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Microbacterium chocolatum and method for preparing (4S,5R)-half ester by using same

A technology of microbacteria and chocolate, which is applied in the direction of bacteria and fermentation, can solve the problems of high cost, insufficient selectivity of pig liver esterase, and restrictions on the large-scale acquisition and use of catalysts, and achieves increased yield, easy preparation, and convenient cultivation Effect

Inactive Publication Date: 2012-08-22
SHANGHAI INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main problem at present is that the selectivity of pig liver esterase is not high enough and expensive, and its animal source limits the large-scale acquisition and use of the catalyst

Method used

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  • Microbacterium chocolatum and method for preparing (4S,5R)-half ester by using same
  • Microbacterium chocolatum and method for preparing (4S,5R)-half ester by using same
  • Microbacterium chocolatum and method for preparing (4S,5R)-half ester by using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Cultivation of Microbacterium chocolate

[0053] Fresh slant medium: glucose 15.0 g / L, yeast extract 5.0 g / L, peptone 10.0 g / L, MgSO 4 1g / L, agar 15-20 g / L, pH 7.0.

[0054] Inclined seed cultivation

[0055] take refrigerator 4 o C preserved Microbacteria chocolate ( Microbacteriumchocolatum ) SIT101 CGMCC NO.4436 strain, after taking it out, inoculate it on the above-mentioned fresh slant medium, at 30 oC cultured for 48 h to obtain Microbacterium chocolate ( Microbacteriumchocolatum ) SIT101 bevel seed, and save it in 4 o C in the refrigerator.

[0056] Fermentation medium: glucose 15.0 g / L, yeast extract 5.0 g / L, peptone 10.0 g / L, MgSO 4 1 g / L, pH 7.0.

[0057] take 4 o C preserved Microbacteria chocolate ( Microbacteriumchocolatum ) SIT101 CGMCC NO.4436 slanted seeds, pick a ring and inoculate into a 250 ml shake flask containing 50 ml fermentation medium, at 30 o C, 160 rpm shaking culture for 48 h, at 10000 g Centrifuge for about 15 min to obtai...

Embodiment 2

[0060] Preparation of meso-dimethyl ester

[0061] The meso-diacid (28.32 g, 0.08 mol), anhydrous methanol (16.2 mL, 0.4 mol),

[0062] Concentrated sulfuric acid (0.4 mL, 7.5 mmol) and benzene (65 mL) were heated to reflux for 6 h with stirring. The solvent was removed under reduced pressure, and water (40 mL) and ethyl acetate (40 mL) were added to the residue. After the organic phase was separated, the aqueous phase was extracted three times with ethyl acetate (10 mL). Combine the ethyl acetate phases and wash with 2 M Na 2 CO 3 (3 times in total, 5 ml each time) and water (4 times in total, 5 ml each time) to wash. Afterwards, it was dried with anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain meso-dimethyl ester (II), with a yield of about 92% and a purity of 99%.

[0063] Get the microbacteria chocolate ( Microbacteriumchocolatum ) SIT101 resting cells, suspended in 9.5ml sodium phosphate buffer solution (0.1 M, pH 8.0), adding...

Embodiment 3

[0065] Get the microbacteria chocolate ( Microbacteriumchocolatum ) SIT101 resting cells, suspended in 9.5 ml of sodium phosphate buffer solution (0.1 M, pH 8.0), adding 0.1 mmol of meso-dimethyl ester obtained in the example, and then adding 0.5 ml of methanol, and the reaction mixture was heated at 30° C, 160 r / min shaking reaction on a constant temperature shaking table, intermittent sampling is extracted with ethyl acetate, and the enantiomeric excess value and the productive rate of the product are analyzed by chiral liquid chromatography. After reacting for 24 hours, (4 S , 5 R )-hemimethyl ester in a yield of 87% with an enantiomeric excess (e.e.) greater than 99%.

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Abstract

The invention discloses microbacterium chocolatum SIT101, CGMCCNO. 4436, and a method for preparing (4S,5R)-half ester by asymmetrical hydrolysis of meso diester (II) in the presence of microbacterium chocolatum serving as a biocatalyst. The method comprises the following steps of: putting microbacterium chocolatum cells in buffer solution, adding the meso diester to the buffer solution, and catalyzing the meso diester to asymmetrically hydrolyze so as to obtain a target product (4S,5R)-half ester. In the method for preparing the (4S,5R)-half ester by taking the microbacterium chocolatum as the biocatalyst, the used biocatalyst is easy to prepare, reaction conditions are mild, the yield of the (4S,5R)-half ester is up to 95 percent, enantiomeric excess is more than 99 percent, production cost is low, and the preparation method has a good industrial application and development prospect.

Description

technical field [0001] The present invention relates to a kind of microbacterium and its application, more specifically relate to a kind of chocolate microbacterium and use this chocolate microbacterium as biocatalyst to prepare as shown in formula (I) (4 S , 5 R )-half-ester method. [0002] [0003] where R is C 1 ~C 3 Alkyl, C 1 ~C 3 Alkenyl; Ar is phenyl. Background technique [0004] Known (4 as shown in formula (I) S , 5 R )-half esters are synthesized d - Biotin ( d -Biotin, also known as the key intermediate of vitamin H and coenzyme R). [0005] d -Biotin, also known as vitamin H, is one of the water-soluble B vitamins. It is a coenzyme of carboxylase and an important coenzyme in the intermediate metabolism of sugar, protein and fat. Except some bacteria, yeast and plants can synthesize biotin by themselves, almost all animals and some microorganisms need to obtain biotin from exogenous sources. Biotin has a wide range of uses, 20-30% is used for me...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P13/02
Inventor 徐毅吴范宏陈建波周音卉吴小梅姚跃良王东升朱婷
Owner SHANGHAI INST OF TECH
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