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Halohydrin dehalogenase mutant, coding gene, plasmid, genetically engineered bacterium and application thereof

A technology of halohydrin dehalogenase and genetically engineered bacteria, applied in genetic engineering, application, halocarbon lyase, etc., can solve the problem that the yield is only 30.7%, achieve stereoselectivity, high purity, and enantiomer selection rate-enhancing effect

Active Publication Date: 2021-12-28
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the research group catalyzed the synthesis of (S)-1-chloro-3-phenoxy -2-propanol, the yield is only 30.7% (Catalysis Letters,2019,149:629–637)

Method used

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  • Halohydrin dehalogenase mutant, coding gene, plasmid, genetically engineered bacterium and application thereof
  • Halohydrin dehalogenase mutant, coding gene, plasmid, genetically engineered bacterium and application thereof
  • Halohydrin dehalogenase mutant, coding gene, plasmid, genetically engineered bacterium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1 A kind of halohydrin dehalogenase mutant

[0023] A halohydrin dehalogenase mutant, wherein the halohydrin dehalogenase mutant takes the original halohydrin dehalogenase as the re-extracted mutation, and the amino acid sequence of the original halohydrin dehalogenase is as follows:

[0024]

[0025] The letters in the above amino acid sequence are the single-letter abbreviations of amino acids, and the names of the amino acids represented by the abbreviations in the specific amino acid sequence are shown in the following table for details:

[0026]

[0027] The gene sequence of the original halohydrin dehalogenase used in this embodiment is as follows:

[0028]

[0029]

[0030] In the halohydrin dehalogenase mutant, combined mutations are performed at positions R1 to R21 in the sequence shown in SEQ ID NO.1, and 6 histidines are added to its N-terminal. Among them, the combined mutation of R1 to R21 is that the 3rd lysine is mutated into aspar...

Embodiment 2

[0038] Example 2 A coding gene encoding a halohydrin dehalogenase mutant and a recombinant plasmid carrying the above-mentioned gene

[0039] Encode the halohydrin dehalogenase mutant in Example 1 to form a coding gene with the halohydrin dehalogenase mutant, specifically, synthesize SEQ ID NO.2 by a total synthesis method through genetic engineering operations, namely The coding gene with mutant enzyme, the coding gene sequence of halohydrin dehalogenase is HHDH mut .

[0040] Both the halohydrin dehalogenase coding gene and the Escherichia coli expression vector pET28a were double-digested with NcoI and XhoI, respectively. After 3-6 hours of digestion, the digested products were recovered and ligated with T4 DNA ligase at 16°C for 16 hours to obtain the recombinant Plasmid pET28a-HHDHmut. The T4 DNA ligase, restriction enzymes Nco I and Xho I used above were purchased from Fermetens.

Embodiment 3

[0041] Embodiment 3 A kind of genetically engineered bacteria

[0042] A genetically engineered bacterium, using Escherichia coli as a host to express the recombinant plasmid described in Example 2.

[0043] The specific method is: the recombinant plasmid pET28a-HHDH mut Transform into E.coliBL21 (DE3) recipient bacteria, smear on LB agar plate containing kanamycin (concentration 50 mg / L), cultivate overnight at 37°C, colonies grow on the plate. Randomly pick a single clone, extract the plasmid for sequencing after culture, and the sequencing results show that the genetically engineered bacteria, that is, the positive clone E.coli BL21(DE3) / pET28a-HHDH mut .

[0044] Cultivate the above-mentioned genetically engineered bacteria in 50mL LB medium of kanamycin with a mass concentration of 50mg / L at 37°C and 200r / min for 10h; then inoculate to a new mass concentration of 1vt.% In 50mL LB medium containing 50mg / L kanamycin, culture at 37°C and 200r / min, and when the optical den...

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Abstract

The invention discloses a halohydrin dehalogenase mutant, a coding gene, a plasmid, a genetically engineered bacterium and application thereof, halohydrin dehalogenase is subjected to sequence mutation to form the halohydrin dehalogenase mutant, the coding gene for coding the halohydrin dehalogenase mutant, the recombinant plasmid carrying the coding gene and the genetically engineered bacterium for expressing the recombinant plasmid, the finally prepared genetically engineered bacterium has high stereoselectivity in catalytic synthesis of (S)-1-chloro-3-phenoxy-2-propanol, has higher purity and yield compared with the catalytic synthesis and resolution of halohydrin dehalogenation original enzyme, and is beneficial to industrial production.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a halohydrin dehalogenase mutant, a coding gene, a recombinant plasmid, a genetically engineered bacterium and applications thereof. Background technique [0002] Chiral 1-chloro-3-phenoxy-2-propanol is an important intermediate in organic synthesis, which can be used to synthesize a large number of bioactive molecules, and is widely used in pharmaceutical chemicals, agricultural chemicals and other industries. The synthesis of chiral o-haloalcohols has been widely concerned by researchers. The prior art has reported asymmetric hydroboration, (transfer) hydrogenation, biocatalytic reduction of R-chloroketones and dynamic kinetic resolution of halohydrins. and other methods. However, the traditional chemical synthesis method uses a metal catalyst, which is highly toxic, seriously pollutes the environment, and the reaction conditions are harsh. It often requires a higher reaction t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P17/02C12P13/00C12R1/19
CPCC12N9/88C12N15/70C12P17/02C12P13/00C12P41/001C12Y405/01C12N2800/101
Inventor 黄和薛锋夏晖徐晴
Owner NANJING NORMAL UNIVERSITY
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