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Immobilized halohydrin dehalogenase and application thereof

A technology of halohydrin dehalogenase and enzyme immobilization, which is applied to the direction of immobilized on/in organic carriers, halocarbon lyase, enzyme, etc., which can solve the problems of inability to meet industrial production requirements, poor stability, etc., and achieve excellent operation Effects of stability and high adsorption efficiency

Inactive Publication Date: 2016-08-10
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are few reports on the immobilization of halohydrin dehalogenase, Pavel Dvorak et al. 48(12):6859–6866) used polyvinyl alcohol as a carrier to embed and immobilize halohydrin dehalogenase, but the operation batch was only 10 batches, the stability was poor, and it could not meet the requirements of industrial production

Method used

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  • Immobilized halohydrin dehalogenase and application thereof
  • Immobilized halohydrin dehalogenase and application thereof
  • Immobilized halohydrin dehalogenase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction and Fermentation of Recombinant Halohydrin Dehalogenase Genetic Engineering Bacteria

[0044] 1) Construction of recombinant halohydrin dehalogenase gene engineering bacteria: the halohydrin dehalogenase gene HHDH (nucleotide sequence shown in SEQ ID NO.1) was ligated with PGEM-T vector and introduced into E.coli JM109 , HHDH / PGEM-T and plasmid pET28b(+)-Nit were double digested, ligated overnight with ligase, and the ligation product pET28b(+)-HHDH was introduced into the host E.coli BL21(DE3), and recombinant E. coli was obtained by screening E. coliBL21(DE3) / pET28b(+)-HHDH.

[0045] 2) Fermentation of recombinant halohydrin dehalogenase genetically engineered bacteria

[0046] (1) Slant culture: Inoculate the recombinant E. coli BL21(DE3) / pET28b(+)-HHDH into the slant medium containing a final concentration of 50 μg / ml kanamycin, and culture at 37°C for 12 hours to obtain slant cells The final concentration of the slant medium consists of: pe...

Embodiment 2

[0049] Example 2 Preparation of Crude Enzyme Liquid of Recombinant Halohydrin Dehalogenase

[0050]Wash the recombinant halohydrin dehalogenase cells obtained in Example 1 with clear water, dissolve them with a certain amount of pH=7.0, 0.1M sodium phosphate buffer, prepare a bacterial suspension with a concentration of 1g / 10mL, and store them on ice Ultrasonic treatment (240W, ultrasonic 1s, interval 1s) in a water bath for 20 minutes to break the cells, the broken solution was centrifuged at 9000rpm at 4°C for 10 minutes, the supernatant was the crude enzyme solution, and the enzyme content was calculated as 0.1 by the weight of the wet cells before breaking. g / ml. Then take 1 ml of the supernatant, and use the BCA protein content detection kit (Nanjing Kaiji Biotechnology Development Co., Ltd.) to determine that the protein content of the crude enzyme solution is about 10 mg / mL.

Embodiment 3

[0051] Example 3 Immobilization of recombinant halohydrin dehalogenase

[0052] 3.1 Selection of carrier

[0053] Carry out preliminary experiments to screen out potential carriers, and then carry out detailed immobilization conditions for the selected carriers.

[0054] Epoxy resins produced by different manufacturers have different parameters such as water content, pore size distribution on the carrier surface, and epoxy density, which will cause certain differences in the final results when immobilizing enzymes. Considering the price difference of epoxy resins at home and abroad, four kinds of epoxy resins, LX-1000EP(C) from Xi’an Lanxiao Technology Co., Ltd. and ES-1, ES-105 and ES-103B from Nankai Technology Co. Alternative immobilization carrier.

[0055] Take 10mL of the crude enzyme solution obtained in Example 2 and add it to 200mL, pH=7.0, 0.1M sodium phosphate buffer solution, mix well to form the enzyme solution to be immobilized; mix the enzyme solution to be im...

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Abstract

The invention discloses immobilized halohydrin dehalogenase and an application thereof. The immobilized halohydrin dehalogenase has high conversion efficiency on a halohydrin substrate, is strong in substrate tolerance, and has excellent catalysis selectivity on the substrate and structural analogues thereof. In the invention, recombinant halohydrin dehalogenase is successfully fixed on the surface of an epoxy resin carrier through multipoint covalence, and through low-salt adsorption and addition of glycerol as a protection agent of an enzyme activity center, enzyme activity loss during immobilization is effectively reduced. The immobilization method is simple in operation, is low in raw materials cost, and is suitable for large-scale operation. The immobilized halohydrin dehalogenase has homogeneous property, high stability and good repeatability. With the immobilized halohydrin dehalogenase,epoxy chloropropane, (R)-4-cyano-3-hydroxylethyl butyrate, (S)-2,3-dichloro-1-propyl alcohol and the like can be synthesized. The immobilized halohydrin dehalogenase can be continuously used for more than 50 batches before significant reduction of activity of the immobilized halohydrin dehalogenase and also has good industrial application prospect.

Description

1. Technical field [0001] The invention relates to a method for preparing an immobilized halohydrin dehalogenase, a method for preparing an immobilized recombinant halohydrin dehalogenase with high operational stability, and the preparation of a chiral ring by catalyzing the immobilized recombinant halohydrin dehalogenase Oxychlorohydrin and other epoxy compound methods. 2. Background technology [0002] Halohydrin dehalogenase, also known as halohydrin-hydrogen halide lyase, catalyzes the conversion of aromatic or aliphatic ortho-halohydrins into epoxides and hydrogen halides through an intramolecular affinity substitution mechanism, and is one of the key enzymes for microbial degradation of organic halogen compounds one. Halohydrin dehalogenase forms a hydrogen bond between the conserved serine in the protein structure and the hydroxyl oxygen atom of the substrate, stabilizing and binding to the substrate, reducing the pKa value of tyrosine through arginine, and tyrosine ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/08C12N9/88C12P17/02C12P13/00C12P41/00C12P7/04
CPCC12N11/08C12N9/88C12P7/04C12P13/002C12P17/02C12P41/001C12Y405/01
Inventor 郑裕国邹树平顾恺薛亚平
Owner ZHEJIANG UNIV OF TECH
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