI endonuclease site), the nucleotide sequences of the primers are shown in SEQ ID NO.3 and SEQ ID NO.4. And the gene was inserted into the pET-24a plasmid by using NdeI and XhoI endonuclease sites, and the ligated vector was transferred into Escherichia coli BL21 (DE3) to establish a carbonyl reductase genetically engineered bacterium. The primers for PCR amplification of the target gene are:

[0017] Forward primer: GGGAATTCCATATGACGACTACTTCAAATGCGCTCG (SEQ ID NO.3)

[0018] Reverse primer: CCGCTCGAGCCTCCTGAGCCAGCTTAATGGCGGA (SEQ ID NO.4)

Embodiment 2

[0019] Embodiment 2 Obtaining of carbonyl reductase mutant gene

[0020] In this study, the method of error-prone PCR random mutation was used to transform the carbonyl reductase gene. Error-prone PCR is to use low-fidelity DNA polymerase to amplify the target gene. By adjusting the reaction conditions, such as increasing the concentration of magnesium ions, adding manganese ions, and changing the concentration of four kinds of dNTPs in the system, the amplification process can be changed. Mutation frequency, so as to randomly insert mutations into the target gene to obtain random mutants of different protein molecules.

[0021] The PCR system used in this study is as follows.

[0022] The 50μl PCR system is as follows: 5×PCR Buffer 10μl, dNTP mixture concentration 2.5mmol / L1μl, MgCl 2 (2.5mmol / L) 2μl, MnCl 2 (2.5mmol / L) 2μl, carbonyl reductase template gene 3μl, Taq DNA polymerase 1μl (2.5U / μl), and the rest were added to 50μl of sterilized double distilled water.

[0023...

Embodiment 3

[0031] The shaking flask culture of embodiment 3 recombinant escherichia coli

[0032] The recombinant Escherichia coli obtained in Examples 1 and 2 was inoculated into 100 mL of LB medium containing kanamycin (50 μg / mL) (peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH7.0 ), cultured at 37° C. in a shaker at 250 rpm for 5 hours. Transfer the 1:200 bacterial culture solution to a 200mL shake flask filled with LB medium (containing 50 μg / mL kanamycin), and place it under the same conditions for shaking culture. When the OD600 value of the bacterial solution reaches 0.6-0.8, add the inducer IPTG with a final concentration of 0.4mmol / L, induce the expression of the culture solution at 25°C for 12-16 hours, and collect the bacterial cells by centrifugation (13000rpm, 30min, 4°C). And washed twice with phosphate buffer (pH7.0), then resuspended with 3 times the volume of buffer, ultrasonically disrupted in an ice bath, centrifuged (13000rpm, 10min, 4°C), and then detected by S...

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Abstract

The invention discloses a carbonyl reductase mutant as well as a coding gene and application thereof. A carbonyl reductase mutant gene has a sequence shown as SEQ ID NO. 1; an amino acid sequence of acarbonyl reductase mutant coded by the carbonyl reductase mutant gene is shown as SEQ ID NO. 2. A genetically engineered bacterium for producing carbonyl reductase contains the carbonyl reductase gene provided by the invention. A preparation method of the carbonyl reductase comprises the following step: culturing the genetically engineered bacterium for producing the carbonyl reductase to obtainthe carbonyl reductase through recombinant expression. The carbonyl reductase prepared by the recombinant expression is used as catalyst, and optical active chiral alcohol is prepared through an asymmetric reducing carbonyl compound; preparation of the recombinant carbonyl reductase is realized, and the recombinant carbonyl reductase is used for asymmetrically synthesizing and catalyzing (R)-2-hydroxyl-4-ethyl phenylbutyrate. The substrate conversion rate reaches 96.8 percent and the ee of a reduction product is greater than 99 percent.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a carbonyl reductase mutant, its coding gene and its application. Background technique [0002] Atorvastatin calcium, whose trade name is Lipitor, is an important cholesterol-lowering drug. Its (3R,5R)-6-cyano-3,5-dihydroxyhexanoic acid tert-butyl ester (molecular formula is C(CH3)3OCOCH2CH(OH)CH2CH(OH)CH2CN), with two chiral structures Hydroxyl is the key chiral intermediate in the synthesis of atorvastatin calcium. It has the activity of potently inhibiting hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase, blocking the reduction of HMG-CoA to hydroxyl The key binding site of mevalonic acid can greatly reduce cholesterol and effectively improve atherosclerosis. There are many ways to synthesize the key chiral intermediate of atorvastatin calcium. The common methods mainly include chemical method and enzymatic method. Compared with the chemical method, the enzymatic method has mi...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/04C12N15/70C12N1/21C12P7/62C12P7/02C12R1/19
CPCC12N9/0006C12P7/02C12P7/62C12Y101/01184
Inventor 陈本顺常斌陈峻青武涛石利平徐春涛刘靖朱伟伟杨莹陈晓佩
Owner 宿迁阿尔法科技有限公司
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