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Bacillus subtilis and method of using same to prepare L-menthol

A technology of Bacillus subtilis and menthol, applied in the field of L-menthol preparation, can solve problems such as affecting catalytic function, inactivation, and variability, and achieve the effects of reducing purification cost, changing substrate specificity, and increasing solubility

Active Publication Date: 2019-10-25
ANHUI FENGLE PERFUME +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, microorganisms perform catalysis in the organic phase, and many factors will adversely affect the catalytic function.
Most microorganisms are more easily denatured and inactivated in the presence of organic solvents

Method used

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  • Bacillus subtilis and method of using same to prepare L-menthol
  • Bacillus subtilis and method of using same to prepare L-menthol
  • Bacillus subtilis and method of using same to prepare L-menthol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: the screening of Bacillus subtilis:

[0033] (1) Primary screening: Bacillus subtilis strains screened from the soil and stored in a -80°C refrigerator. Take the strains stored in the -80°C refrigerator and melt in 40°C water; 250mL shake flask, 20% liquid volume (seed medium: (g / L): peptone 10.0, yeast powder 5.0, NaCl 10.0, pH 7.0, 121°C , 20min, high-pressure steam sterilization), inoculum size 50μL, 30°C, 150rpm culture overnight activation;

[0034] Take 100 μL of the culture solution and add it to a 250mL shake flask, the filling volume is 20% (fermentation medium: (g / L): peptone 5.0, yeast powder 2.0, (NH4) 2 SO 4 2.0, sucrose 3.0, anhydrous K 2 HPO 4 2.0, NaCl 1.0, anhydrous MgSO 4 0.2, olive oil 10 (add separately after subpackaging), pH 7.0, 115°C, 20min, high-pressure steam sterilization), 30°C, 150rpm for 24h;

[0035]Get 25mL of fermentation broth and centrifuge (8000rpm, 5min), remove the supernatant, and the gained thalline precipit...

Embodiment 2

[0053] Embodiment 2: the solvent selection of preparing L-menthol by catalytic hydrolysis

[0054] Centrifuge (7000rpm, 10min) 25mL of the fermented culture solution of Example 1, remove the supernatant, and the gained thallus is placed in a 50mL Erlenmeyer flask, adding 20ml concentration is the buffer solution disodium hydrogen phosphate-sodium dihydrogen phosphate of 0.1mol / L With substrate DL-mentha acetate, so that the substrate concentration is 2000ppm, add water, organic reaction solvent methanol, ethanol, isopropanol, n-butanol, acetone, or DMSO respectively, so that their final concentration is 20wt%, Seal with parafilm, hydrolyze at 30°C and 150rpm. During the reaction, use 0.1mol / L KOH solution to control the reaction pH to 6.5. After 20h, add twice the volume of organic solvent ethyl acetate for extraction, gas phase CP chiral column detection, Determination of enzyme activity and e.e. p . The results are shown in Table 2.

[0055] The influence of table 2 diffe...

Embodiment 3

[0061] Embodiment 3: add the preparation process of activating ion

[0062] Centrifuge (7000rpm, 10min) 25mL of the fermented culture solution of Example 1, remove the supernatant, and the gained thallus is placed in a 50mL Erlenmeyer flask, adding 20ml concentration is the buffer solution disodium hydrogen phosphate-sodium dihydrogen phosphate of 0.1mol / L and substrate DL-menth acetate, so that the concentration of the substrate is 2000ppm, add organic reaction solvent methanol, so that the concentration is 20wt%, and then add the activation ion K + 、Na + , NH 4 + etc., the amount of activating ions added is the final concentration of 5mmol / L; the sealing film is sealed, 30°C, 150rpm for hydrolysis, during the reaction, use 0.1mol / L KOH solution to control the reaction pH to 6.5. After 20h, add twice the volume Organic solvent ethyl acetate extraction, gas phase CP chiral column detection, determination of enzyme activity and e.e. p . The results are shown in Table 3.

...

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Abstract

The invention provides bacillus subtilis IFE122 and a method of using the same to prepare L-menthol. Bacillus subtilis IFE122 is collected at the China General Microbiological Culture Collection Center at 3# Yard One, Beichen West Road, Chaoyang District, Beijing on June 06th, 2019 under the number of CGMCC No.17904. The method is stable in process, ee value during preparation reaches 99.49%, sideeffects like hydrolysis and racemization caused by polar solvent conversion are overcome, and product purifying difficulty is lowered.

Description

technical field [0001] The invention relates to the technical field of L-menthol preparation, in particular to Bacillus subtilis and a method for preparing L-menthol by Bacillus subtilis. Background technique [0002] Menthol (C 10 h 20 O, molecular weight 156.4), has another name called menthol, and its scientific name is 5-methyl-2-isopropyl cyclohexanol, which is the main component of peppermint essential oil, and its existing state is in the form of free state and ester. host. As a more important terpene chiral flavor, menthol has a wide range of applications in fine chemicals, beverages, food and medicine. There are 3 chiral centers in the molecular structure of menthol, so there will be 8 stereoisomers after chemical synthesis. Among the 8 stereoisomers, L-menthol has a unique minty aroma and a strong cooling effect, and the taste is fresh and light; Aids digestion and promotes penetration. [0003] Generally speaking, the industrial preparation of single-configu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P41/00C12P7/02C12R1/125
CPCC12P7/02C12P41/005C12N1/205C12R2001/125
Inventor 朱林江章保华方靖怡王广强朱俊峰陆跃乐陈小龙
Owner ANHUI FENGLE PERFUME
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