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Method for preparing L-menthol from stereo-selective hydrolysis of DL fatty acid menthyl ester by whole-cell biological process

A stereoselective, whole-cell technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of difficult recycling, low reaction equilibrium conversion rate, low product purity, etc. Scope and operational stability, reduction of production cost, high reaction stability effect

Inactive Publication Date: 2007-06-13
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0012] South African CSIR organization (Chaplin J A, Gardiner N S et al., WO 0236795, 2002) utilizes Pseudomonas fluorescens lipase selective transesterification resolution DL-menthol to prepare L-menthol, the reaction equilibrium conversion rate is low (about 20%), The reaction products are complex, including L-menthyl acetate, organic solvent, unconverted stereoisomers, excess esterification agent and reaction by-products, which bring difficulties to the separation of L-menthyl acetate, the separation procedure is cumbersome and the product purity Not high; and using lye to hydrolyze L-menthyl acetate, the conversion rate is low, less than 50%
[0013] Germany Haarmann&Reimer company (Gatfield IL, Hilmer J M et al., US 6706500, 2002) utilizes recombinant Candida Rugosa lipase to selectively hydrolyze DL-menthyl benzoate to prepare L-menthol. This enzyme is not stable in aqueous solution and is difficult to recover Utilization, although the stability of the enzyme can be improved after immobilization, the enzyme inactivation caused by immobilization is serious, and the highest activity yield is only 43%; and the substrate concentration of the enzyme-catalyzed hydrolysis reaction is not high, the highest is about 0.8%

Method used

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  • Method for preparing L-menthol from stereo-selective hydrolysis of DL fatty acid menthyl ester by whole-cell biological process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1D

[0063] The preparation of embodiment 1DL-menthyl acetate

[0064] Add 0.02mol menthol and 0.02mol acetic anhydride to a 100ml three-necked flask equipped with a condensing reflux device and a thermometer, and add 0.4mmol p-toluenesulfonic acid as a catalyst, add n-heptane as a reaction solvent until the reaction volume is 10ml, stir and heat Reflux reaction 4h. After the reaction is completed, cool down to below 40°C, add 5% NaHCO 3 Neutralize the excess acid until no more bubbles are produced, let it stand for stratification, remove the water phase, wash the organic phase with saturated brine, separate the water phase and use anhydrous MgSO 4 Let dry overnight. After filtration, the mixed solution was rotatably evaporated to remove the solvent to obtain pure DL-menthyl acetate. The purity of DL-menthyl acetate detected by gas chromatography was 99.9%, and the yield (calculated as menthol) was 98.5%.

Embodiment 2

[0065] The hydrolysis situation of embodiment 2 different bacterial strains to DL-menthyl acetate

[0066] In 1ml, 50mmol / L potassium phosphate buffer solution (pH7.0), add 13mg of dry cells and 10mg of DL-menthyl acetate, shake and react on a constant temperature shaker at 30°C for 24 hours, after the reaction, the mixture is centrifuged to remove Bacteria, the supernatant was extracted with ethyl acetate to extract the product and substrate, and the e.e. value and conversion rate of the product were analyzed by chiral gas chromatography. The transformation status of each strain is shown in Table 1.

[0067] Table 1

[0068] microbial strain

Embodiment 3

[0069] Example 3 Asymmetric hydrolysis of DL-menthyl acetate with CCTCC M201021 whole cells

[0070] In 1ml, 50mmol / L potassium phosphate buffer solution (pH 7.0), add 13mg of dried bacteria and 10mg of DL-menthyl acetate, shake the reaction on a constant temperature shaker at 30°C for 12, 16, 20, 24, 36 , 48 hours, after the reaction, the mixture was centrifuged to remove bacteria, the supernatant was extracted with ethyl acetate to extract the product and substrate, and the e.e. value and conversion rate of the product were analyzed by chiral gas chromatography. The results are shown in Table 2.

[0071] Table 2

[0072] Response time (hours)

[0073] Table 2 shows that after 24 hours of reaction, the conversion rate is close to 50% of the theory, and the reaction time can be extended to 48 hours, and the optical purity of the product can still reach more than 90% e.e.

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Abstract

The invention relates to the method used to hydrolyze DL-fat acid mint ester and produce L-menthol by full cell biological method stereo-selectivity. It supplies the high stereo-selectivity strain, produces full cell, uses the DL-fat acid mint ester as substrate, hydrolyzes and produces L-menthol by full cell biological method stereo-selectivity. The by-product D-fat acid mint ester can be hydrolyzed to high purity D-menthol of which optical purity is 80%e.e-100%e.e; yield is 30%-60% while producing L-menthol of which optical purity is 90%e.e-100%e.e; conversion rate is 40%-50%. Compared with the international L-menthol preparation method, the adopted full cell catalysis can omit fussy enzyme purification, reduce enzyme activity damage, increase enzyme action substrate range and operation stability, and reduce L-menthol production cost.

Description

technical field [0001] The invention discloses a method for preparing L-menthol by stereoselectively hydrolyzing DL-fatty acid menthyl ester with a whole-cell biological method, and belongs to the technical field of biological method resolution of racemic compounds. Background technique [0002] Menthol is currently one of the most industrially valuable chiral compounds and is widely used in the fields of medicine, food, cosmetics and chiral reagent preparation. Natural menthol is affected by weather, region, etc., and its output and quality fluctuate greatly. Synthetic menthol can solve the above problems. People have carried out a large amount of researches to the synthetic approach of menthol, and many have been applied to industrialized production (Fan Cunliang, Yang Zhongbao, "Perfume Flavor Cosmetics", 2002, the 4th phase, p28). What usually adopt these methods to synthesize is the racemic mixture of L-menthol, and the existence of D-menthol greatly affects the smell ...

Claims

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Application Information

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IPC IPC(8): C12P39/00C12P7/06C12N1/14C12N1/16C12N1/20C12R1/39C12R1/72C12R1/645
CPCY02E50/17Y02E50/10
Inventor 徐岩于丽娟
Owner JIANGNAN UNIV
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