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Recombinant halohydrin dehalogenase, and mutant and engineering strain and applications thereof

A halohydrin dehalogenase, mutant technology, applied in application, genetic engineering, recombinant DNA technology and other directions, can solve problems such as difficult to obtain, low product ee value, and inability to industrial production.

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

However, the halohydrin dehalogenases discovered so far are not easy to obtain, and halohydrin dehalogenases with excellent catalytic properties make most asymmetric dehalogenation reactions have low yields and low ee values ​​of products, which cannot be really applied to industrial production.

Method used

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  • Recombinant halohydrin dehalogenase, and mutant and engineering strain and applications thereof
  • Recombinant halohydrin dehalogenase, and mutant and engineering strain and applications thereof
  • Recombinant halohydrin dehalogenase, and mutant and engineering strain and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The acquisition of embodiment 1 parental gene and the preparation of recombinant expression plasmid and recombinant engineered bacteria

[0029] According to the amino acid sequence predicted to be Sneathiella glossodoripedis (Genbank No.WP_037493663) contained in GenBank, the amino acid sequence is shown in SEQ ID No.2, the codon-biased synthetic gene was designed in Escherichia coli, and submitted to Shanghai Xuguan Biotechnology Development Ltd Synthetics. The base sequence is the gene shown in SEQ ID No.1, and Xba I and Xho I restriction sites are introduced at both ends, and it is synthesized by Shanghai Xuguan Biotechnology Co., Ltd. ℃ double digestion with restriction endonucleases Xba I and Xho I for 5 h, purified by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit (Aisijin Biotechnology (Hangzhou) Co., Ltd.). Under the action of T4 DNA ligase, the target fragment was ligated with the vector plasmid pET28b...

Embodiment 3

[0033] Embodiment 3 Expression of recombinant halohydrin dehalogenase

[0034]The recombinant escherichia coli obtained in Example 1 and Example 2 were inoculated into the LB medium containing kanamycin sulfate at a final concentration of 50 mg / L respectively, cultured with shaking at 37° C. overnight, and inoculated at 1% (v / v) Put the amount into a 500mL Erlenmeyer flask containing 100mL LB medium, place it on a shaker at 37°C and 180rpm for shaking culture, when the OD 600 of the culture medium reaches 0.6, add IPTG with a final concentration of 0.5mM as an inducer, and induce at 28°C After 10 hours, the culture solution was centrifuged to collect the cells (that is, wet cells), and washed twice with phosphate buffered saline to obtain resting cells. The resulting resting cells were suspended in 20 mL of pH 8.0 buffer, ultrasonically disrupted in an ice bath for 15 min, centrifuged, and the supernatant was collected, which was the crude enzyme solution of the recombinant ha...

Embodiment 4

[0035] Example 4: Halohydrin dehalogenase and mutant V137I catalyze the synthesis of (S)-epichlorohydrin from 1,3-dichloropropanol

[0036] The composition of the wild-type transformation system and the transformation operation are as follows: 0.2 g of wild-type halohydrin dehalogenase wet cells (prepared in Example 3) and 30 mM of 1,3 - Dichloropropanol, reacted on a shaking table at 35°C and 150r / min. After reacting for 2.5 minutes, the reaction solution was extracted with 2 times the volume of ethyl acetate, extracted twice, the extracts were combined, and 1-chloro For n-hexane, the conversion rate and ee value of the substrate were determined by gas chromatographic analysis. After reacting for 2.5 minutes, the conversion rate of 1,3-dichloropropanol reached over 95%, the yield of (S)-epichlorohydrin reached 88.6%, and the ee value reached 84.5%.

[0037] The composition of the mutant transformation system and the transformation operation are as follows: 0.2 g of mutant V1...

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Abstract

The invention discloses a recombinant halohydrin dehalogenase and a mutant, an engineering strain and applications thereof. The amino acid sequence of the recombinant halohydrin dehalogenase is shown in SEQ ID No: 2. The invention also discloses an application of the recombinant halohydrin dehalogenase and the mutant of recombinant halohydrin dehalogenase in catalyzing asymmetric dehalogenation of 1,3-dichloropropanol so as to synthesize (S)-epoxy chloropropane and in preparing other chiral epoxides and beta-substituted alcohol. Compared with other halohydrin dehalogenases, the halohydrin dehalogenase obtained according to the invention and the mutant thereof have higher enantioselectivity and have an extremely good industrial application prospect.

Description

(1) Technical field [0001] The invention relates to a halohydrin dehalogenase, its gene and mutant, a recombinant expression vector containing the gene and the mutant, a recombinant bacterium, a method for preparing the recombinant enzyme by using the recombinant bacterium, and the preparation of the recombinant halohydrin dehalogenase. Oxides, Chiral Epoxides, β-Substituted Alcohols Methods. (2) Background technology [0002] Halohydrin dehalogenases, also known as halohydrin-hydrohalide lyases, catalyze the conversion of aromatic or aliphatic ortho-halohydrins to epoxides and hydrogen halides through an intramolecular nucleophilic substitution mechanism. Halohydrin dehalogenase can not only catalyze the cleavage of carbon-halogen bonds for dehalogenation reactions, but also catalyze and accept a series of unnatural nucleophiles other than halide ions with high selectivity, such as N 3 - , NO 2 - 、CN - etc. mediated epoxide ring-opening reactions. Halohydrin dehalogen...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P41/00C12P17/02C12P13/00C12P7/62C12R1/19
CPCC12N9/14C12P17/02C12Y308/01
Inventor 柳志强郑裕国薛锋朱杭芹王亚军沈寅初
Owner ZHEJIANG UNIV OF TECH
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