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Enzymatic method for preparing statins intermediates

A compound and statin technology, which is applied in the field of enzymatic preparation of statin compound intermediates, can solve the problems of large amount of enzyme used, affecting extraction yield, high production cost, etc.

Active Publication Date: 2015-08-12
SHANGHAI RES & DEV CENT OF INDAL BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, the intermediates of atorvastatin are usually synthesized by chemical methods. Because this method has the disadvantages of high production cost and large pollution, enzyme-chemical methods have recently been used to replace this chemical method.
However, the enzymes currently used generally have low catalytic activity, resulting in a large amount of enzyme used in the catalytic reaction, affecting the extraction yield, and the production cost is still high

Method used

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  • Enzymatic method for preparing statins intermediates
  • Enzymatic method for preparing statins intermediates
  • Enzymatic method for preparing statins intermediates

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Effect test

preparation example Construction

[0097] Preparation of formula I compound and statin compound

[0098] As used herein, the structural formula of the compound of formula I is It is the most important intermediate of statins and is usually synthesized by chemical methods.

[0099] The present invention provides a kind of method of biosynthetic formula I compound, specifically, described method comprises the steps: (a) in the regeneration system of coenzyme factor (as NADPH) and coenzyme factor (as glucose dehydrogenase GDH and glucose) In the presence of alcohol dehydrogenase CmADH3, the compound of formula III is catalytically reduced to generate the compound of formula II; (b) the compound of formula II is reacted with 2,2-dimethoxypropane to form the compound of formula I. The compound of formula I obtained by this method has very high purity, the content of diastereomer is only 0.0106%, and the content of enantiomer is only 0.01%. Wherein, R of the compounds of formula I, formula II, and formula III is a...

Embodiment 1

[0124] Construction of embodiment 1 CmADH3 expression strain

[0125] For the protein sequence of alcohol dehydrogenase CmADH3 derived from Candida magnoliae (NCBI accession number: ABB91667), the codons suitable for expression in Escherichia coli were optimized, and the gene sequence was synthesized completely, and enzymes were designed at both ends NdeI and BamHI sites were cut, and subcloned into the corresponding sites on the vector pET24a (purchased from Novagen) to obtain the recombinant plasmid pET24a-CmADH3.

[0126] The constructed recombinant plasmid pET24a-CmADH3 was transformed into Escherichia coli expression host BL21(DE3) by calcium chloride method to obtain BL21(DE3) / pET24a-CmADH3.

[0127] Codon-optimized gene sequence (SEQ ID NO.:3):

[0128]

[0129]

[0130] Protein sequence (SEQ ID NO.1):

[0131]

Embodiment 2

[0132] Example 2 Construction of KRED316 expression strain

[0133] According to the gene sequence of the reported carbonyl reductase mutant derived from Saccharomyces cerevisiae (Chinese patent application number: 200780036841.6, SEQ ID NO.315), the sequence was synthesized from the whole gene, and restriction sites NdeI and BamHI were designed at both ends, and It was subcloned into the corresponding site on the vector pET24a (purchased from Novagen) to obtain the recombinant plasmid pET24a-KRED316.

[0134] The constructed recombinant plasmid pET24a-KRED316 was transformed into Escherichia coli expression host BL21(DE3) by calcium chloride method to obtain BL21(DE3) / pET24a-KRED316.

[0135] The gene sequence is as follows:

[0136]

[0137]

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Abstract

The invention discloses a method for preparing statins intermediates through biosynthesis, and specifically discloses an enzymatic method for preparing statins intermediates. The enzymatic method comprises the following steps: (a) carrying out catalytic reduction reactions on a compound (III) in the presence of alcohol dehydrogenase CmADH3, co-enzyme factors, and a regeneration system of an optional co-enzyme factor to produce a compound (II); (b) reacting the compound (II) with 2,2-dimethoxy propane to generate a compound (I). The provided method can reduce the production cost and environmental pollution, and thus has a great application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for enzymatically preparing statin intermediates. Background technique [0002] Atorvastatin is a statin blood lipid regulating drug, which is an HMG-CoA reductase inhibitor. The hydrolyzate after oral absorption competitively inhibits the rate-limiting enzyme hydroxymethylglutaryl-CoA reductase in the process of cholesterol synthesis in vivo, thereby reducing the synthesis of cholesterol and increasing the synthesis of low-density lipoprotein receptors. Prevention and treatment of hypercholesterolemia, mixed hyperlipidemia, coronary heart disease and cerebral apoplexy. [0003] At present, the intermediates of atorvastatin are usually synthesized by chemical methods. Because this method has the disadvantages of high production cost and large pollution, enzyme-chemical methods have recently been used to replace this chemical method. However, the enzymes currently used gene...

Claims

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Application Information

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IPC IPC(8): C12P13/00C12N9/04C12N15/53C12N15/63C12N1/19C12N1/21C12R1/19
Inventor 陶荣盛孙梁栋朱傅赟杨晟蒋宇
Owner SHANGHAI RES & DEV CENT OF INDAL BIOTECH
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