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Method for utilizing halohydrin dehalogenase engineering fungi to prepare R-phenyl glycidyl ether

A technology of phenyl glycidyl ether and halohydrin dehalogenase is applied in the field of preparation of chiral drug intermediate R-phenyl glycidyl ether, and can solve the problem of few chiral epoxides, wild-type whole cells or recombinant halohydrin Dehalogenase activity or low enantioselectivity, etc., to achieve the effect of environmental friendliness, good enantioselectivity, and high catalytic activity

Active Publication Date: 2018-04-06
YANCHENG INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, so far, few reports have been reported on enzymatic resolution of chiral epoxides by halohydrin dehalogenases.
This is mainly due to the lower activity or enantioselectivity of wild-type whole-cell or recombinant halohydrin dehalogenases
On the other hand, it has not been reported that halohydrin dehalogenase preferentially catalyzes the hydrolysis of (S)-aryl glycidyl ether

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Synthesis of Halohydrin Dehalogenase Gene

[0041] Synthesize the halohydrin dehalogenase gene sequence HHDH shown in SEQ ID NO.2 with a total synthesis method through the conventional operation of genetic engineering Ab , the amino acid sequence of the halohydrin dehalogenase encoded by the gene is shown in SEQ ID NO.1.

Embodiment 2

[0043] Construction of Halohydrin Dehalogenase Gene Expression Vector and Its Recombinant Transformant

[0044] The synthesized halohydrin dehalogenase gene and pET28a were both digested with NcoI and XhoI respectively. After digestion for about 5 hours, the digested products were recovered and ligated with T4 ligase at 16°C for 16 hours to obtain the recombinant expression plasmid pET28a- HHDH Ab .

[0045] The expression vector pET28a-HHDH Ab Transformed into E.coli BL21 (DE3) recipient bacteria, spread on LB agar plate containing kanamycin (final concentration 50 mg / L), and cultured overnight at 37°C, colonies grew on the plate.

[0046] Randomly pick a single clone, extract the plasmid for sequencing after culture, and the sequencing results show that a positive clone E.coliBL21(DE3) / pET28b-HHDH was obtained Ab .

Embodiment 3

[0048] Expression of halohydrin dehalogenase

[0049] The recombinant transformant E.coli BL21(DE3) / pET28a-HHDH obtained in Example 2 Ab Inoculate into LB liquid medium, culture at 37°C, 150rpm for 10-12h;

[0050]Then inoculate to the LB liquid culture medium that contains kanamycin (50mg / L of final concentration) by volume concentration 2% inoculum size and carry out expansion culture, 37 ℃, 150rpm cultivates to the OD of culture fluid 600 Between 0.6-0.8, add IPTG to a final concentration of 0.1mM, induce culture at 28°C, 180rpm for 10h, collect the cells by centrifugation of the culture solution, wash twice with normal saline to obtain wet cells.

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PUM

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Abstract

The invention discloses a method for utilizing halohydrin dehalogenase engineering fungi to prepare R-phenyl glycidyl ether, and relates to the field of preparing chiral drug midbody R-phenyl glycidylether. By means of the method for utilizing the halohydrin dehalogenase engineering fungi to prepare R-phenyl glycidyl ether, an engineering fungus thalli containing a halohydrin dehalogenas gene isused as a catalyst, a phenyl glycidyl ether substrate is catalyzed and converted to obtain R-phenyl glycidyl ether, and the method has high enantiomer selectivity and a very high catalysis activity and is environmentally friendly in the producing process.

Description

technical field [0001] The invention relates to the field of preparation of chiral drug intermediate R-phenyl glycidyl ether, in particular to a method for preparing R-phenyl glycidyl ether by using halohydrin dehalogenase engineering bacteria. Background technique [0002] Chiral epoxides are very important chiral synthetic building blocks in organic synthesis, which contain at least one chiral carbon atom, and many important biologically active compounds can be synthesized by selective ring opening. Optically pure aryl glycidyl ethers are important intermediates for the synthesis of chiral aminoalcohols and β-blockers. Therefore, the synthesis of optically active epoxides has become a research hotspot today. [0003] Halohydrin dehalogenase (HHDH for short), also known as halohydrin epoxidase or halohydrin-hydrogen halide lyase, plays a vital role in the degradation of organic halides, which can be metabolized by microorganisms get. HHDH can catalyze the dehalogenation ...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N1/21C12P17/02C12R1/19
CPCC12N9/88C12P17/02C12Y405/01
Inventor 薛峰亚香菊修元松丁鸽高健
Owner YANCHENG INST OF TECH
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