Rhodococcus and application thereof in preparation of (S)-(+)-2,2-dimethylcyclopropane carboxylic acid

A technology of dimethyl cyclopropanecarboxylate and dimethyl cyclopropane, which is applied in the field of Rhodococcus ECU 1013, can solve the problem of high price, the enantioselectivity of Rhodococcus cannot meet the requirements of the pharmaceutical industry, and the number of repeated use is small, etc. question

Inactive Publication Date: 2012-10-31
EAST CHINA UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] Therefore, the technical problem to be solved in the present invention is exactly to use highly toxic cyanide in the process of preparing (S)-(+)-DMCPA for prior art catalytic substrate 2,2-dimethylcyclopropanecarbonitrile, to The environment has adverse effects; the existing commercial esterase is expensive, the activity of the enzyme is relatively low, the number of repeated use is not much, and the existing rhodococcus can not meet the requirements of the pharmaceutical industry in terms of enantioselectivity. Provide A strain of Rhodococcus sp. ECU 1013, the preservation number of which is CGMCC No.5911

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0098] Example 1 Shake flask fermentation culture of Rhodococcus sp. ECU 1013 CGMCC No. 5911

[0099] Shake flask fermentation medium formula (g / L): glucose 5, yeast extract 5, NaCl 1, KH 2 PO 4 1. MgSO 4 0.2, CaCl 2 0.1, pH 6.0, prepared with tap water, sterilized at 121°C for 20 minutes. Take the Rhodococcus slant preserved at 4°C, pick a loop and inoculate it into a 500mL Erlenmeyer flask containing a 100mL shake flask fermentation medium, shake culture at 30°C and 180rpm for 36h, the cell viability of the fermentation broth is about 3.2u / L. After the cultivation, the cells were collected by centrifugation at 8800 rpm for 10 minutes, washed twice with saline, and stored at 4°C for later use. The cell viability unit (u) is defined as: under the conditions of 30°C and pH 7.0, the hydrolysis of methyl 2,2-dimethylcyclopropane formate to 1.0 μmol of 2,2-dimethylcyclopropane formic acid is produced every hour. The amount of cells needed.

Embodiment 2

[0100] Example 2 Fermentation tank culture of Rhodococcus sp. ECU 1013 CGMCC No. 5911 and preparation of crude Rhodococcus esterase powder

[0101] Fermentation medium formula (g / L): glucose 20, yeast extract 10, NaCl5, KH 2 PO 4 5. MgSO 4 2. CaCl 2 1, pH 7.5, 121 ℃ high temperature sterilization for 20 minutes. The seed liquid medium formula is the same as the fermentation medium. Take the Rhodococcus slant preserved at 4°C, pick a loop and inoculate it into a 500mL Erlenmeyer flask containing 100mL seed liquid culture medium, and culture it for 12h at 35°C and 180rpm with shaking. Three bottles of seed liquid (300ml) were connected to a 5L fermentor containing 3L of fermentation medium, and fermentation was carried out at 30°C. Aeration: 0.8~1.2vvm, pH is not controlled during fermentation. After culturing for 25 hours, the cell viability of the fermentation broth reached 11u / L, and the cells were collected by centrifugation at 9000 rpm, and the wet weight of the bacteria rea...

Embodiment 3

[0103] Example 3 Rhodococcus sp. ECU 1013 CGMCC No. 5911 cells catalyze the enzymatic hydrolysis of ethyl 2,2-dimethylcyclopropane carboxylate

[0104] Take 0.5 g of the wet cells obtained in Example 1 and suspend them in 10 mL of phosphate buffer (100 mM, pH 7.0), and add 5 mM 2,2-dimethylcyclopropane ethyl carboxylate. The reaction mixture is kept at 30°C, 180 rpm Shake for 72h. After the reaction is over, take 100μL of the reaction solution and add 20% H 2 SO 4 Adjust the pH of the solution to <2, add an equal volume of ethyl acetate to extract the product and substrate, and analyze the ee value and yield of the product through the gas phase. The ee value of the product was 83.9%, and the yield was 21.2%.

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Abstract

The invention discloses a rhodococcus (Rhodococcus sp.) bacterial strain ECU 1013, and the preservation serial number of the bacterial strain is CGMCC No.5911. The invention also discloses a preparation method of esterase of the rhodococcus and a method for catalyzing and preparing the (S)-(+)-2,2-dimethylcyclopropane carboxylic acid [(S)-(+)-DMPCA for short]. The rhodococcus bacterial strain ECU 1013 and the methods have the advantages that 2,2- dimethylcyclopropane carboxylic acid ester is catalyzed by using the rhodococcus and the esterase of the rhodococcus, enantioselective hydrolysis is performed and the (S)-(+)-DMPCA is prepared, the process has the advantages of being high in specifity, good in the catalyzing effect, mild in reaction condition, and the like. The optical purity of the target product (S)-(+)-DMPCA is 98% ee, and the single splitting is above 40%.

Description

Technical field [0001] The present invention belongs to the field of bioengineering, and particularly relates to a strain of Rhodococcus sp. ECU 1013, the preservation number of which is CGMCC No. 5911. The method for preparing the Rhodococcus esterase, and the method for preparing (S)-(+)-2,2-dimethylcyclopropanecarboxylic acid by using the Rhodococcus cell and its esterase to catalyze the preparation. Background technique [0002] (S)-(+)-2,2-Dimethylcyclopropanecarboxylic acid [(S)-(+)-DMCPA] is an important chiral intermediate for the synthesis of cilastatin. Cilsatatin is a renal dehydropeptidase inhibitor developed by Merck (Merck) in the United States. It has no antibacterial effect and is only used as a specific enzyme inhibitor; Imipenem ) Is a β-lactam antibiotic with high antibacterial activity, but it is easily destroyed by kidney dehydropeptidase. Tienam, a composite preparation made of cilastatin and imipenem at a ratio of 1:1, is the first new-type carbapenem ant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/18C12P7/40C12R1/01
Inventor 许建和刘朝红潘江郁惠蕾
Owner EAST CHINA UNIV OF SCI & TECH
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