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Halohydrin dehalogenase mutant from parvibaculum lavamentivorans and application of halohydrin dehalogenase mutant

A technology of halohydrin dehalogenase and mutants, which is applied in the field of preparation of atorvastatin intermediate-4-cyano-3-hydroxybutyrate ethyl ester, can solve the problem of heavy drug burden for patients, high price, and Problems such as staying high

Active Publication Date: 2015-07-01
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This variety has been monopolized by foreign pharmaceutical giants for a long time, resulting in high prices
A piece of "Lipitor" costs as much as 10 yuan, and the burden of medication is heavy for patients

Method used

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  • Halohydrin dehalogenase mutant from parvibaculum lavamentivorans and application of halohydrin dehalogenase mutant
  • Halohydrin dehalogenase mutant from parvibaculum lavamentivorans and application of halohydrin dehalogenase mutant
  • Halohydrin dehalogenase mutant from parvibaculum lavamentivorans and application of halohydrin dehalogenase mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Construction of random mutation library of halohydrin dehalogenase

[0039] Random mutation technology reference (Current Protocols in Protein Science 26.6.1-26.6.10, 2011; Anal. Biochem. 2008, 375: 376-378) description. Primer P1 and primer P2 were designed according to GenBank ABS64560.1 gene sequence (see Table 1). First, an error-prone PCR experiment is performed on the parental HHDH-PL gene (the nucleotide sequence is shown in SEQ ID NO.1) with primers P1 and P2. 50μL error-prone PCR reaction system: 25μL 2×GoTaq Master Mix, 5 μL 50 mM MgCl 2 buffer, 5μL 1mM MnCl 2 , 0.5 μL P1 (50 μM) and P2 (50 μM), 1 μL (100-200 ng) HHDH-PL plasmid template, 13 μL deionized water. PCR program: pre-denaturation at 95°C for 3 minutes, 25 cycles: 94°C for 30s, 55°C for 30s, 72°C for 60s, and finally extension at 72°C for 10 minutes. After the PCR products were verified by agarose nucleic acid electrophoresis, the products were purified using PCR cleanup Kit. MEGAWH...

Embodiment 2

[0040] Example 2: Establishment of a high-throughput method for screening halohydrin dehalogenase mutants

[0041] Since the purpose of this experiment is to screen a halohydrin dehalogenase mutant that can efficiently catalyze (S)-CHBE to synthesize HN, establishing an efficient high-throughput screening method is a key step to improve the realization of this invention. The methods reported in the literature to detect the activity of halohydrin dehalogenases are based on protons and halide ions produced by the dehalogenation process, but cannot detect the ring-opening process, so these methods are not suitable for screening halohydrin dehalogenation of (S)-CHBE to HN enzyme. In order to establish a suitable screening method, the present invention has established a new high-throughput screening method based on azide detection, such as image 3 shown.

[0042] Pick a single colony clone (the mutant library constructed in Example 1) and culture it in a 2 mL deep 96-well plate ...

Embodiment 3

[0043] Example 3: Construction of double mutants F176M / A187R and F176M / A187S

[0044] The plasmid of mutant F176M was extracted, and mutation primers P3 and P4 (see Table 1) were designed for A187R mutation, and mutation primers P5 and P6 (see Table 1) were designed for A187S mutation. Mutation system 50μL: 10μL 5×PS buffer, 4μL dNTPMix (0.8mM), 1μL (100-200ng) F176M plasmid, 0.5μL PrimerStar, 1μL (50μM) mutation primer and 32.5μL deionized water. PCR program: 72°C for 10 min, 95°C for 5 min, 30 cycles: 98°C for 10 s, 55°C for 15 s, 72°C for 8 min and 72°C for 10 min. After PCR was positive by 0.9% agarose gel electrophoresis analysis, take 20 μL of PCR solution, add 1 μL DpnI, digest at 37°C for 2 hours to remove template plasmid DNA, inactivate at 65°C for 10 minutes, and transform into competent cells E.coli BL21(DE3) , Spread LB plates containing ampicillin (50 mg / L). A single colony was picked and sequenced to analyze that the mutants F176M / A187R and F176M / A187S were su...

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Abstract

The invention provides a halohydrin dehalogenase mutant from parvibaculum lavamentivorans and an application of the mutant in synthesizing atorvastatin midbody. A high-throughput screening technology is established to directionally deform halohydrin dehalogenase to obtain the mutant F176M / A187R, wherein the activity of the mutant F176M / A187R is improved by 3.11 times relative to wild type alohydrin dehalogenase. The mutant F176M / A187R performs whole-cell catalysis on 100g / L (S)-4-chlo-3-hydroxyl ethyl butyrate to synthesize (R)-4- cyanogroup-3-hydroxyl ethyl butyrate, wherein the molar yield is 86%.

Description

(1) Technical field [0001] The invention relates to the fields of molecular biology and biotechnology, in particular to a method for preparing halohydrin dehalogenase mutants using gene mutation technology, a high-throughput screening method for a library of halohydrin dehalogenase mutants, and the obtained mutation Body and its application in the preparation of atorvastatin intermediate (R)-4-cyano-3-hydroxybutyric acid ethyl ester. (2) Background technology [0002] Cardiovascular disease (CVD) is the leading cause of death worldwide. Cardiovascular diseases such as coronary heart disease, stroke, and hypertension have become the number one killer of the Chinese population. In the primary and secondary prevention of cardiovascular and cerebrovascular diseases, statins can effectively inhibit cholesterol synthesis and significantly reduce the incidence of cardiovascular and cerebrovascular diseases, and have become the basic drugs for the treatment of atherosclerotic diseas...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12P7/62C12R1/01
CPCC12N9/88C12P7/62
Inventor 柳志强郑裕国万南微沈寅初
Owner ZHEJIANG UNIV OF TECH
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