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Halohydrin dehalogenase mutant derived from ADI (Agrobacterium radiobacter) and application of halohydrin dehalogenase mutant to preparation of (S)-epichlorohydrin

A halohydrin dehalogenase, epichlorohydrin technology, applied in the application, halocarbon lyase, enzyme and other directions, can solve the stereoselectivity and reaction system obstacles of halohydrin dehalogenase, cannot achieve optical purity, cannot satisfy Industrial application and other issues, to achieve the effect of easy control of the operation process, good industrial application value, and low production cost

Active Publication Date: 2019-04-09
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Limited by the stereoselectivity and reaction system barriers of halohydrin dehalogenase, the chiral ECH products prepared by the current halohydrin dehalogenase method cannot reach optical purity and cannot meet the requirements of industrial applications

Method used

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  • Halohydrin dehalogenase mutant derived from ADI (Agrobacterium radiobacter) and application of halohydrin dehalogenase mutant to preparation of (S)-epichlorohydrin
  • Halohydrin dehalogenase mutant derived from ADI (Agrobacterium radiobacter) and application of halohydrin dehalogenase mutant to preparation of (S)-epichlorohydrin
  • Halohydrin dehalogenase mutant derived from ADI (Agrobacterium radiobacter) and application of halohydrin dehalogenase mutant to preparation of (S)-epichlorohydrin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Construction of Halohydrin Dehalogenase Site-Directed Saturation Mutation Library

[0032] Primers were designed according to the GenBank AAK92099 gene sequence (see Table 1). Use primers I81X-F and I81X-R, F86X-F and F86X-R, V94X-F and V94X-R, respectively, to carry out site-directed saturation mutation on the parental HheC gene (nucleotide sequence shown in SEQ ID NO.1) experiment. 50 μL PCR reaction system: 25 μL 2×Phanta Max Buffer, 1 μL dNTP, 1 μL P1 (50 μM) and P2 (50 μM), 2 μL (100-200ng) parental plasmid template, 1 μL Phanta Max Super-Fidelity DNApolymerase, 19 μL deionized water.

[0033] PCR program: pre-denaturation at 95°C for 3min, 30 cycles: 95°C for 15s, holding at a suitable annealing temperature for 15s, 72°C for 3min30s, and finally extending at 72°C for 10min.

[0034] After the PCR products were verified by agarose nucleic acid electrophoresis, the products were purified using PCR cleanup Kit. After PCR was positive by 0.9% agarose gel...

Embodiment 2

[0035] Example 2: Screening of Halohydrin Dehalogenase Single-point Saturation Mutation Library

[0036] Pick a single colony clone (the mutant library constructed in Example 1) and culture it in a 2 mL deep 96-well plate containing 1 mL LB and 50 μg / mL kana resistance. At the same time, three parents were selected as controls. A 2 mL deep 96-well plate was cultured at 37°C for 5 hours, then 100 μL of the bacterial solution was transferred to another sterile 2 mL 96-well plate, and 100 μL of 30% (wt / vol) sterile glycerol was added to it. Add 100 μL of LB medium containing 50 μg / mL kana-resistance and 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) to the remaining 900 μL bacterial liquid, and place at 28°C for induction for 12-14 hours. The induced strain was centrifuged at 3,000×g and 4°C for 30 min, the supernatant was discarded, and the bacterial cells were collected. 500 μL of reaction system (200 mM phosphate buffer, 1.3-dichloro-2-propanol) was added to each well, the ...

Embodiment 3

[0038] Example 3: Construction of Halohydrin Dehalogenase Iterative Saturation Mutation Library

[0039] Primers were designed according to the GenBank AAK92099 gene sequence (see Table 1). Use primers F86X-F and F86X-R, V94X-F and V94X-R, respectively, and use the 81st position preferred mutant plasmid as a template to carry out iterative saturation mutations at positions 86 and 94. 50 μL PCR reaction system: 25 μL 2 ×Phanta Max Buffer, 1 μL dNTP, 1 μL P1 (50 μM) and P2 (50 μM), 2 μL (100-200ng) parental plasmid template, 1 μL Phanta Max Super-Fidelity DNA polymerase, 19 μL deionized water. PCR program: pre-denaturation at 95°C for 3min, 30 cycles: 95°C for 15s, holding at a suitable annealing temperature for 15s, 72°C for 3min30s, and finally extending at 72°C for 10min. After the PCR products were verified by agarose nucleic acid electrophoresis, the products were purified using PCR cleanupKit. After the PCR was positive by 0.9% agarose gel electrophoresis analysis, take ...

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Abstract

The invention relates to a halohydrin dehalogenase mutant derived from ADI (Agrobacterium radiobacter) and an application of the halohydrin dehalogenase mutant to preparation of (S)-epichlorohydrin. The mutation site is one of the following: (1) 81st site, (2) 86th site and (3) 94th site. A plurality of mutant strains are obtained by modification of halohydrin dehalogenase, (S)-ECH prepared from 1,3-dichloro-2-propanol by conversion has extremely high stereoselectivity. In a phosphate buffer system, (S)-ECH with optical purity (e.e. value) larger than 99% can be prepared by catalysis. The technology has outstanding advantages of excellent enzyme stereoselectivity, simple reaction system, easy-to-control operation process, low production cost and the like, and has good industrial application value.

Description

(1) Technical field [0001] The invention relates to a halohydrin dehalogenase mutant derived from Agrobacterium radiata and its application in preparing (S)-epichlorohydrin. (2) Background technology [0002] Chiral epichlorohydrin (ECH) is an important three-carbon synthon, which has important applications in the preparation of chiral drugs; among them, the lipid-lowering drugs atorvastatin, aryloxypropanol β-adrenergic blocker Many drugs, such as atenolol, weight-loss drug L-carnitine, angina pectoris drug metoprolol, and antibiotic algazoline, all require (S)-epichlorohydrin as a chiral intermediate to complete chiral drugs. The preparation of (S)-ECH has a wide market demand. The traditional (S)-ECH chemical synthesis process starts from racemic epichlorohydrin, and the Salan reagent catalyzes the hydrolysis of racemic epichlorohydrin to obtain an e.e. value of 99%. However, the production of (S)-ECH The rate is 43%, and its catalyst cost is extremely high, and the env...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12P17/02
CPCC12N9/88C12P17/02C12Y405/01
Inventor 柳志强张晓健郑裕国邓涵中
Owner ZHEJIANG UNIV OF TECH
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