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Carbonyl reductase genetically-engineered bacteria immobilized cells and application thereof

A technology of genetically engineered bacteria and immobilized cells, applied to methods based on microorganisms, biochemical equipment and methods fixed on or in inorganic carriers, can solve the problems of low substrate concentration, poor stability, and resistance to organic solvents. Poor acceptability and other problems, to achieve the effects of high product yield and purity, reduction of "three wastes" emissions, and simplification of process steps

Active Publication Date: 2018-02-02
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are few studies on the synthesis of immobilized cell / enzyme catalyzed chiral intermediates of statins at home and abroad. The related researches have low recovery rate of total enzyme activity, high preparation cost of immobilized enzyme, unsatisfactory catalytic activity, poor stability, low However, problems such as low concentration of the compound and poor tolerance to organic solvents seriously limit the application of immobilized biocatalysts in the key intermediates of statin drugs (3R,5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester and (3R ,5R)-6-cyano-3,5-dihydroxyhexanoic acid tert-butyl ester production

Method used

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  • Carbonyl reductase genetically-engineered bacteria immobilized cells and application thereof
  • Carbonyl reductase genetically-engineered bacteria immobilized cells and application thereof
  • Carbonyl reductase genetically-engineered bacteria immobilized cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: Activated carbon pretreatment

[0025] Activated carbon (purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd., pharmaceutical grade) was sieved through a 40-mesh sieve. Weigh 10 g of sieved activated carbon and add it to 100 mL of 1M hydrochloric acid, and stir at 50°C for 1 h. Suction filtration, wash the activated carbon with distilled water and rinse until the filtrate is close to neutral (pH value is 6.9-7.1), put the activated carbon into an oven to dry, that is, 10 g of pretreated activated carbon, and store it for later use.

Embodiment 2

[0026] Embodiment 2: the cultivation of carbonyl reductase genetic engineering bacteria cell

[0027] (1) Construction of carbonyl reductase genetically engineered bacteria:

[0028] Primer 1 (CCG CATATG ACTGATCGTTTAAAAG), Primer 2 (TTG CTCGAG TTATTGAGCAGTGTATCC), and Nde I and Xho I restriction enzyme sites (underlined) were introduced into primer 1 and primer 2, respectively. Using the genomic DNA of Lactobacillus parabuchneri as a template, under the priming of primer 1 and primer 2, high-fidelity Pfu DNA polymerase was used for PCR amplification to obtain the carbonyl reductase CR1 gene sequence, which was sequenced using Nde The amplified fragment was treated with I and Xho I restriction enzymes (TaKaRa) and ligated with the commercial vector pET28b (Invitrogen) treated with the same restriction enzymes using T4 DNA ligase (TaKaRa) , to construct the expression vector pET28b-CR1 ( Liu Z-Q, et al. Biotechnology Progress, 2017, DOI 10.1002 / btpr.2460. )( image 3 ). ...

Embodiment 3

[0030] Example 3: Immobilization of Carbonyl Reductase Genetically Engineered Bacteria Cells

[0031] Potassium phosphate salt (K 2 HPO 4 -KH 2 PO 4 ) buffer solution (molar concentration is 100mM), weigh 100g of the carbonyl reductase genetically engineered bacterium wet thallus prepared by the method in Example 2 and add it to 1L, pH 7.0, 100mM potassium phosphate buffer solution to obtain 1L of bacterial suspension. Accurately weigh 18g (dry weight) of activated carbon (18g / L) pretreated in Example 1 and add it to 1L of bacterial suspension for mixing. At room temperature (20-30°C) and 500rpm, stir and adsorb with a water bath stirring paddle for 30min; Then add 40mL of 5% polyethyleneimine aqueous solution (according to 4% of the total system volume 1L), at room temperature, 500rpm, stir and cross-link with a water bath stirring blade for 1h; then add 30mL of 50% glutaraldehyde aqueous solution (according to 3% of the total system volume 1L added), at room temperature,...

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Abstract

The invention provides a method of using immobilized Escherichia coli engineered bacterial cells to catalytically synthesize statin drug chiral intermediates, tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate and (3R,5R)-tert-butyl-6-cyano-3,5-dihydroxyhexanoate. The method comprises: culturing carbonyl reductase-containing Escherichia coli engineered bacteria, preparing immobilized cells, and synthesizing the novel drug chiral intermediates, tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate and (3R,5R)-tert-butyl-6-cyano-3,5-dihydroxyhexanoate by catalysis of the immobilized cells. The immobilized biological Escherichia coli engineered bacterial cells are used in the method as the catalyst, the stability is good, the service life is long, tolerance to organic solvents is good, the catalyst is reusable, adding expensive external coenzymes is not required, the catalyst is capable of catalyzing the synthesis of statin drug intermediates in an organic phase reaction system, the productsare high in yield and purity, the production cost can be greatly reduced, the process steps can be simplified, emission of three wastes is reduced, and the method is very worthy of application in theindustrial production of statin drug chiral intermediates.

Description

(1) Technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for immobilizing microbial cells containing carbonyl reductase and using immobilized cells as catalysts to synthesize statin intermediates (3R,5S)-6-chloro-3,5- Methods for tert-butyl dihydroxyhexanoate and (3R,5R)-6-cyano-3,5-dihydroxyhexanoate tert-butyl. (2) Background technology [0002] Since the beginning of the new century, cardiovascular and cerebrovascular diseases have become the primary diseases that endanger human life and health in my country and even in the world. Statins can effectively reduce blood lipid levels and prevent the occurrence of atherosclerosis and coronary heart disease by inhibiting HMG-CoA reductase. They are important basic drugs for the prevention and treatment of cardiovascular and cerebrovascular diseases at home and abroad. (3R,5S)-6-Chloro-3,5-dihydroxyhexanoic acid tert-butyl ester and (3R,5R)-6-cyano-3,5-dihy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12P7/62C12P13/00C12R1/19
CPCC12N11/14C12P7/62C12P13/004
Inventor 柳志强郑裕国张晓健姚丹凯王亚军郑玲王文重
Owner ZHEJIANG UNIV OF TECH
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