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Method for preparing chiral amine through asymmetric reduction under catalysis of marine strain

An asymmetric and chiral amine technology, which is applied in the field of preparation of chiral amines by catalyzing asymmetric reduction of marine strains, can solve the problems of low reaction selectivity, no gene and protein structure, etc., and achieve high yield, simple equipment, and optical purity high effect

Inactive Publication Date: 2015-04-22
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the reaction selectivity of the enzyme is still low, and there is no report on gene and protein structure information.

Method used

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  • Method for preparing chiral amine through asymmetric reduction under catalysis of marine strain
  • Method for preparing chiral amine through asymmetric reduction under catalysis of marine strain
  • Method for preparing chiral amine through asymmetric reduction under catalysis of marine strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] (1) Cultivation of Pseudomonas kilonsis: Inoculate the strain into 50L216L medium with an inoculation volume of 1%. The composition of 216L medium is 10.0g / L peptone, 5.0g / L yeast powder, 0.5g / L beef extract, 0.5g / L sodium citrate, 0.2g / L NH 4 NO 3 , 1.0g / L NaAc, prepared with sea water. The culture conditions are as follows: initial pH 7.0, volume fraction of liquid volume 10%, culture temperature 30°C, shaker speed 150 rpm, culture time 72 hours.

[0017] Preparation of cells: The fermentation broth obtained at the end of the culture was centrifuged in a refrigerated centrifuge (4°C, 10,000rpm, 15min) to obtain the cells, the supernatant was discarded, the pellet was resuspended with Tris-HCl buffer (pH7.6), and washed thoroughly After centrifugation, the operation was repeated 3 times to obtain cells.

[0018] (2) Whole-cell catalytic reaction: the reaction system is 0.5mol / L 4-methyl-2-pentanone, 1mol / L alanine, 1mol / L glycerol, 0.01mol / L Tris-hydrochloric acid b...

Embodiment 2

[0021] (1) Experimental procedures are as in (1) to (2) of Example 1.

[0022] (2) Whole-cell catalytic reaction: the reaction system is 0.02mol / L 2-butanone, 0.2mol / L NH 4 Cl, 0.2mol / L glucose, 0.01mol / L sodium acetate buffer, 1.5g / L cells, reaction system pH 13, reaction volume 500mL, at 50°C, 200rpm shaking reaction, reaction time 30h.

[0023] (3) Separation and detection: Use 6mol / L NaOH to adjust the pH to 14, then extract with 250mL of methyl tert-butyl ether, the separated methyl tert-butyl ether layer is dehydrated with anhydrous sodium sulfate, vacuum at low temperature Dry, dissolve the residual solid with a mixed solution of n-hexane and isopropanol (volume ratio is 9:1), detect with high performance liquid chromatography, calculate the yield of product amine R-2-butylamine to be 72.1%, e.e. value was 92.7%.

Embodiment 3

[0025] (1) Experimental procedures are as in (1) to (2) of Example 1.

[0026] (2) Whole-cell catalytic reaction: the reaction system is 0.01mol / L acetophenone, 0.02mol / L ammonia water, 0.01mol / L isopropanol, 0.2mol / L Tris-hydrochloric acid buffer, 0.025g / L cells , the reaction volume is 20L, the pH of the reaction system is 7, the reaction volume is 5L, the reaction is shaken at 10°C at 300rpm, and the reaction time is 180h.

[0027] (3) Separation and detection: Use 6mol / L NaOH to adjust the pH to 14, and then extract with 10L methyl tert-butyl ether. The separated methyl tert-butyl ether layer is dehydrated with anhydrous sodium sulfate, and vacuumed at low temperature After drying, the residual solid was dissolved with a mixture of n-hexane and isopropanol (9:1 by volume), and detected by high performance liquid chromatography. The yield of the product S-phenylethylamine was 80.7%, and the e.e. value was 90.2%.

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Abstract

The invention provides a method for preparing chiral amine through asymmetric reduction under the catalysis of a marine strain, and relates to chiral amine. The method comprises the following steps: inoculating a strain into a 216L culture medium, culturing, then centrifuging the obtained fermentation liquid to obtain cells, and redissolving and washing with a buffer solution, thus preparing cell sap; and adding ketone used as a substrate into the obtained cell sap, then adding an amino donor and a cosubstrate for cyclic regeneration of coenzyme, and performing asymmetric reduction and amination under the catalysis of the cells, thus obtaining the chiral amine product. The new strain which contains NADH (nicotinamide adenine dinucleotide)-dependent amine dehydrogenase and is different from strains reported before is obtained through the screening of marine microorganisms, and the reaction system and reaction conditions of preparing chiral amine through the asymmetric reduction and amination of ketone under the catalysis of full cells of the strain are optimized. The optical purity of the obtained chiral amine product is up to 90% or above, and the yield is up to 70% or above. The method provided by the invention is convenient to operate, has the advantages of high optical purity of the product, high yield and the like, is simple in equipment and has better industrial application prospects in the field of preparing chiral amine under biological catalysis.

Description

technical field [0001] The invention relates to a chiral amine, in particular to a method for preparing a chiral amine by catalytic asymmetric reduction of a marine strain. Background technique [0002] Chiral amines are important pharmaceutical and fine chemical intermediates, and can also be used as chiral sources of metal chiral ligands and chiral auxiliaries [Applied Organometallic Chemistry, 2011,25:110-116], which can catalyze the asymmetric reduction. Chiral amines can also be used as chiral resolution reagents for racemate resolution. Chiral amines can be prepared by chemical synthesis and biocatalysis. The chemical preparation methods of chiral amines mainly include: the reduction of amino acids or their derivatives, the coupling of aldehydes or ketones with imines, and the amination and ring opening of epoxy compounds. Biological preparation of chiral amines is mainly achieved by enzymatic resolution or asymmetric reduction. [0003] The enzymes used to prepare...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/00C12R1/38
Inventor 王世珍方柏山
Owner XIAMEN UNIV
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