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Microbacterium oxydans and method for preparing chiral bis(trifluoromethyl) phenyl ethanol by using same

A technology for oxidizing microbacteria and isopropanol, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of only 15% yield, low enzyme selectivity, and low production capacity, and achieve Easy extraction and separation, high product concentration and low cost

Active Publication Date: 2012-03-21
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are some problems in obtaining the chiral alcohol by whole-cell biocatalysis at present: such as low production capacity, and often contain a variety of ketoreductases in microorganisms, resulting in low selectivity of the final enzyme and many by-products (Homann screened 4 strains of product R-type microorganisms, the highest ee value is 92%
[0007] 2006, 17, 2000-2005) By screening alcohol dehydrogenase to convert substrate S, the alcohol dehydrogenase derived from Lactobacillus kefir can obtain P (ee>99%), but NAD(P)H needs to be added for coenzyme recycles with only 15% yield

Method used

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  • Microbacterium oxydans and method for preparing chiral bis(trifluoromethyl) phenyl ethanol by using same
  • Microbacterium oxydans and method for preparing chiral bis(trifluoromethyl) phenyl ethanol by using same

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Embodiment 1

[0024] Example 1 strain screening

[0025] Screening of strains producing carbonyl reductase: soil samples were collected from orchards, enriched in MSM medium containing acetophenone (concentration: 1 mM), and cultured at 30° C. and 220 rpm for 7 days. 1% of the enriched solution was added to MSM medium containing acetophenone (concentration: 1 mM) for further enrichment, and culture was continued for 7 days at 30° C. and 220 rpm. The formula of MSM medium is Na2HPO42g / L, KH2PO41g / L, NH4Cl0.4g / L, MgCl20.4g / L. Dilute the second enrichment culture solution to an appropriate concentration, spread it on a screening plate (MSM+acetophenone 1mM+yeast powder 0.1g / L+agar powder 15g / L), culture at 30°C for 1-3 days, each plate Many single colonies of microorganisms can be seen on the surface, and these strains may be carbonyl reductase-producing strains. The substrate 3,5-bis-trifluoromethyl acetophenone (substrate S) was further screened for biocatalysis in order to obtain microbial ...

Embodiment 2

[0028] The identification of embodiment 2 screening bacterial strains

[0029] In order to confirm the taxonomic status of the strain and understand its performance in detail, the strain was initially identified. This strain has the following characteristics:

[0030] Colony: light yellow or yellow colony, smooth surface, neat edges, opaque.

[0031] Physiological and biochemical characteristics: Gram-positive bacteria, rod-shaped, aerobic, producing carbonyl reductase.

[0032] 16S rRNA identification: Genomic DNA was used as a template to amplify the 16S rRNA sequence with bacterial universal primers 27f and 1492r. Its sequence is as follows (1486bp):

[0033] AGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGTGAACACGGAGCTTGCTCTGTGGGATCAGTGGCGAACGGGTGAGTAACACGTGAGCAACCTGCCCCTGACTCTGGGATAAGCGCTGGAAACGGCGTCTAATACTGGATATGCGACGTGATCGCATGGTCTGCGTCTGGAAAGAATTTCGGTTGGGGATGGGCTCGCGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGTCGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAG...

Embodiment 3～5

[0035] Examples 3-5 CCTCC M 2010179 growing cells transformed substrate S into product P.

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Abstract

The invention belongs to the technical field of biological catalysis, and relates to microbacterium oxydans and a method for preparing optically pure (R)-1-[3,5-bis(trifluoromethyl) phenyl] ethanol by using the microbacterium oxydans. The preservation number of the microbacterium oxydans C3 is CCTCC M2010179. The microbacterium oxydans is cultured at the temperature of 27-33 DEG C for 24-48 hours, and then substrate 3,5-bis-trifluoromethyl acetophenone is added to convert auxocyte; or the culture bacterium is collected and is resuspended in a buffer solution, and then the substrate is added to convert resting cells so as to generate a target product, namely the optically pure (R)-1-[3,5-bis(trifluoromethyl) phenyl] ethanol. The microbacterium oxydans has the advantages of easiness in industrialization, easiness in preparation of a catalyst thereof, mild reaction conditions and high conversion efficiency.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, and relates to microbacteria oxidizing and a method for preparing optically pure (R)-1-[3,5-bis(trifluoromethyl)phenyl]ethanol using the bacteria. Background technique [0002] Optically active (R)-1-[3,5-bis(trifluoromethyl)phenyl]ethanol (Chinese name is (R)-1-3,5-bistrifluoromethylphenylethanol, CAS number is 127852-28-2 , hereinafter abbreviated as P) is the key chiral intermediate of the chiral drugs Aprepitant (aripitant, trade name Emend) and fosaprepitant dimeglumine developed and produced by Merck, wherein fosaprepitant dimeglumine is the injection form of Aprepitant. The chiral drug Aprepitant is a selective high-affinity receptor blocker for human substance P / neurokinin 1 (NK-1), which has a new pharmacological mechanism of action, mainly through the novel mechanism of blocking the brain's nausea and vomiting signals It has a good effect on the prevention of acute or delayed vomi...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P41/00C12P7/22C12R1/01
Inventor 吴中柳刘艳张超汤传根田小亮裴小琼
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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