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A method for enzymatic resolution and preparation of n-methyl-d-aspartic acid

A technology for aspartic acid and enzymatic separation, applied in the field of bioengineering

Active Publication Date: 2021-08-24
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, Pseudomonas is potentially pathogenic

Method used

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  • A method for enzymatic resolution and preparation of n-methyl-d-aspartic acid
  • A method for enzymatic resolution and preparation of n-methyl-d-aspartic acid

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Embodiment 1

[0024] A method for preparing N-methyl-D-aspartic acid by enzymatic resolution, said method comprising the steps of:

[0025] 1) Bacillus subtilis WB600 cells transformed into the pMA5-YwbN-trsox plasmid (SEQ ID NO.1) were inserted into 5L TB medium at an inoculum size of 5%, at a temperature of 30° C., and a stirring paddle speed of 150 rpm, and cultured for 36 hours; fermentation The TrSOX activity of the liquid supernatant reached 1200U / mL;

[0026] 2) Mix the above 5L of the fermentation supernatant containing 1200U / mL TrSOX with 5L of 20% N-methyl-DL-aspartic acid solution, the impeller speed is 150rpm, the pH value is maintained at 6, and the reaction temperature is maintained at 40°C, react for 12 hours, use HPLC and TLC to detect the content of N-methyl-L-aspartic acid and L-aspartic acid, the demethylation conversion rate of N-methyl-L-aspartic acid reaches About 48%.

[0027] 3) After the enzymatic conversion, 0.2% (mass volume ratio) of alum was added to flocculat...

Embodiment 2

[0029] A method for preparing N-methyl-D-aspartic acid by enzymatic resolution, said method comprising the steps of:

[0030] 1) Bacillus subtilis WB600 cells transformed into the pMA5-YwbN-trsox plasmid (SEQ ID NO.1) were inserted into 5L TB medium at an inoculum size of 5%, at a temperature of 40° C., and a stirring paddle speed of 150 rpm, and cultured for 36 hours; fermentation The TrSOX activity of the liquid supernatant reached 2100U / mL;

[0031] 2) Mix the above 5L of the fermentation supernatant containing 2100U / mL TrSOX with 5L of 20% N-methyl-DL-aspartic acid solution, the impeller speed is 150rpm, the pH value is maintained at 7, and the reaction temperature is maintained at 60°C, react for 24 hours, use HPLC and TLC to detect the content of N-methyl-L-aspartic acid and L-aspartic acid, the demethylation conversion rate of N-methyl-L-aspartic acid reaches About 80%.

[0032] 3) After the enzyme conversion is completed, add 0.3% (mass volume ratio) of alum to flocc...

Embodiment 3

[0034] A method for preparing N-methyl-D-aspartic acid by enzymatic resolution, said method comprising the steps of:

[0035]1) Bacillus subtilis WB600 cells transformed into the pMA5-YwbN-trsox plasmid (SEQ ID NO.1) were inserted into 5L TB medium at an inoculum size of 5%, at a temperature of 40° C., and a stirring blade rotating speed of 150 rpm, and cultured for 48 hours; fermentation The TrSOX activity of the liquid supernatant reached 3200U / mL;

[0036] 2) Mix the above 5L of the fermentation supernatant containing 3200U / mL TrSOX with 5L of 20% N-methyl-DL-aspartic acid solution, the impeller speed is 150rpm, the pH value is maintained at 8, and the reaction temperature is maintained at 70°C, react for 36 hours, use HPLC and TLC to detect the content of N-methyl-L-aspartic acid and L-aspartic acid, the demethylation conversion rate of N-methyl-L-aspartic acid reaches About 95%.

[0037] 3) After the enzyme conversion is completed, add 0.5% (mass volume ratio) of alum t...

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Abstract

The invention discloses a method for enzymatic resolution and preparation of N-methyl-D-aspartic acid, the method comprising the following steps: (1) constructing the Thermomicrobium roseum sarcosine oxidase gene in pMA5-YwbN- trsox plasmid, and transformed into Bacillus subtilis WB600 cells for secretory expression; (2) Bacillus subtilis WB600 cells transferred to pMA5-YwbN-trsox gene plasmid were fermented, and the culture supernatant obtained was mixed with N-methyl-DL- The aspartic acid solution is mixed, and the biocatalysis with chiral selectivity is carried out for N-methyl-L-aspartic acid; (3) N-methyl-D aspartic acid is separated by isoelectric point crystallization .

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for enzymatic resolution and preparation of N-methyl-D-aspartic acid. Background technique [0002] N-methyl-D-aspartic acid (NMDA) is an amino acid derivative naturally occurring in the animal body, the molecular formula is C 5 h 9 NO 4 , with a molecular weight of 147.13, is an important excitatory neurotransmitter L-glutamate homologue in the mammalian central nervous system. NMDA generally only exists in the nervous and endocrine tissues of humans and animals, participates in the regulation and secretion of the hypothalamus-pituitary growth axis, and can significantly increase the intracellular Ca 2+ NMDA can also play an important role in the regulation of neuroendocrine, significantly promoting the secretion of growth hormone (GH) in animals; by inhibiting the DNA synthesis of PC12 cells and affecting the proliferation of cells, it can be used for anti-tu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/20C12P41/00
CPCC12P13/20C12P41/001
Inventor 辛瑜张梁张瑶高秋月
Owner JIANGNAN UNIV
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