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Acyltransferase for catalytic synthesis of simvastatin and mutant thereof

An acyltransferase and simvastatin technology, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of complicated product purification steps and low selectivity, and achieves the effects of short conversion time, high activity and friendly reaction environment.

Active Publication Date: 2017-10-20
杭州泓友医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The biotransformation method currently uses the existing commercial esterase to catalyze the reaction with low regioselectivity, which still causes problems such as complicated purification steps of the reaction product

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Screening of acyltransferases

[0032] Using the reported acyltransferase LovD (derived from A. terreus) as a template, BLAST alignment was performed on NCBI, and the sequences with a similarity between 40-70% were selected for gene synthesis and induced expression, and used in the Enzymatic screening of vastatin. The screening system is as follows: Reaction system (substrate concentration 1g / L)

[0033] An enzyme that can be used for the enzymatic synthesis of simvastatin was successfully screened. The enzyme is an unknown protein derived from fungal sp. NO.14919. The encoded nucleotide sequence is shown in SEQ NO: 2, and the encoded amino acid sequence As shown in SEQ NO:1.

[0034] However, the reactivity of the enzyme is low, and directed evolution is required to meet the needs of industrial applications.

Embodiment 2

[0035] Embodiment 2, establishment of mutant library

[0036] Using the expression plasmid synthesized by the whole gene (synthesized by Changzhou Jiyu Biotechnology Co., Ltd., the amino acid sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 2) was cloned on the plasmid, and the Primers F1 and R1 are forward and reverse primers respectively, and error-prone PCR is performed to construct a mutant library.

[0037] The primer sequences are as follows: F1: 5'-GGAATTCCATATGCAGGATATCGAAC-3'; R1: 5'-CCCAAGCTTTCAGTCGTTGCGCAGT-3', with Nde I and Hind III Restriction site.

[0038] The error-prone PCR reaction system is as follows: 10* PCR buffer 2.5μL, 10mM dGTP 0.5μL, 10mM dTTP 0.5μL, 10mM dCTP 2.5μL, 10mM dATP 2.5μL, 1mM MnCl 2 2.5 μL, 55 mM MgCl 2 2.5 μL, 10 μM F11 μL, 10 μM R1 1 μL, Template (10ng / μL) 1 μL, Taq DNA polymerase (5U / μL, takara) 0.2 μL, ddH 2 O to make up to 25 μL.

[0039] Error-prone PCR was performed on a Bio-Rad T100 thermal c...

Embodiment 3

[0041] Example 3 Activation and Induced Expression of Mutant Library

[0042]Pick a single clone from the plate cultured overnight to a 96-deep well plate filled with 1 mL of LB liquid medium with a final concentration of 50 μg / mL kanamycin sulfate, and culture overnight at 37°C and 220 rpm with shaking. On the next day, draw 100 μL of the culture solution from the 96 empty plates of the above overnight culture, and add it to a fresh 1 mL 96 deep-well plate containing LB liquid medium with a final concentration of 50 μg / mL kanamycin sulfate, at 37 ° C, 220 rpm After 4 hours of shaking culture, IPTG with a final concentration of 1 mM was added for induction, and then culture was continued at 30° C. for 20 hours. A total of 400 single clones were picked for activity screening.

[0043] Centrifuge the induced bacterial solution at 4°C and 4000 rpm for 15 min, and discard the supernatant. The cells were suspended in 100 μL of 50 mM sodium phosphate buffer (pH 7.0), added with ly...

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PUM

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Abstract

The invention provides acyltransferase for catalytic synthesis of simvastatin and a preparation method thereof. The acyltransferase has an amino acid sequence as shown in SEQ ID No. 2, and the site 161, site 163, site 180 and site 235 of the amino acid sequence undergo mutation so as to obtain a mutant. The enzyme activity of the novel acyltransferase in the invention is 12 times the enzyme activity of a wild enzyme; and the acyltransferase provided by the invention has the characteristics of low cost, short conversion time, simple operation, etc. and has good large-scale industrial application prospects.

Description

technical field [0001] The invention belongs to the field of genetic engineering and enzyme engineering, and relates to an acyltransferase for catalyzing the synthesis of simvastatin and a mutant thereof. Background technique [0002] Simvastatin, trade name Zocor (Zocor), English name Simvastatin, simvastatin is a statin (statin) blood lipid-lowering drug that inhibits hydroxymethylglutaryl coenzyme A (HMG-COA) reductase Agent, inhibits the synthesis of endogenous cholesterol, and is a blood lipid regulator. Literature data show that it can reduce the content of total cholesterol (TC) in serum, liver and aorta of rabbits with hyperlipidemia, reduce very low-density lipoprotein cholesterol (VLDL-C), low-density lipoprotein cholesterol (LDL-C) level effect. It is clinically used to treat hypercholesterolemia, coronary heart disease, control the content of protein alcohol in the blood and prevent vascular diseases. [0003] Simvastatin was developed by Merck of the United S...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12P17/06
CPCC12N9/1025C12N15/70C12P17/06
Inventor 祝俊黄科学吴锋余玉奎巫佳张晨晨刘双喜邢小飞徐飞
Owner 杭州泓友医药科技有限公司
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