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Method for preparing bacillus thuringiensis and L-menthol thereof

A technology of Bacillus aureus and menthol, applied in the field of Bacillus thuringiensis and its L-menthol preparation, which can solve the problems of unseen separation process and results

Inactive Publication Date: 2011-05-25
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CSIR Company of South Africa used eight-isomer menthol as a substrate to carry out enzymatic asymmetric transesterification resolution, focusing on the separation of alcohol esters and the reuse of invalid bodies, which improved the utilization efficiency of the substrate, but in the example No specific resolution process and results have been seen in (Jennifer Ann Chaplin. Method for preparing (-) menthol and similar compounds: CN, 1452603 [P]. 2003-10-29)

Method used

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  • Method for preparing bacillus thuringiensis and L-menthol thereof
  • Method for preparing bacillus thuringiensis and L-menthol thereof
  • Method for preparing bacillus thuringiensis and L-menthol thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0044] The screening of embodiment 1 bacterial classification

[0045] Through the isolation and screening of 8 soil samples around mint fields, mint potted plants, and daily chemical products factories, 120 strains with different traits and good growth were obtained on the plate, and then 120 strains were screened and re-screened. The 120 isolated strains were first screened by double-plate chromogenic method, and 8 strains were selected that could preferentially hydrolyze L-menthyl acetate. The obtained 8 bacterial strains were re-screened, separated and purified to obtain the bacterial strains of the present invention.

Embodiment 2

[0046] The identification of embodiment 2 strains

[0047] The bacterial species of the present invention is obtained by extracting the genome sequence and performing 16sRNA sequencing. The obtained sequence was compared with the sequence in RCSB Protein Data Bank, and the bacterial species with a similarity of more than 99% were Bacillus thuringiensis and Bacillus cereus. Then, through nitrate reduction, V-P, xylose utilization, mannitol utilization, egg yolk utilization, casein utilization and other physiological and biochemical experiments, the strain was identified as Bacillus thuringiensis.

Embodiment 3

[0048] The investigation of embodiment 3 strains producing enzyme site and enzymes

[0049] Centrifuge the cultured bacterial solution for 24 hours at high speed to remove the bacterial cells, resuspend the centrifuged bacterial cells in the same volume of normal saline to make a bacterial suspension, add octaisomeric mint propionate to the supernatant and bacterial suspension respectively Alcohol ester, the substrate concentration is 50mM, after 24 hours of reaction, the product is detected by GC. The effective conversion rate in the bacterial suspension was 30%, the ee value was >99%, and the de value was 97.9%; the effective conversion rate in the supernatant was 72%, the ee value was >99%, and the de value was 98.5%. It can be seen that this hydrolase is an extracellular enzyme. Add 5ml of phosphate buffer (pH=7.5, 0.02 5mol / l) and 4ml of polyvinyl alcohol olive oil emulsion into a 50ml Erlenmeyer flask, place it in a water bath at 37°C for 5min, and then add 1ml of the e...

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Abstract

The invention discloses a method for preparing bacillus thuringiensis and L-menthol thereof. The preservation number of the bacillus thuringiensis is CGMCC NO.3189. The method comprises the following steps: 1) inoculating the bacillus thuringiensis in a shaking culture seed liquid from an inclined surface; 2) inoculating the seed liquid into 1-2L of fermentation medium for culturing; 3) removing the cell by centrifuging the culture liquid obtained after the fermentation culturing so as to obtain the fermentation supernatant containing extracellular lipase; and adjusting the pH value by hydrochloric acid solution or sodium hydroxide solution; 4) adding octo-isomer racemic menthyl acrylate in the fermentation supernatant to carry out asymmetric hydrolysis reaction, thus obtaining the L-menthol and non-hydrolyzed menthyl acrylate; and 5) extracting by a n-hexane to obtain a mixture; and separating the mixture by column chromatography, thus obtaining the L-menthol. The bacillus thuringiensis is extracted; and an efficient lipase being able to be excreted by the bacillus thuringiensis is also extracted. The octo-isomer racemic menthyl acrylate or the octo-isomer racemic menthyl acrylate mixture can be efficiently and selectively catalyzed and hydrolyzed, thus obtaining the L-menthol with high purity.

Description

technical field [0001] The invention relates to a bacillus thuringiensis and a preparation method of L-menthol thereof. Background technique [0002] Menthol is a compound with important industrial value at present. It has eight isomers of stereo configuration, but there are great differences in odor and efficacy among them. L-menthol has a fresh, brisk cool air and a strong cooling effect, while several other isomers basically do not have these two effects. Due to seasonal changes and poor cultivation, as well as the impact of the amount of mint planting itself, natural L-menthol sometimes cannot meet the demand, and the insufficient part needs to be met by synthetic menthol. [0003] If the synthetic menthol is racemic menthol, its odor and taste are obviously inferior to naturally occurring L-menthol, so the method for separating L-menthol from racemic menthol is very important. The splitting of menthol mainly includes chemical splitting and biological splitting. Among...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P7/02C12R1/07
Inventor 杨立荣孟彦叶琼吴坚平徐刚
Owner ZHEJIANG UNIV