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Halogenohydrin dehalogenase mutant strain, halogenohydrin dehalogenase mutant and preparation method and application thereof

A technology of halohydrin dehalogenase and mutant, applied in the field of microorganisms, can solve the problem of not finding industrialized production of halohydrin dehalogenase and the like

Active Publication Date: 2010-06-30
ANGELYEAST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, for the dehalogenation reaction of halohydrin dehalogenase in ortho-halohydrins, the applicant has not found relevant reports on the industrial production of halohydrin dehalogenase by genetic engineering methods in China

Method used

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  • Halogenohydrin dehalogenase mutant strain, halogenohydrin dehalogenase mutant and preparation method and application thereof
  • Halogenohydrin dehalogenase mutant strain, halogenohydrin dehalogenase mutant and preparation method and application thereof
  • Halogenohydrin dehalogenase mutant strain, halogenohydrin dehalogenase mutant and preparation method and application thereof

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preparation example Construction

[0027] The method for preparing a halohydrin dehalogenase mutant according to one embodiment of the present invention comprises the following steps:

[0028] A) carrying out shake flask fermentation according to the strain of the embodiment of the present invention;

[0029] B) seed tank fermentation;

[0030] C) commercial tank fermentation; and

[0031] D) Treating the bacterial cells obtained from commercial tank fermentation to obtain liquid halohydrin dehalogenase mutants.

[0032] The method for preparing a halohydrin dehalogenase mutant according to an embodiment of the present invention may further include:

[0033] E) drying the obtained liquid halohydrin dehalogenase mutant to obtain a solid halohydrin dehalogenase mutant.

[0034] Each step of the present invention is described in detail below.

[0035] 1. Construction process of genetically engineered bacteria:

[0036] 1. Extraction of Agrobacterium radiobacter AD1 genomic DNA:

[0037] 1) 5ml LB culture med...

Embodiment

[0132] I. the construction of genetically engineered bacteria of the present invention

[0133] Relying on large primer PCR technology, mutate the halide ion binding region (Pro175-Tyr187) of halohydrin dehalogenase, construct the mutant and vector pBAD, transform into the host MC1061 to construct a mutant library, and screen the mutant library , found that the effect of P175H and Y177W mutations is relatively ideal, the amino acid sequence of the mutant is SEQID NO.2, and a genetically engineered bacterium capable of producing the mutant was further obtained, and the preservation number of the genetically engineered bacterium is CCTCC NO: M208089.

[0134] II. Fermentation of genetically engineered bacteria

[0135] The bacterial strain of the present invention is fermented according to the following conditions.

[0136] A) Shake flask fermentation: take 5g yeast extract, 10g peptone, 10gNaCl, dissolve in 800ml distilled water, adjust the pH to 7, use distilled water to set ...

example 1

[0151] Example 1: Catalytic synthesis of (R)-4-cyano-3-hydroxybutyric acid ethyl ester

[0152] The reaction buffer (50mM Tris·H 2 SO 4 , pH: 8) heated to a constant temperature of 30°C; the substrate [(S)-4-chloro-3-hydroxybutyric acid ethyl ester] was added to the reaction buffer, and the content of the substrate in the aqueous solution was controlled to be 10g / l, Stir well. First dilute the 30% NaCN solution by 10-20 times, then adjust the pH of the NaCN solution to 8.0 with dilute sulfuric acid, add the diluted NaCN according to the molar ratio of the substrate and NaCN in a ratio of 1:2, and stir evenly; add the haloalcohol for dehalogenation The enzyme mutant (halohydrin dehalogenase mutant: (S)-4-chloro-3-hydroxybutyrate ethyl ester = 1:100 (w / w)) catalyzes the reaction, and the reaction is regulated by adding NaCN during the reaction Liquid pH. After reacting at 30°C for 24 hours, the catalytic efficiency reaches 97%, and the optical purity (ee) value of the target...

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Abstract

The invention provides a gene engineering bacteria for producing dehalogenase and realizes the industrial production of dehalogenase industrialised by culturing the gene engineering bacteria via fermentation technology. The invention also provides the use of the gene engineering bacteria; and the halogenohydrin dehalogenase produced by the gene engineering bacteria can be applied to ring-opening reaction of ortho-halogenohydrin dehalogenation epoxidation and epoxide.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a halohydrin dehalogenase gene mutant, the construction, production and application of a genetically engineered bacterium for producing the halohydrin dehalogenase mutant. Background technique [0002] Epoxides are recognized as one of the most important and widely used synthetic intermediates in organic synthesis. Such compounds are easy to prepare by chemical synthesis, but the stereoselectivity of the chemical reaction is poor. In the process of dehalogenation and epoxidation for the hydroxyl group and halogen linked to the chiral carbon, the chemical reaction generally has no stereoselectivity, although it can be used with the help of some chemical catalysts. Stereoselective reactions are carried out under the following conditions, but the general reaction conditions are harsh and the results are not ideal. With the help of the use of halohydrin dehalogenase biocatalyst, high s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N1/21C12P17/02C12P7/02C12R1/19
Inventor 俞学锋李知洪余明华姚鹃余华顺杨海珍熊茂盛石雨
Owner ANGELYEAST CO LTD
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