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Pseudomoas umsongensis YC1612 for producing halohydrin dehalogenases and application and preparing method thereof

A kind of halohydrin dehalogenase, Pseudomonas technology, applied in the field of microorganisms

Active Publication Date: 2017-09-22
YANCHENG INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Halohydrin dehalogenases have received extensive attention because of their application prospects in sewage treatment and in the synthesis of chiral drug intermediates, but the research on halohydrin dehalogenases is in its infancy at home and abroad.

Method used

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  • Pseudomoas umsongensis YC1612 for producing halohydrin dehalogenases and application and preparing method thereof
  • Pseudomoas umsongensis YC1612 for producing halohydrin dehalogenases and application and preparing method thereof
  • Pseudomoas umsongensis YC1612 for producing halohydrin dehalogenases and application and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Screening of Pseudomonas umsongensis YC1612

[0037] The present invention takes soil samples from tidal flats in Yancheng, Jiangsu, and takes 40 soil samples altogether. The specific method of screening:

[0038] Weigh 5 g of the soil sample, add 50 mL of sterile water to make a soil suspension, take 1.0 mL of the supernatant, add it to the screening medium, and culture it on a shaker at 30°C and 150 rpm / min for 4 days.

[0039] Select the shake flask whose color changes from blue to yellow, and then take the culture solution for gradient dilution, and take 10 -4 、10 -5 ,1 0-6 Gradient dilutions of the above samples were spread on solid LB medium and cultured in a constant temperature incubator at 30°C for 3 days.

[0040] The grown single colony was picked and inoculated into the liquid fermentation medium for cultivation. Cultured on a shaker at 30°C for 2 days, the cells were collected by centrifugation, and washed with phosphate buffered saline.

[0041] The ce...

Embodiment 2

[0048] Identification of Pseudomonas umsongensis YC1612

[0049] 2.1 The bacterial strain YC1612 screened in Example 1 was cultured on LB plates and beef extract peptone plates at 30°C for 24 hours, and the morphology was observed. The results were as follows: figure 1 As shown, the colony is round and smooth, moist, opaque, with neat edges, milky white, and easy to pick.

[0050] 2.2 The screened bacterial strain YC1612 was subjected to physiological, biochemical and BIOLOG identification (see Table 1 and Table 2), and the chromosomal DNA was extracted according to the method of the refined molecular biology experiment guide, and the extracted total cell DNA was used as a template, using primer: p16S -8:5'-agagtttgatcctggctcag-3'(SEQ ID NO:2) and p16S-1541:5'-aaggaggtgatccagccgca-3'(SEQ ID NO:3) amplify the 16S rDNA gene of the strain, and the gene product is the same as the T vector After the connection, Shanghai Sangong was commissioned to amplify and sequence the 16S rDNA...

Embodiment 3

[0059] Preparation of Wet Cells of Pseudomonas umsongensis YC1612

[0060] The wild-type strain Pseudomonas yincasti YC1612 obtained in Example 1 containing halohydrin dehalogenase was inoculated into LB liquid medium, cultivated at 30°C for 24 hours, and then inoculated into fresh After culturing at 30°C for 48 hours in the fermentation medium, centrifuge the culture solution at 4°C and 10,000 rpm for 10 minutes, discard the supernatant, and collect the precipitate to obtain wet cells containing halohydrin dehalogenase.

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Abstract

The invention discloses a pseudomoas umsongensis YC1612 for producing halohydrin dehalogenases and an application and preparing method thereof and belongs to the field of microorganisms. The preservation number of the pseudomoas umsongensis YC1612 is CCTCC NO:M 2017173. The pseudomoas umsongensis YC1612 can produce the halohydrin dehalogenases, and can catalyze epoxide to be subjected to ring opening and prepare chirality epoxide and beta- to replace alcohol in the presence of nucleophilic reagents. When N3- is used as the nucleophilic reagents and o-methyl phenyl glycidyl ether is catalyzed to conduct ring opening reaction, the ee value of (S)-o-methyl phenyl glycidyl ether can reach 98%, and the ee value of generated (R)-1-nitrine-3-(2- methylphenoxy)-2- propyl alcohol reaches 96.8%. The pseudomoas umsongensis YC1612 can be applied to the situation that the epoxide is catalyzed to be subjected to selective ring opening and the chirality epoxide and the beta- are prepared to replace the alcohol, and has a significant application prospect.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a halohydrin dehalogenase-producing Pseudomonas yinchengii YC1612 and its application and preparation method. 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 and is mostly found in 1,3-dichloropropanol , epihalohydrin and many other important environmental pollutants in the biodegradation pathway. Halohydrin dehalogenases can be widely used in the synthesis of epoxides and β-substituted alcohols. Among them, azide alcohols, cyano-substituted alcohols and nitro alcohols are precursors for the synthesis of amino alcohols; chiral amino alcohols are a very important class of compounds in the field of biopharmaceuticals, and can be used to synthesize a variety of biologically active substances. Isothiocyanate-substituted alcohols ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/88C12P13/00C12R1/38
CPCC12N9/88C12P13/00C12P13/004C12Y405/01C12N1/205C12R2001/38
Inventor 薛锋窦琳霞童琦修元松高健黄和
Owner YANCHENG INST OF TECH
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