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Rhodococcus ZJPH1003 and application thereof in preparing S-(+)-2,2-dimethylcyclopropane carboxylic acid

A technology of ethyl dimethylcyclopropanecarboxylate and dimethylcyclopropane is applied to Rhodococcus ZJPH1003 and its application field in the preparation of S-(+)-2,2-dimethylcyclopropanecarboxylic acid, which can Solve the problems of limited industrial application and high price of commercial lipase, and achieve the effects of shortened substrate conversion reaction time, easy cultivation and low preparation cost

Active Publication Date: 2011-08-24
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although adopting lipase Novozyme 435 also can selectively hydrolyze racemic 2,2-dimethylcyclopropane formic acid ethyl ester synthetic S-(+)-2,2-dimethylcyclopropane formic acid (Wang Pu etc., Journal of Catalysis , 2010, 31(6): 651-655), but the high price of commercial lipase also limits its industrial application to a certain extent

Method used

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  • Rhodococcus ZJPH1003 and application thereof in preparing S-(+)-2,2-dimethylcyclopropane carboxylic acid
  • Rhodococcus ZJPH1003 and application thereof in preparing S-(+)-2,2-dimethylcyclopropane carboxylic acid
  • Rhodococcus ZJPH1003 and application thereof in preparing S-(+)-2,2-dimethylcyclopropane carboxylic acid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: the detection method of product

[0049] The optical purity and yield of the target product were determined by GC: the concentration of the product and residual substrate in the reaction extract was analyzed by gas chromatography, and quantified by the internal standard method. The internal standard is dodecane. Take 1 mL of the extract and add 1 μL of dodecane for analysis. Gas chromatography conditions: Japan Shimadzu GC-2014 gas chromatograph; American Varian CP-Chirasil-Dex chiral capillary gas chromatography column (25m×0.25mm×0.25μm). The carrier gas is high-purity nitrogen, the flow rate is 2mL / min; the injection volume is 1 μL, and the split ratio is 15:1; the temperature of the detector and the injection port are both 250°C; the temperature of the chromatographic column is 80-180°C; the heating rate: 8°C / min; the detector is FID.

[0050] The concentrations of substrate and product in the reaction solution were calculated using relative correc...

Embodiment 2

[0056] Embodiment 2: the acquisition of wet fungus cells

[0057] Seed medium formula: glucose 10g / L, peptone 5g / L, yeast extract 3g / L, (NH 4 ) 2 SO 4 4g / L, K 2 HPO 4 2g / L, KH 2 PO 4 1g / L, NaCl 0.5g / L, MgSO 4 ·7H 2 O0.5g / L, solvent is water, pH 7.0.

[0058] Fermentation medium formula: glycerol 15g / L, peptone 25g / L, K 2 HPO 4 2g / L, KH 2 PO 4 1g / L, NaCl 1.5g / L, MgSO 4 ·7H 2 O 0.5g / L, solvent is water, pH 7.0.

[0059] Pick a ring of bacteria (CCTCC NO: M 2010371) from the mature slant and insert it into a 250mL shake flask with 100mL seed medium, cultivate it at 30°C and 200rpm for 24 hours to obtain a seed solution, and then inoculate it with a volume ratio of 10%. Transfer the seed solution to a 250mL shake flask containing 100mL of fermentation medium, and culture at 30°C and 200rpm for 48 hours. After the cultivation, the fermented liquid was centrifuged and washed once with normal saline, and the wet bacterial cells were collected for future use.

Embodiment 3

[0061] The wet thalline obtained in Example 2 is suspended in 10 mL of disodium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 6.98), and the wet thalline is 400 g / L by wet weight; add 10 mmol / L of 2,2 -Ethyl dimethylcyclopropanecarboxylate was used as a substrate, and placed in a shaker at 20° C. at 200 rpm for 24 hours. Using the detection method of Example 1, the concentration of the product S-(+)-2,2-dimethylcyclopropanecarboxylic acid was 4.65mmol / L, the optical purity e.e. value was 80.1%, and the yield was 46.5%.

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Abstract

The invention provides a new strain-Rhodococcus sp. ZJPH1003 and an application thereof in preparing S-(+)-2,2-dimethylcyclopropane carboxylic acid through microbial catalytic asymmetric hydrolysis of 2,2-dimethylcyclopropane ethyl formate. The strain is conserved at China Center for Type Culture Collection, the conservation address is Wuhan University, Wuhan, China, postcode 430072, the conservation number is CCTCC M 2010371, and the conservation date is December 29, 2010. In the invention, the adopted method for preparing the S-(+)-2,2-dimethylcyclopropane carboxylic acid through microbial catalytic asymmetric hydrolysis of the 2,2-dimethylcyclopropane ethyl formate by utilizing the new microbial strain has the advantages of novel route, high yield, environmental friendliness and the like; and by utilizing a chiral biocatalysis method which takes a Rhodococcus ZJPH1003 cell as a catalyst, for the target product-S-(+)-2,2-dimethylcyclopropane carboxylic acid, the e.e can reach 82.5% and the yield can reach 41.3% when the substrate concentration is 30mmol / L.

Description

(1) Technical field [0001] The present invention relates to a new bacterial strain that can be used for microbial catalyzed asymmetric hydrolysis of 2,2-dimethylcyclopropane formic acid ethyl ester to prepare S-(+)-2,2-dimethylcyclopropane formic acid-Rhodococcus (Rhodococcus sp.) ZJPH1003 and its application in microbial catalyzed asymmetric hydrolysis of ethyl 2,2-dimethylcyclopropanecarboxylate to prepare S-(+)-2,2-dimethylcyclopropanecarboxylic acid. (2) Background technology [0002] The chemical name of cilastatin is (+)-(Z)-7-[(2R)-(2-amino-2-carboxyethyl)sulfur]-2-[(1S)-(2,2-di Methylcyclopropanecarboxamido)]-2-heptenoic acid was the first renal dehydrodipeptidase inhibitor to be used clinically. Taineng, a composite agent made of cilastatin and imipenem at a ratio of 1:1 , is a carbapenem antibiotic, which has both strong broad-spectrum antibacterial activity and β-lactamase inhibitory effect, and is especially suitable for combined infection of multiple bacteria...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P7/40C12R1/01
Inventor 王普何军邀许江丽钟成刚吕亚萍
Owner ZHEJIANG UNIV OF TECH
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