An improved ketoreductase and its application

A reductase and mutant technology, applied in the field of biocatalytic synthesis, can solve the problems of large enzyme consumption, unfavorable scale-up production, and inability to use on a large scale

Active Publication Date: 2021-05-04
天津迪嘉医药技术开发有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] He Qinting Catalyzes 5-((4S)-2-oxo-4-phenyl(1,3-oxazolidin-3-yl))-1-(4-fluoro Synthesis of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-dione from phenyl)pentane-1,5-dione In oxazolane-2-one, although the conversion rate and product purity are high, the substrate concentration is low, which is not conducive to scale-up production, and the production cost is also high
In the Escherichia coli whole-cell catalytic process used by Zheng Guojun, the substrate conversion rate is low and cannot be used on a large scale
The catalytic effect of Emily Mudever's mutant enzyme has been improved, but there are still problems such as large amount of enzyme used and insufficient conversion efficiency, resulting in (4S)-3-[(5S)-5-(4-fluorophenyl) The enzymatic production of -5-hydroxypentanoyl]-4-phenyl-1,3-oxazolan-2-one is expensive

Method used

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  • An improved ketoreductase and its application
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  • An improved ketoreductase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020]Example 1: Obtaining the recombinant plasmid of the wild-type ketoreductase parent of the T.brockii bacterial strain

[0021] The ketoreductase maternal coding gene of T.brockii strain (GenBank: X64841.1) was obtained through the NCBI Benbank nucleic acid database, and the codons and restriction sites were optimized, and a service provider was entrusted to artificially synthesize the full-length gene and restriction sites at both ends point, and pET28a (+) expression plasmid were digested respectively, gel cutting recovery, connection and transformation Escherichia coli BL21 (DE3) competent cells were spread on the LB agar plate containing 50mg / L kanamycin sulfate, 37 Cultivate overnight. Select several single colonies to LB medium (containing 50mg / L kanamycin sulfate), culture overnight at 37°C, and use a plasmid extraction kit to extract recombinant plasmids, perform PCR and sequencing verification, and obtain the recombination of the positive ketoreductase parent pla...

Embodiment 2

[0022] Example 2: Random mutation of the wild-type ketoreductase parental gene

[0023] According to the content described in Example 1, using the recombinant plasmid containing the wild-type ketoreductase female coding gene of the T.brockii strain as a template, and using Primer 5.0 to design and synthesize two ketoreductase female coding genes according to the T.brockii strain end primers (Table 1), using error-prone PCR technology (see Table 2 for materials and concentrations, and Table 3 for reaction conditions) to obtain linear gene fragments containing a large number of base mutations, these PCR products and pET28a(+) expression plasmids were respectively Enzyme digestion, gel recovery, connection and transformation of Escherichia coli BL21 (DE3) competent cells, spread on LB agar plates containing 50 mg / L kanamycin sulfate, and culture overnight at 37°C.

[0024] Table 1 Random Mutagenesis Primer Sequence

[0025]

[0026] Table 2 50μL error-prone PCR material syste...

Embodiment 3

[0030] Example 3: Cloning and expression of ketoreductase mutants

[0031] In order to facilitate the cloning, expression and identification of ketoreductase mutants, compatible restriction endonuclease sites were designed at the 5' and 3' ends of the gene, and Nde I and Xho I restriction enzymes can be used to separate the target The gene and pET28a(+) (other expression plasmids that can express proteins in E. coli can also be used) are digested and DNA gel is recovered at the same time, and the recovered target gene and the larger fragment of the plasmid are ligated with T4 DNA ligase For the reaction, the ligation product was transformed into Escherichia coli BL21 (DE3) competent cells, and then the transformed competent cells were spread on LB agar plates containing 50 mg / L kanamycin sulfate, and cultured overnight at 37°C.

[0032] Pick a single colony grown on the above-mentioned petri dish and inoculate it in LB liquid medium containing 50mg / L kanamycin sulfate, culture...

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Abstract

The invention relates to an improved ketoreductase and belongs to the technical field of biocatalytic synthesis. The ketoreductase mutant of the present invention, the amino acid sequence of the ketoreductase mutant is an amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 1 is mutated, and the mutation sites of the mutated amino acid sequence are respectively: position 140 I is mutated to S, and the optional mutation points include at least one of the following points: the 205th D mutation to E, the 125th H mutation to Q, the 329th N mutation to S, and the 294th L mutation to I. The 150th D is mutated to Q, and the 36th P is mutated to S. The invention provides a ketoreductase and application thereof, which can effectively reduce (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1 , 3-oxazolane-2-one and the cost of industrial production of ezetimibe.

Description

technical field [0001] The invention belongs to the technical field of biocatalytic synthesis, in particular, relates to an improved ketoreductase and its use in the preparation of ezetimibe chiral intermediate (4S)-3-[(5S)-5-(4-fluoro phenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolane-2-one method. Background technique [0002] Ezetimibe, chemical name: (3R,4S)-1-(4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3-hydroxypropyl ]-4-(4-hydroxyphenyl)-2-azetidinone is the first cholesterol selective absorption inhibitor drug jointly developed by Schering-Plough and Merck and launched in 2002. The trade name is Ezetrol (Yi Shi Chun). (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolane-2-one as The key chiral intermediate in the synthesis of wheat cloth raw materials, the most advantageous preparation process reported so far is the use of carbonyl reductase to catalyze 5-((4S)-2-oxo-4-phenyl(1,3 -Oxazolidin-3-yl))-1-(4-fluorophenyl)pentane-1,5-dione can be ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12P17/14
CPCC12N9/0004C12P17/14
Inventor 夏俊刚金宁
Owner 天津迪嘉医药技术开发有限公司
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