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Halohydrin dehalogenase mutant for improving enantioselectivity and application thereof

A halohydrin dehalogenase, enantioselective technology, applied in the fields of genetic engineering and enzyme engineering

Active Publication Date: 2019-11-08
YANCHENG INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, there are few reports on improving the stereoselectivity of the ring-opening reaction of epoxides catalyzed by halohydrin dehalogenases.

Method used

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  • Halohydrin dehalogenase mutant for improving enantioselectivity and application thereof
  • Halohydrin dehalogenase mutant for improving enantioselectivity and application thereof
  • Halohydrin dehalogenase mutant for improving enantioselectivity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Implementation of Site-Directed Saturation Mutagenesis

[0114] Recombinant plasmid pET28a-HHDH Ab Design appropriate mutation primers for the mutation template (see Patent Publication No. CN107881182A for the construction method).

[0115] The primers used for site-directed mutagenesis were:

[0116]

[0117] The PCR amplification system is: 10 μL of 5×PS Buffer, 4 μL of dNTP (2.5 mM each), 0.5 μL of forward and reverse mutation primers, 0.5 μL of template plasmid, 0.5 μL of PrimeSTAR DNA polymerase, and make up to 50 μL.

[0118] PCR conditions were pre-denaturation at 98°C for 2 minutes, 25 cycles: 98°C for 10s, 65°C for 10s, 72°C for 6min, and finally 72°C for 10min. Take 20 μL of PCR solution, add 1 μL Dpn I, digest at 37°C for 2-3 hours to remove plasmid DNA as a template, inactivate at 65°C for 10 minutes, immediately transform competent cells E.coli BL21(DE3), and coat with kanamycin (50mg / L) LB plates, cultivated at 37°C, picked positive transformants for...

Embodiment 2

[0121] Inducible expression of mutants and parental

[0122] The recombinant engineered strain prepared in Example 1 was cultured in 50 mL LB medium with a final mass concentration of 50 mg / L kanamycin at 37° C. and 200 r / min for 10 h. Then inoculate into the new 50mL LB culture medium that contains the kanamycin of 50mg / L with the inoculum size of 1vt.%, still with 37 ℃, 200r / min culture, treat to be cultivated to optical density (OD 600 ) is 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) inducer to a final concentration of 0.15mM, and induce expression at 28°C and 200r / min for 12h. 5000 × g, 5min centrifugation to collect the bacterial cells, and NaH with pH 8.0 2 PO 4 -Na 2 HPO 4 The buffer was resuspended and washed, centrifuged at 5000×g for 5 min, and E. coli cells were collected and stored at -20°C for future use.

Embodiment 3

[0124] Determination of enantioselectivity of mutant enzymes

[0125] Substrate benzyl glycidyl ether (structural formula sees figure 1 ) HPLC analysis method: using Agilent-1220 system, column type: Chiralcel OD-H column (Daicel Co., Japan; 4.6×250mM, 5μm); chromatographic conditions: column temperature 30°C; mobile phase: n-hexane : Isopropanol=9:1 (v / v); Flow rate: 0.8mL / min; UV wavelength is 254nm; (S)- and (R)-substrate retention times (min) are about 9.3 and 10.1 respectively. Substrate phenyl glycidyl ether (structural formula sees figure 2 ) HPLC analysis method: using Agilent-1220 system, column type: Chiralcel OD-H column (Daicel Co., Japan; 4.6×250mM, 5μm); chromatographic conditions: column temperature 30°C; mobile phase: n-hexane : Virahol=83:17 (v / v); Flow rate: 0.8mL / min; UV wavelength is 220nm; (R)- and (S)-substrate retention times (min) are about 8.1 and 12.4 respectively. Substrate o-nitrophenyl glycidyl ether (structural formula sees image 3 ) HPLC an...

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Abstract

The invention relates to a halohydrin dehalogenase mutant for improving enantioselectivity and application thereof, and belongs to the technical field of enzyme engineering and biocatalysis. The halohydrin dehalogenase mutant is obtained by carrying out single-point mutation or combined mutation on 89th arginine, 137th valine, 178th proline, 179th asparagine and 187th phenylalanine in a sequence as shown in SEQ ID NO.1. Compared with wild type halohydrin dehalogenase, the mutant obtained by the invention obviously improves enantioselectivity (E value) during the preparation of (S)-o-nitrophenyl glycidyl ether, (R)-benzyl glycidyl ether and (R)-phenyl glycidyl ether, the maximum improvement is 44.6, 2.9 and 9.4 times of the original enzyme respectively, and has good industrial application prospect.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and enzyme engineering, and in particular relates to a halohydrin dehalogenase mutant with improved enantioselectivity and application thereof. Background technique [0002] Halohydrin dehalogenase (Halohydrin Dehalogenase, HHDHs, EC 4.5.1.X), also known as halohydrin-hydrogen halide lyase, belongs to the short-chain dehydrogenase / reductase family. The reason why halohydrin dehalogenase has attracted much attention is its application in the field of biocatalysis. The biocatalytic reaction mediated by it has the advantages of mild reaction conditions, high stereoselectivity and no need for coenzyme. Halohydrin dehalogenase can not only catalyze the cleavage of carbon-halogen bonds for dehalogenation reaction to synthesize chiral halohydrins and epoxides, but also catalyze and accept a series of unnatural nucleophiles with high selectivity, such as N 3 - , NO 2 - 、CN - The epoxide ...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/00C12P17/02C12R1/19
CPCC12N9/88C12N15/70C12P13/00C12P17/02C12Y405/01
Inventor 薛锋张丽李凤伟梁慧星李寒
Owner YANCHENG INST OF TECH
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