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Carbonyl reductase, mutant and application of carbonyl reductase to preparation of statin synthetic intermediate

A technology of carbonyl reductase and reductase, which is applied in the direction of oxidoreductase, application, genetic engineering, etc., can solve the problems of increased cost, low catalytic activity, poor substrate tolerance, etc.

Active Publication Date: 2017-07-07
EAST CHINA UNIV OF SCI & TECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the above reports, although KRED from Codexis has the advantages of high catalytic efficiency and stereoselectivity, it only has a conversion rate of 98%, and the remaining unreacted substrate increases the cost of subsequent separation and purification of the product, making the biological The catalytic synthesis process is not economical and efficient enough. The reactions catalyzed by whole cells of wild bacteria or carbonyl reductases have problems such as low catalytic activity, poor substrate tolerance, and long reaction time, which are far from the requirements of industrial applications. Therefore, it is necessary to explore A new type of carbonyl reductase with better performance to develop an economical, efficient and environmentally friendly biocatalytic synthesis process

Method used

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  • Carbonyl reductase, mutant and application of carbonyl reductase to preparation of statin synthetic intermediate
  • Carbonyl reductase, mutant and application of carbonyl reductase to preparation of statin synthetic intermediate
  • Carbonyl reductase, mutant and application of carbonyl reductase to preparation of statin synthetic intermediate

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Embodiment 1

[0069] The gene cloning of embodiment 1 carbonyl reductase LbCR

[0070] According to the open reading frame of carbonyl reductase LbCR, design upstream and downstream primers as follows:

[0071] The upstream primer, whose sequence is shown in SEQ ID No.3, specifically:

[0072] 5'-CGC GGATCC ATGACAGATCGTTTGAAAGATAAAGTGGCA-3';

[0073] The downstream primer, whose sequence is shown in SEQ ID No.4, is specifically:

[0074] 5'-CCC AAGCTT TTAAGCGCGTTGACCACCGTCAACCGTA-3';

[0075] Wherein, the underlined part of the upstream primer is the BamHI restriction site, and the underlined part of the downstream primer is the HindIII restriction site.

[0076] The genomic DNA of Lactobacillus brevis CGMCC 1.3258 was used as a template for PCR amplification. PCR system: 2×Taq PCR MasterMix 25μl, upstream primer and downstream primer (10ng / μl) each 2.5μl, genomic DNA (100ng / μl) 1μl and ddH 2 O 19 μl. The PCR amplification program was as follows: 32 cycles of denaturation at 95°C...

Embodiment 2

[0077] Example 2 Preparation of Carbonyl Reductase LbCR Recombinant Expression Plasmid and Recombinant Expression Transformant

[0078] The carbonyl reductase target DNA fragment obtained by PCR amplification in Example 1 and pET 28a empty plasmid were double-digested overnight with restriction endonucleases BamHI and HindIII at the same time, then purified by agarose gel electrophoresis and recovered with a DNA kit. Ligate the recovered target fragment and the empty vector under the action of T4 DNA ligase at 4°C for 12 hours to obtain the recombinant plasmid pET28a-LbCR. The recombinant expression vector construction strategy of the gene is as follows: figure 1 shown.

[0079] Transform the resulting recombinant plasmid into E.coli DH5α, spread it on an LB medium plate containing 50 μg / ml kanamycin, and incubate it at 37°C for 8 hours. The grown colonies are verified by colony PCR and successfully amplified. Positive clones with a target band of about 750bp in length. Aft...

Embodiment 3

[0080] Example 3 Carbonyl reductase LbCR mutant construction

[0081] CASTing technology was used to construct the mutation library of carbonyl reductase LbCR: select non-conserved residues in the substrate binding pocket of LbCR for combinatorial saturation mutation, use degenerate codon NDT to design mutation primers, use pET28a-LbCR as a template, and use high-fidelity polymerization Enzyme PrimeSTAR for PCR. The PCR reaction conditions are as follows: to a PCR reaction system with a total volume of 20 μL, add 0.5–20 ng of template, 10 μL of 2×PrimeSTAR (Premix), 0.4 μL (10 μM) of each pair of mutant primers, and add sterilized distilled water to 20 μL. PCR reaction program: (1) denaturation at 98°C for 10 sec, (2) annealing at 55°C for 5 sec, (3) extension at 72°C for 60 sec, steps (1) to (3) were performed for 30 cycles in total, and the product was stored at 4°C. After the PCR product was verified by agarose gel electrophoresis analysis, the restriction enzyme DpnI was ...

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Abstract

The invention relates to carbonyl reductase derived from lactobacillus brevis CGMCC 1.3258, an activity-improved mutant of the carbonyl reductase, a gene of the carbonyl reductase, a recombinant expression vector and a recombinant expression transformant containing the gene, as well as application of the recombinant carbonyl reductase or the recombinant expression transformant serving as a catalyst to catalysis of asymmetric reduction preparation of a chiral hydroxyl compound, particularly application in biological catalytic synthesis aspect of an atorvastatin chiral intermediate 6-cyan-(3R,5R)-dihydroxyhexanoate. Compared with other methods for performing asymmetric reduction preparation on the 6-cyan-(3R,5R)-dihydroxyhexanoate, the invention has the advantages of high substrate concentration, mild reaction condition, environmental friendliness, high yield, high optical purity of products and the like, so good application prospect in the industrialized production of atorvastatin chiral side chains is achieved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a carbonyl reductase derived from Lactobacillus brevis CGMCC 1.3258 and a mutant with improved activity thereof, its gene, a recombinant expression vector containing the gene and a recombinant expression transformant, And the application of using the recombinant carbonyl reductase or recombinant expression transformant as a catalyst to catalyze asymmetric reduction to prepare chiral hydroxyl compounds, especially the atorvastatin chiral intermediate 6-cyano-(3R,5R)-dihydroxyhexyl Applications in the biocatalytic synthesis of tert-butyl esters. Background technique [0002] Atorvastatin is a kind of statin drugs, mainly used for the treatment of hypercholesterolemia and coronary heart disease, the reaction mechanism is to reduce the low density Lipoprotein (LDL) and triglyceride levels. Pfizer's drug Lipitor, whose active ingredient is atorvastatin, had annual sales of mo...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/70C12P7/02C12P13/00
Inventor 郑高伟宫绪敏许建和潘江钱小龙
Owner EAST CHINA UNIV OF SCI & TECH
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