Biological catalysis method for preparing D-amino acid through deracemizing DL-amino acid

An amino acid, deracemization technology, applied in the biological field, can solve the problems of high price, poor process universality, complex separation process, etc., and achieve the effect of good versatility, easy separation, high chemical purity and optical purity

Inactive Publication Date: 2012-07-18
CHONGQING CHIRAL BIOCATALYSIS TECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The biological resolution method uses enzymes such as acylase and lipase as biocatalysts to split the derivatized DL-amino acid. The theoretical yield of D-amino acid is 50%, which involves steps such as derivatization and derivatization, and the process is not universal.
Biological asymmetric synthesis methods such as transaminase method use D-aminotransferase as a biocatalyst, expensive keto acid as raw material, and another D-amino acid as ammonia donor, and the separation process is quite complicated

Method used

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  • Biological catalysis method for preparing D-amino acid through deracemizing DL-amino acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Cell Culture

[0027] 1) Incline culture: put Alcaligens faecali 1. The 1799 strain was inoculated on sterilized solid medium and cultured statically at 30°C for 48h. Solid medium (g / L): peptone 10, beef extract 3, sodium chloride 5, agar 15, pH 7.4~7.6.

[0028] 2) Primary culture: Inoculate the cells cultured on the slant into a test tube containing 5ml of sterilized medium, and culture on a shaker at 30°C for 24 hours. Medium (g / L): peptone 10, beef extract 3, sodium chloride 5, pH 7.0.

[0029] 3) Expansion culture: Inoculate the primary cultured cells in 50ml sterile expansion medium containing inducer at 10% inoculum, and culture on a shaker at 30°C for 24h. Medium (g / L): peptone 10, beef extract 3, sodium chloride 5, L-alanine 10, pH 7.0. The culture solution was centrifuged for 15min (8000rpm) to collect the wet cells.

[0030] In step 1) above, replace with Alcaligens faecali 1.767, 1.924, 1.7686, 1.2006, 1.1837, according to the same steps...

Embodiment 2

[0031] Example 2: Cell Permeabilization Treatment

[0032] Add 50ml of 30% acetone aqueous solution to 50g of wet cells, treat at room temperature for 10min, centrifuge for 15min (8000rpm), wash twice with normal saline, and obtain 48g of permeable cells.

Embodiment 3

[0033] Embodiment 3: D-alanine preparation

[0034] The permeable cells obtained in Examples 1-2 were used as biocatalysts. Take 50g of permeabilized wet cells and suspend them in 1L of distilled water, add 17.8g (0.2mol) DL-alanine, react at 30°C for 24h, monitor the reaction with chiral HPLC until the complete conversion of L-alanine. After the reaction is completed, centrifuge for 15min (8000rpm) to remove the cells. The centrifugate was acidified with concentrated hydrochloric acid to pH 2~3, concentrated to dryness under reduced pressure, added 100ml of absolute ethanol to the residue, filtered to remove insoluble matter, added 7.0ml (0.1mol) propylene oxide to the filtrate, stirred at room temperature for 30min, filtered , the solid was washed with absolute ethanol and dried to obtain 8.0 g of D-alanine with a yield of 45% and ee of 99.8%.

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Abstract

The invention provides a biological catalysis method for preparing D-amino acid through deracemizing DL-amino acid. According to the method, manure production base coli cells subjected to permeability are used as a biological catalyst, L-amino acid oxidase in the biological catalyst is utilized to oxidize and denitrify the specificity of L-antipode into corresponding ketonic acid under the existence of air, and D-antipode is retained; and meanwhile, hydrogen peroxide generated in a decomposition reaction process of co-expressed catalase is utilized. The method avoids derivation and derivation removal steps of a traditional split method, has the characteristics of simple process, low cost, high product optical purity and good environmental protection and is suitable for the industrial production of the D-amino acid.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a new process for preparing D-amino acid by biocatalysis. In particular, a biocatalytic method for preparing D-amino acid by catalyzing the deracemization of DL-amino acid by Alcaligenes faecalis cells. Background technique [0002] In nature, D-amino acid is a rare non-protein source amino acid, and its quantity accounts for about 10% of the more than 300 kinds of amino acids that have been discovered. With the development of medicine and biology, it is found that D-amino acid is closely related to certain diseases such as cataract, early dementia, kidney disease and so on. In addition, D-amino acids have been increasingly used as chiral intermediates in the synthesis of chiral drugs, chiral pesticides and chiral food additives, and are widely used in the fields of medicine, pesticides and food. [0003] The preparation method of D-amino acid mainly includes chemical method and biol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P41/00C12R1/05
Inventor 夏仕文方国兰何从林徐红梅
Owner CHONGQING CHIRAL BIOCATALYSIS TECH
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