Method for enzymatic resolution of N-methyl-D-aspartic acid
A technology for aspartic acid and enzymatic separation, applied in the field of bioengineering
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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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