Tsukamurella-tyrosinosolvens and application thereof in catalysis preparation of (S) -alpha - ethyl -2-oxo-1-pyrrolidine acetic acid prepared by catalysis

A technology resistant to T. tyrosinii and T. tyrosinii, applied in biochemical equipment and methods, bacteria, methods based on microorganisms, etc., can solve the problem of large amount of active Raney nickel and high cost of raw materials , Raw materials and reagents are expensive, etc.

Active Publication Date: 2010-06-23
ZHEJIANG UNIV OF TECH
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  • Abstract
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AI Technical Summary

Problems solved by technology

However, in the reduction reaction, the sulfur-containing compound is easy to poison the catalyst, so the active Raney nickel is used in a large amount, usually 7 to 10 times the weight of the reduced compound.
Because active Raney nickel is flammable when exposed to air in a dry state, there are certain safety hazards in the industrial application of this process
The starting material of this process is a sulfur-containing chiral amino acid, which has high raw material cost and is prone to heavy metal pollution.
Ates C et al. (Oxopyrrolidine compounds, preparation of said compounds and their use in the manufacturing of levetircetam and analogues [P]. WO: 03041080A2, 2003-2-20) use (S)-2-aminobutyric acid as a raw material to obtain Amino esters undergo intermolecular nucleophilic substitution with ethyl 4-bromobutyrate molecules, and are synthesized by ring closure in the presence of 2-hydroxypyridine. This method also has the problems of expensive raw material reagents, large losses, and low yields

Method used

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  • Tsukamurella-tyrosinosolvens and application thereof in catalysis preparation of (S) -alpha - ethyl -2-oxo-1-pyrrolidine acetic acid prepared by catalysis
  • Tsukamurella-tyrosinosolvens and application thereof in catalysis preparation of (S) -alpha - ethyl -2-oxo-1-pyrrolidine acetic acid prepared by catalysis
  • Tsukamurella-tyrosinosolvens and application thereof in catalysis preparation of (S) -alpha - ethyl -2-oxo-1-pyrrolidine acetic acid prepared by catalysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1: the isolation of bacterial strain

[0062] Add 1g of soil sample to 10mL of 0.85% normal saline, shake well to make a uniform suspension; draw 0.5mL of soil suspension and inoculate it into a 250mL Erlenmeyer flask containing 50mL of enrichment medium, shake the flask at 30°C and 200rpm Cultivate for 4 to 5 days. After the culture medium becomes turbid, take 1 mL of the culture medium and transfer it to a fresh enrichment medium, continue to cultivate for 4 to 5 days, and repeat the enrichment culture for 3 to 4 times. The enrichment solution was serially diluted and applied to the separation plate, and a single colony was obtained after multiple separations.

[0063] The enrichment medium uses racemic α-ethyl-2-oxo-1-pyrrolidine ethyl ester as the sole carbon source, and its formula is as follows: racemic α-ethyl-2-oxo-1-pyrrolidine ethyl ester Ester 10mmol / L, (NH 4 ) 2 SO 4 2g / L, K 2 HPO 4 2g / L, KH 2 PO 4 1g / L, NaCl 0.5g / L, MgSO 4 ·7H 2 O 0.5g / ...

Embodiment 2

[0065] Embodiment 2: the acquisition of wet fungus cells

[0066] Seed medium formula: glucose 15g / L, yeast powder 12.1g / L, NH 4 Cl 9.5g / L, KH 2 PO 4 1g / L, K 2 HPO 4 2g / L, MgSO 40.6g / L, NaCl 0.6g / L, pH 7.0;

[0067] Fermentation medium formula: glucose 15g / L, yeast powder 12.1g / L, NH 4 Cl 9.5g / L, KH 2 PO 4 1g / L, K 2 HPO 4 2g / L, MgSO 4 0.6g / L, NaCl 0.6g / L, pH 7.0;

[0068] Pick a ring of bacteria from the mature slant and insert it into a 250mL shake flask with 50mL seed medium, cultivate it at 30°C and 200rpm for 24 hours to obtain a seed solution, and then transfer the seed solution to In a 250mL shake flask containing 100mL fermentation medium, culture at 30°C and 200rpm for 36 hours. After the cultivation, the fermentation broth was centrifuged and washed once with a phosphate buffer solution of pH 8.0, and the wet bacterial cells were collected for future use.

Embodiment 3

[0070] The wet thallus obtained in Example 2 was suspended in phosphate buffer (0.1mol / L, pH 6); 10.4g / L of racemate α-ethyl-2-oxo-1-pyrrolidine was added The ester was used as a substrate, and the cell concentration (by dry weight) was 20 g / L, and placed in a shaker at 30° C. at 200 rpm for 24 hours. After the reaction, the reaction solution was centrifuged, and the supernatant was taken, and the yield and optical purity of the product (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid were measured by the aforementioned detection method, and the yield was 37.7%. Optical purity 64% e.e.

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Abstract

The present invention provides a novel strain, tsukamurella-tyrosinosolvens E105, and an application thereof in the chiral biocatalysis preparation of (S)-alpha-ethyl-2-oxo-1-pyrrolidine acetic acid. The tsukamurella-tyrosinosolvens E105 is stored in China Center for Type Culture Collection (CCTCC) at Wuhan University of Science and Technology (postal code: 430072) on Dec.16th, 2009. The preservation series number is CCTCC NO:M209306. The present invention adopts a microorganism preparation method of (S)-alpha-ethyl-2-oxo-1-pyrrolidine acetic acid for the first time and provides a novel strain of high stereoselectivity and capability of preparing (S)-alpha-ethyl-2-oxo-1-pyrrolidine acetic acid of high optical purity. The present invention can make the optical purity of the target product (S)-alpha-ethyl-2-oxo-1-pyrrolidine acetic acid prepared by the chiral biocatalysis of tsukamurella-tyrosinosolvens E105 cells reach 99.4%, and make productivity reach 48.1%. The novel strain obtained by screening provides helpful references in the aspects of research in the microorganism preparation of the key chiral intermediate of levetiracetam and the technique optimization of bioconversion.

Description

(1) Technical field [0001] The invention belongs to the technical field of biocatalysis, and relates to a new bacterial strain - tyrosine-resistant Tsukamurella (Tsukamurella tyrosinosolvens) E105, and its use in the preparation of (S)-α-ethyl-2- Application of oxy-1-pyrrolidine acetic acid. (2) Background technology [0002] The chemical structural formula of (S)-α-ethyl-2-oxo-1-pyrrolidine acetic acid is: [0003] [0004] (S)-α-Ethyl-2-oxo-1-pyrrolidineacetic acid is synthesized from levetiracetam (LEV, trade name: Keppra ) is an important chiral intermediate. The chemical name of levetiracetam is: (S)-α-ethyl-2-oxo-1-pyrrolidineacetamide, which is a nootropic drug with a pyrrolidone structure developed by Belgian Union Chemical Company (UCB). It has high activity on memory impairment caused by scopolamine, and has anticonvulsant and antiepileptic effects at the same time. Its S-isomer (levetiracetam) has biological activity, while the R-isomer has no activity. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P17/10C12R1/01
Inventor 王普何军邀袁帅
Owner ZHEJIANG UNIV OF TECH
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