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Parvibaculum lavamentivorans ZJB 14001, halohydrin dehalogenase enzyme gene, enzyme, engineered bacterium and application

A technology of halohydrin dehalogenase and genetically engineered bacteria, which can be used in genetic engineering, applications, bacteria, etc., and can solve problems such as limiting the application of halohydrin dehalogenase

Inactive Publication Date: 2015-02-18
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although halohydrin dehalogenases have important applications in the preparation of many optically pure epoxides and β-substituted alcohols, the scarcity of halohydrin dehalogenase genes greatly limits the application of halohydrin dehalogenases

Method used

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  • Parvibaculum lavamentivorans ZJB 14001, halohydrin dehalogenase enzyme gene, enzyme, engineered bacterium and application
  • Parvibaculum lavamentivorans ZJB 14001, halohydrin dehalogenase enzyme gene, enzyme, engineered bacterium and application
  • Parvibaculum lavamentivorans ZJB 14001, halohydrin dehalogenase enzyme gene, enzyme, engineered bacterium and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Screening and Identification of Example 1 Bacterial Strains

[0049] The soil and seawater from the coastal area of ​​Zhoushan were collected, enriched with the enrichment medium, spread on the plate of the enrichment medium, and cultured at 30°C for one week. The single bacterium colony of the different form that separates obtains with fermentation medium (composition of fermentation medium: NaCl 5g / L, beef extract 10g / L, peptone 5g / L, yeast powder 5g / L, solvent is deionized water, pH value natural .) Cultivated at 30°C to obtain a large amount of wet bacteria.

[0050] Using 0.2g of wet bacteria as a catalyst, 20mmol / L 1,3-dichloropropanol as a substrate, and 20ml of 50mM, pH7.0 PBS as a reaction medium, the conversion reaction was carried out at 37°C for 30min. After the reaction After the reaction solution was extracted with ethyl acetate, the ethyl acetate layer was taken to analyze the content of the product in the reaction solution by GC. A strain with halohydr...

Embodiment 2

[0055] The culture condition of embodiment 2 food cleaning agent parvobacterium ZJB14001

[0056] (1) Incline cultivation

[0057] Inoculate the slant culture medium with Escherichia coli ZJB14001 and culture it at 30°C for 7 days to obtain slant bacteria;

[0058] The composition of the slant medium is: NaCl 5g / L, peptone 10g / L, CaCl 2 0.02g / L and agar 2.0g / L, the solvent is deionized water, and the pH value is natural.

[0059] (2) Fermentation culture

[0060] Bacterial colonies were picked from the slant and inoculated into the fermentation medium, and cultured at 30° C. and 150 rpm for 5 days to obtain a bacterium-containing fermentation broth.

[0061] The components of the fermentation medium are: NaCl 5g / L, peptone 10g / L, CaCl 2 0.02g / L, the solvent is water, and the pH value is natural.

Embodiment 3

[0062] Embodiment 3: the cloning of halohydrin dehalogenase HHDH-PL gene

[0063] The total genomic DNA of the Parvibaculum lavamentivorans ZJB14001 cells obtained in Example 2 was extracted with a rapid nucleic acid extractor (purchased from eppendorf), and PCR was performed using the genomic DNA as a template and primers 3 and 4 as amplification primers. 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 3 and primer 4 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.

[0064] Primer 3: 3'- CCATGG CGCGCAGCATTCTC-5′;

[0065] Primer 4: 3'- CTCGAG CTAGCGCGCGGTGGCCC-5′

[0066] The PCR instrument of Biorad was used, and the PCR reaction conditions were: pre-denaturation at 94°C for 3min, followed by a temperature cycle of 94°C ...

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Abstract

The invention provides a halohydrin dehalogenase encoded gene, a recombinant vector containing the gene, and a recombinant genetically engineered bacterium, and provides a novel bacterial strain, namely parvibaculum lavamentivorans ZJB 14001, of the gene. The recombinant halohydrin dehalogenase is regarded as an invertase to synthesize epoxy chloropropane by taking catalysis of 1,3-dichloro-2-propanol as an example, to prepare (S)-4-chloro-3-hydroxybutyronitrile by taking catalysis of CN-mediated (S)-epoxy chloropropane with a ring-opening reaction as an example, and to respectively prepare (S)-4-chloro-3-hydroxybutyronitrile and ethyl-(R)-4-cyano-3-hydroxybutyrate by taking catalysis of 1,3-dichloro-2-propanol and (S)-4-chloro-3-hydroxybutyronitrile as an example with CN-mediated ring opening.

Description

(1) Technical field [0001] The invention relates to a halohydrin dehalogenase gene derived from a food cleaner Parvibaculum lavamentivorans ZJB14001 strain, a recombinant expression vector thereof, a genetically engineered bacterium, and the preparation of a recombinant halohydrin dehalogenase and chiral Applications in halohydrins, epoxides and β-substituted alcohols. (2) Background technology [0002] Halohydrin dehalogenase (EC 4.5.1.X), also known as halohydrin lyase, can catalyze the conversion of aliphatic and aromatic ortho-halohydrins into epoxides and release halide ions through an intramolecular nucleophilic substitution mechanism ( figure 1 ); meanwhile, in ionic nucleophiles (such as N 3 - , NO 2 - 、CN - 、SCN - and OCN - ) in the presence of halohydrin dehalogenase can highly selectively catalyze the ring-opening reaction of epoxides mediated by nucleophiles to generate corresponding optically pure β-substituted alcohols ( figure 2 ), where azide alcoho...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/70C12N15/81C12N1/21C12N1/19C12N1/20C12P13/00C12P17/02C12R1/01
Inventor 柳志强郑裕国万南微沈寅初
Owner ZHEJIANG UNIV OF TECH
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