Bacillus WZZ006 and application thereof

A Bacillus, culture technology, applied in Bacillus WZZ006 and its application fields, can solve the problems of high single enantiomer, violent chemical reaction, environmental organic pollution, etc., and achieve mild enzyme reaction conditions and strong regioselectivity , the effect of little environmental pollution

Active Publication Date: 2019-03-22
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Although the current chemical synthesis method can achieve rapid synthesis, it is difficult to obtain a single enantiomer with high optical purity due to the complex racemization competition factors in the chemical resolution method
Among them, the asymmetric hydroxylation reaction step not only requires a large amount of chiral catalyst,

Method used

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  • Bacillus WZZ006 and application thereof
  • Bacillus WZZ006 and application thereof
  • Bacillus WZZ006 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 strain WZZ006 screening and identification

[0026] 1. Screening of bacteria producing stereoselective ester hydrolase

[0027]Primary screening of strains: Weigh 1g of wet soil sample (obtained from farmland soil in Zhejiang, Anhui, Henan and other places) and suspend it in 10mL sterile saline, shake and mix well; add 3mL suspension to 50mL containing 10mmol / L racemic 5-chloro-2,3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester liquid enrichment medium, at 30°C, 200r / min After culturing for 5-7 days, aseptically transfer 1 mL of the turbid bacterial solution to a fresh solution containing 10 mmol / L racemic 5-chloro-2,3-dihydro-2-hydroxy-1-oxo-1H-indene-2 - Liquid enrichment medium for methyl carboxylate, enriched continuously for 3 to 4 times. In the enrichment medium, racemic methyl 5-chloro-2,3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylate is the only carbon source, and the formula of the enrichment medium is as follows: NaNO 3 3.0g...

Embodiment 2

[0038] Bacillus WZZ006 was inoculated into the slant culture medium and cultured at 30°C for 2 days to obtain slant cells. Slant medium composition (g / L): beef extract 3, peptone 10, Nacl 1, agar powder 15-20, solvent is deionized water, pH 7.0.

[0039] The slant bacteria were inoculated into 50 mL of seed culture medium, and cultured at 30° C. and 180 r / min for 24 hours to obtain seed liquid. Seed medium composition (g / L): beef extract 3, peptone 10, Nacl 1, solvent is deionized water, pH 7.0.

[0040] Under sterile conditions, the seed solution was inoculated in the fermentation medium with an inoculum volume concentration of 2%, and cultured at 30° C. and 180 r / min for 24 hours to obtain a fermentation solution. The formula of the fermentation medium is (g / L): glucose 15, beef extract 15, K 2 HPO 4 ·3H 2 O 6, KH 2 PO 4 3. MgSO 4 ·7H 2 O 1.0, NaCl 0.5, solvent is deionized water, pH 6.5. Take 10 mL of fermentation broth and centrifuge at 10,000 rpm for 10 minutes,...

Embodiment 3

[0043]Take 0.05 g of the dried bacillus WZZ006 prepared by the method in Example 2, and add it to the mixture containing racemic 5-chloro-2,3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic 1mL pH 5.0 citric acid-sodium citrate buffer solution with a final methyl ester concentration of 20mM, react at 30°C and 180rpm for 24h, centrifuge the reaction solution, take the supernatant, adjust the pH to 2 with 4M HCl, and then use an equal volume of acetic acid Ethyl ester extraction, take 50 μL of organic layer, after vacuum drying, add 1mL mobile phase to dissolve, detect by the HPLC method of Example 1, obtain S-(+)-5-chloro-2,3-dihydro-2-hydroxyl- 1-Oxo-1H-indene-2-carboxylic acid methyl ester, the conversion rate is 49.58%, and the substrate e.e.s can reach 90.01%.

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Abstract

The invention discloses bacillus WZZ006 and application thereof. The application comprises the step: with lyophilized powder obtained by freeze drying wet thalli acquired by performing fermentation culture on bacillus WZZ006 as a catalyst, racemic 5-chlorine-2,3-dihydrogen-2-hydroxy-1-oxo-1H-indene-2-carboxylic methyl ether as substrate, and a buffer solution with pH value of 5.0-10.0 as a reaction medium, performing resolution at 20-45 DEG C and 100-300 r/min. The regioselectivity of the bacillus WZZ006 which is the enzyme-produced microorganism bacterial strain is strong, the prepared product enantiomeric excess is not less than 93.01%, the reaction conversion percent is high, the enzyme reaction condition is mild, and the byproduct is less, the environment pollution is small, and a method for efficiently preparing the high-purity enantiomer is suitable for industrial production.

Description

(1) Technical field [0001] The present invention relates to a new bacterial strain-Bacillus WZZ006 and its stereoselective resolution of 5-chloro-2,3-dihydro-2-hydroxyl-1-oxo-1H-indene-2-carboxylic acid methyl ester The application of the reaction to generate optically pure S-(+)-5-chloro-2,3-dihydro-2-hydroxyl-1-oxo-1H-indene-2-carboxylate. (2) Background technology [0002] Indoxacar (Indoxacard), also known as Avatar (hit), Ammate (home run), Avaunt Steward, is a new type of carbamate insecticide developed by DuPont. Its structure is a complex oxadiazine compound, chemical The name is 7-chloro-2,3,4a,5-tetrahydro-2-[methoxycarbonyl(4-trifluoromethoxyphenyl)carbamoyl]indeno[1,2-e][1, 3,4] Methyl diazine-4a-carboxylate, CAS accession numbers: DPX-JW062: [144171-61-9] and DPX-KN128: [173584-44-6]. There is only one chiral center in the structure of indoxacarb, and there are two isomers, R and S. Studies have shown that only the S-isomer is active, and the R-isomer is inact...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P41/00C12P7/62C12R1/07
CPCC12N1/20C12P7/62C12P41/001C12N1/205C12R2001/07
Inventor 章银军张宏云张全郑建永程菲菲汪钊
Owner ZHEJIANG UNIV OF TECH
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