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Halogenohydrin dehalogenation enzyme and encoding gene and vector and bacterial strain and application

A halohydrin dehalogenase, gene technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of poor self-degradation ability, carcinogenicity, high mutagenesis of xenobiotic halides

Active Publication Date: 2013-03-20
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In nature, most xenobiotic halides have poor self-degradation ability, and many compounds are suspected to be carcinogenic or highly mutagenic substances

Method used

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  • Halogenohydrin dehalogenation enzyme and encoding gene and vector and bacterial strain and application
  • Halogenohydrin dehalogenation enzyme and encoding gene and vector and bacterial strain and application
  • Halogenohydrin dehalogenation enzyme and encoding gene and vector and bacterial strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The total genomic DNA of Agromyces sp. CCTCC No: M 2012299 was extracted with a rapid nucleic acid extractor, and PCR was performed under the action of primer 1 (ATGMGNATCGCCCTCGTGACTC) and primer 2 (TTAGGGCAGATAGCC ACCG) using the genomic DNA as a template Amplify.

[0048] The amount of each component in the PCR reaction system (total volume 100 μL): 10×Pfu DNA Polymerase Buffer 10 μL (Mg 2+ ), 0.5 μL of 10 mM dNTP mixture (2.5 mM each of dATP, dCTP, dGTP, and dTTP), 0.5 μL of each cloning primer 1 and primer 2 at a concentration of 50 μM, 1 μL of genomic DNA, 1 μL of Pfu Taq DNA Polymerase, and 86.5 μL of nucleic acid-free water.

[0049] Using Biorad’s PCR instrument, the PCR reaction conditions were: pre-denaturation at 94°C for 3 minutes, then entering into a temperature cycle of 94°C for 30 s, 60°C for 30 s, and 72°C for 1.5 min, a total of 30 cycles, and finally extending at 72°C for 10 min, with a termination temperature of 8 ℃.

[0050] Take 10 μL of the PCR...

Embodiment 2

[0052] Design primer 3 according to embodiment 1 analysis result (CGC CATATG CGCATCGCCCTC GTGACTC) and Primer 4 (CCG CTCGAG TTAGGGCAGATAGCCACCG), and NdeI and XhoI restriction enzyme sites were introduced into primer 3 and primer 4, respectively. Initiated by primer 4 and primer 4, high-fidelity Pfu DNA polymerase (fermentas) was used to amplify to obtain a 735bp halohydrin dehalogenase gene fragment (SEQ ID NO: 1), which was sequenced using Nde I and Treat the amplified fragment with XhoI restriction endonuclease (fermentas), and use T4 DNA ligase (TaKaRa) to connect the fragment with the commercial vector pET28b treated with the same restriction endonuclease to construct the expression vector pET28b - Deh. The constructed intracellular expression vector pET28b-Deh was electrotransformed into Escherichia coli BL21 (Invitrogen), plated and cultured overnight at 37°C, and clones were randomly selected to extract plasmids for enzyme digestion identification. The identificatio...

Embodiment 3

[0054] The recombinant Escherichia coli BL21 / pET28b-Deh containing the intracellular expression recombinant plasmid pET28b-Deh after verification in Example 2 was cultured with LB liquid medium containing 50 μg / ml kanamycin resistance for 12 hours, and then treated with 1% The inoculum amount (v / v) was inoculated into fresh LB liquid medium containing 50 μg / ml kanamycin resistance, and cultivated to the cell concentration OD 600 About 0.6, then add IPTG with a final concentration of 0.5mM to the LB liquid medium, induce culture for 10 hours, centrifuge at 10,000 rpm for 10 minutes at 4°C, and collect the bacterial cells containing the recombinant halohydrin dehalogenase.

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Abstract

The invention provides a halogenohydrin dehalogenation enzyme originating from Agromyces sp., an encoding gene and a vector, and provides application of the halogenohydrin dehalogenation enzyme, the encoding gene and the vector in the process of preparing epoxy chloropropane and (R)-4- cyano-cn-3- hydroxybutyric acid ethyl ester. The halogenohydrin dehalogenation enzyme amino acid sequence is shown in SEQ ID NO.2, and the encoding gene sequence is shown in SEQ ID NO.1. The halogenohydrin dehalogenation enzyme, the encoding gene, the vector and the application have the advantages that the halogenohydrin dehalogenation enzyme originating from Agromyces sp. CCTCC NO. M 2012299 and the encoding gene of the halogenohydrin dehalogenation enzyme are provided; and the encoding gene of the halogenohydrin dehalogenation enzyme can be connected and constructed with an expression vector to obtain expression recombinant plasmid pET28b-Deh of the encoding gene, then can be transformed to escherichia coli BL21, obtained and transformed to escherichia coli bacterial strain respectively and correspondingly to obtain recombinant escherichia coli, and the recombinant escherichia coli has the halogenohydrin dehalogenation enzyme and can be utilized for carrying out biotransformation and catalysis for an enzyme source.

Description

(1) Technical field [0001] The present invention relates to a halohydrin dehalogenase, a coding gene and a carrier, a new bacterial strain producing halohydrin dehalogenase, and its use in the preparation of epichlorohydrin and (R)-4-cyano-3-hydroxybutyric acid ethyl application in esters. (2) Background technology [0002] Halohydrin dehalogenase, also called halohydrin-hydrogen halide lyase, catalyzes the conversion of aromatic or aliphatic o-halohydrins into epoxides and hydrogen halides through an intramolecular nucleophilic substitution mechanism, and is a key enzyme for microbial degradation of organic halogen compounds one. According to their sequence homology, they are divided into 3 types: HheA, HheB, and HheC. Organohalogen compounds have become one of the important environmental pollutants today, mainly due to waste discharge and the wide application of artificially synthesized halides. In nature, most xenobiotic halides have poor self-degradation ability, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N1/14C12N15/63C12N15/70C12P17/02C12P13/00C12R1/645
Inventor 郑裕国薛锋柳志强万南微高爱存沈寅初
Owner ZHEJIANG UNIV OF TECH
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