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A method for preparing r-mandelic acid by a two-step microbial transformation method

A technology for microbial transformation and mandelic acid, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of large pollution, low utilization rate of raw materials, high cost, etc., achieve high molar conversion rate, easy operation, The effect of increasing productivity

Active Publication Date: 2019-05-28
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the invention solves the problems of low raw material utilization rate, large pollution and high cost in the prior art

Method used

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  • A method for preparing r-mandelic acid by a two-step microbial transformation method
  • A method for preparing r-mandelic acid by a two-step microbial transformation method
  • A method for preparing r-mandelic acid by a two-step microbial transformation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] (1) Slant culture: Saccharomyces cerevisiae CGMCC No.3361 was inoculated into the slant medium, and cultured at 30° C. for 3 days. The composition of the slant medium: wort juice 10g / L, yeast powder 3g / L, peptone 5g / L, glucose 10g / L, agar 20g / L, solvent is water, pH is natural; After cooling, make a slope.

[0028] (2) Seed culture: use an inoculation needle to take an inoculation loop of bacteria from the slant culture medium and inoculate it into 4 bottles of 250ml Erlenmeyer flasks containing 100ml of seed culture medium, and cultivate at 30°C and 180rpm for 24 hours to obtain seed liquid. Seed medium composition: glucose 30g / L, yeast powder 3g / L, ammonium sulfate 5g / L, KH 2 PO 4 1g / L, K 2 HPO 4 ·3H 2 O 1g / L, MgSO 4 0.25g / L, the solvent is water, the pH is natural, and sterilized at 120°C for 20min.

[0029] (3) Fermentation culture: Inoculate the seed liquid in 8 bottles of 1000ml Erlenmeyer flasks containing 500ml fermentation medium with an inoculum size ...

Embodiment 2

[0037] In the Erlenmeyer flask containing the phosphate buffer of 7.0 with 20ml pH, add embodiment 1 method gained wet thallus to 0.04, 0.06, 0.08, 0.10, 0.12, 0.14 and 0.16g / ml (dry weight), add respectively final concentration 0.11 mol / L ethyl benzoylformate, respectively placed in a shaker at 30°C and 180rpm for 48 hours. After the reaction, the transformation liquid was centrifuged, and the supernatant was extracted with ethyl acetate to detect the transformation. The ratio and the enantiomeric excess value of ethyl mandelate are shown in Table 2. Separation and purification are the same as in Example 1.

[0038] The results show that the conversion rate tends to increase with the increase of the bacterial concentration, and the increase of the bacterial mass indicates that the amount of carbonyl reductase as a biological reaction catalyst increases. For the microbial conversion reaction accompanied by coenzyme regeneration, the bacterial The increase in the amount not on...

Embodiment 3

[0042] In the Erlenmeyer flask containing the phosphate buffer of 7.0 with 20ml pH, add embodiment 1 method gained wet thallus to 0.10g / ml (dry weight), add 0.11mol / L ethyl benzoylformate respectively, place respectively at 30 ℃, 180rpm shaker for 12, 24, 36, 48 and 60h. After the reaction, the conversion solution was centrifuged, and the supernatant was extracted with ethyl acetate to extract the substrate and product, and the conversion rate and R-mandelic acid were detected by gas chromatography. Ethyl ester enantiomeric excess value, the results are shown in Table 3. Separation and purification are the same as in Example 1.

[0043] The results showed that the conversion rate tended to increase with the increase of the conversion time, and the conversion rate basically reached the maximum value when the reaction time was 48 hours. Saccharomyces cerevisiae CGMCC No.3361 reduction substrate ethyl benzoylformate can obtain highly optically pure R-mandelic acid ethyl ester, a...

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Abstract

The invention discloses a method for preparing R-mandelic acid by the aid of two-step microbial transformation processes. The method includes carrying out transformation reaction on ethyl benzoylformate, first thalli and buffer solution with a pH (potential of hydrogen) value of 6-8 under conditions of the temperature of 20-45 DEG C and the speed of 100-200 rpm to obtain R-ethyl mandelate; carrying out reaction on the R-ethyl mandelate, second thalli, organic solvents and buffer solution with a pH value of 7-9 under conditions of the temperature of 20-40 DEG C and the speed of 100-200 rpm to obtain the R-mandelic acid. The ethyl benzoylformate is used as a substrate during the transformation reaction, the first thalli are obtained by means of fermentation cultivation by the aid of saccharomyces cerevisiae and are used as catalysts during the transformation reaction, and the buffer solution with the pH value of 6-8 is used as a reaction medium during the transformation reaction. The R-ethyl mandelate is used as a substrate during the reaction, the second thalli are obtained by means of fermentation cultivation by the aid of bacillus cereus and are used as catalysts during the reaction, the organic solvents are used as complex solubilizers during the reaction, and the buffer solution with the pH value of 7-9 is used as a reaction medium during the reaction. The method has the advantages that the method is low in cost and is environmentally friendly, and the reaction conditions are mild; the R-mandelic acid is easy to industrially produce on a large scale; the method is easy and convenient to implement and high in molar transformation rate; the yield can be increased and can reach 99.8%, and the ee value can be increased and can reach 100%.

Description

(1) Technical field [0001] The present invention relates to the preparation of a kind of R-mandelic acid, particularly a kind of new technology that uses ethyl benzoylformate as raw material to realize reduction and hydrolysis respectively through two-step microbial conversion method to synthesize R-mandelic acid. Saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.3361 converts ethyl benzoylformate to produce R-mandelic acid ethyl ester and Bacillus cereus (Bacillus cereus) CGMCC No.12336 hydrolyzes R-mandelic acid ethyl ester to produce R-mandelic acid . (2) Background technology [0002] Mandelic acid (Mandelic acid, MA), scientific name α-hydroxyphenylacetic acid, also known as mandelic acid, phenylglycolic acid. Chiral mandelic acid is an important amino acid synthesis precursor and drug intermediate. R-mandelic acid is an important intermediate in the synthesis of antibiotics such as penicillin and cefradine, and can also be used in the synthesis of antitumo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/42C12R1/865C12R1/085
CPCC12P7/42
Inventor 欧志敏马兰
Owner ZHEJIANG UNIV OF TECH
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