Production of chiral alpha-hydroxy acid and its strain by using biological catalysis method

A technology for strains and hydroxy acids, applied in the field of biocatalytic production of chiral alpha-hydroxy acids and strains thereof, can solve the problems of low atom economy, low conversion rate and high equipment cost

Active Publication Date: 2012-09-12
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the preparation of optically active mandelic acid and its derivatives mainly adopts the chemical resolution method of the racemate: first synthesize the racemate of mandelic acid, and then use a certain method to resolve it. The method of resolution is mainly There are the following types: ①Diastereomeric salt crystallization resolution method, the common problem faced is that it is expensive and has certain toxicity, which causes waste of resources and environmental pollution to a certain extent. At present, the large-scale production of R in my country -Mandelic acid is produced by this method
② Chromatographic separation method, the equipment cost of this method is too high, the consumption is large, the cost is too high, and the processing capacity is small, so it is limi

Method used

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  • Production of chiral alpha-hydroxy acid and its strain by using biological catalysis method
  • Production of chiral alpha-hydroxy acid and its strain by using biological catalysis method
  • Production of chiral alpha-hydroxy acid and its strain by using biological catalysis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1: Screening of strains capable of resolving racemic alpha-hydroxy acids to produce single enantiomers

[0070] Collect soil samples around chemical plants all over the country, take 1g of soil samples and dissolve them in 10ml of 0.9% normal saline, break the glass beads and let stand for 10 minutes, pipette 1-2ml of the suspension to contain 50mL of enrichment medium (Preparation: Mandelic acid 10g, (NH 4 ) 2 SO 4 10g, KH 2 PO 4 2.5g, K 2 HPO 4 2.5g, MgSO 4 .7H 2 O 0.2g, FeSO 4 .7H 2 0 0.03g, 1g of NaCl, make up to 1L with water, adjust the pH to 7.0) in a 250mL Erlenmeyer flask, culture on a shaker for 2 to 3 days (30°C, 150r / min) until the culture medium is turbid, and the turbid enrichment culture The inoculum was transferred to fresh enriched culture medium with 2% inoculum for culture, repeated 2 to 3 times, the concentration of racemic mandelic acid could be increased correspondingly to increase the selection pressure, and then the culture m...

Embodiment 2

[0073] Embodiment 2: the fermentation culture of Pseudomonas aeruginosa CCTCC NO: M 2011394

[0074] Fermentation medium: mannitol 10g / L, yeast extract powder 10g / L, K 2 HPO 4 2.5g / L, KH 2 PO 4 2.5g / L, CuCl 2 0.01g / L, NaCl 1g / L, inducer o-chloromandelic acid 2.5g / L, prepared in tap water, pH 7.0, sterilized at 121°C for 20min. After sterilization, cool down and inoculate. Fill a 250mL Erlenmeyer flask with 20% liquid, inoculate a ring of Pseudomonas aeruginosa CCTCC NO: M 2011394, culture at 30°C and 150rpm for 48 hours, centrifuge the fermentation broth and wash it twice with normal saline, and collect the wet bacteria after centrifugation somatic cells. The maximum specific growth rate μ of Pseudomonas aeruginosa CCTCCNO: M 2011394 was found through experimental research m =0.2322h -1 , the biomass doubling time is t d = 2.98h.

Embodiment 3

[0075] Embodiment 3: the fermentation culture of Pseudomonas aeruginosa CCTCC NO: M 2011394

[0076] Medium: mannitol 10g / L, yeast extract powder 10g / L, K 2 HPO 4 2.5g / L, KH 2 PO 4 2.5g / L, CuCl 2 0.01g / L, NaCl 1g / L, inducer o-chloromandelic acid 2.5g / L, prepared in tap water, pH 7.0, sterilized at 121°C for 20min. Cool after sterilization. Fill a 250mL Erlenmeyer shaker flask with 20% liquid, inoculate a ring of Pseudomonas aeruginosa CCTCC NO: M 2011394, culture at 30°C and 150rpm for 24 hours as seed liquid, and receive sterilized fresh medium with 2% inoculum As a fermentation broth, culture at 30°C and 150rpm for 48 hours with shaking. After the end, the fermentation broth was centrifuged and washed twice with saline. After centrifugation, the wet bacterial cells were collected, and the dry weight of the bacterial cells reached 4.2g / L.

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PUM

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Abstract

The invention provides a novel strain of (S) producing-mandelic acid dehydrogenase, that is, Pseudomonas aeruginosa ZJB1125, which is preserved in the China Center for Type Culture Collection with the preservation number CCTCC No:M 2011394 on 13 November 2011 in Wuhan University, Wuhan, China, 430072. (R)-mandelic acid and related derivatives with high optical purity prepared by the strain of the invention have the high optical purity of more than 99 %, and both the yield and corresponding oxidation product ketonic acid yield of being close to 50 %. By using the strain of the invention to split alpha-hydroxy acid raceme, the advantages of simple and safe operation, low cost, mind reaction conditions and the like are achieved, more importantly, the atom economy of the process is significantly raised, and the invention has good industrial application prospects.

Description

(1) Technical field [0001] The present invention relates to a new bacterial strain producing (S)-mandelate dehydrogenase - Pseudomonas aeruginosa (Pseudomonas aeruginosa) ZJB1125, and its preparation of corresponding (R) - Alpha-hydroxyacids and their use in ketoacids. (2) Background technology [0002] Optically active α-hydroxy acids refer to a class of compounds with hydroxyl and carboxyl groups on the chiral carbon. They are important chiral "building blocks" and can be used for the asymmetric synthesis of a large number of bioactive molecules. Among these compounds, optically pure mandelic acid is considered to be the most important representative from a commercial point of view. Mandelic acid is also known as a-hydroxyphenylacetic acid, mandelic acid, and mandelic acid. The structural formulas of its R-type and S-type are as follows: [0003] [0004] Optically active mandelic acid and its derivatives are extremely important pharmaceutical intermediates: such as R...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P41/00C12P7/42C12P7/40C12R1/385
Inventor 郑裕国薛亚平王威沈寅初
Owner ZHEJIANG UNIV OF TECH
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