Method for preparing L-2-aminobutyric acid by enzyme method
A technology for aminobutyric acid and enzymatic preparation, applied in the direction of fermentation, etc., can solve the problems of low process yield, high cost, unsuitable process conditions, etc., and achieve the effects of low process cost, mild reaction conditions, and easy directional control.
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[0013] Example 1
[0014] In a 5ml reaction flask, add 20mg L-threonine, 32mg ammonium formate (about 2.5 times equivalent), 0.2mgNAD + And 2ml phosphate buffer (Na 2 HPO 4 / NaH 2 PO 4 , 0.1M, pH 7.5), adjust the pH of the solution to 7.5 with concentrated ammonia, and then add 1 mg of crude threonine deaminase enzyme, 2 mg of leucine dehydrogenase crude enzyme and 2 mg of formate dehydrogenase crude enzyme. After 3 hours of reaction with magnetic stirring at 25°C, samples were taken for HPLC analysis, and the conversion rate was >95% (C18 column; UV 200nm detection; mobile phase: 20mM phosphate buffer (pH 3.0) / acetonitrile=95 / 5, v / v ), ee value>99% (CR(+) column; UV 200nm detection; mobile phase: aqueous perchloric acid (pH 1.5)).
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[0015] Example 2
[0016] In a 25ml Erlenmeyer flask, add 50mg L-threonine, 80mg ammonium formate (about 2.5 times equivalent), 2.5mgNAD + And 5ml phosphate buffer (Na 2 HPO 4 / NaH 2 PO 4 , 0.1M, pH 8.0), adjust the pH of the solution to 8.0 with concentrated ammonia, then add 5mg crude threonine deaminase enzyme, 10mg crude leucine dehydrogenase enzyme and 20mg crude formate dehydrogenase enzyme. After reacting on a 160rpm rotary shaker at 37°C for 20 hours, samples were taken for HPLC analysis, and the conversion rate was >99%, and the ee value was >99%.
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[0017] Example 3
[0018] In a 25ml Erlenmeyer flask, add 150mg L-threonine, 480mg ammonium formate (about 2.5 times equivalent), 2.5mgNAD + And 5ml phosphate buffer (Na 2 HPO 4 / NaH 2 PO 4 , 0.1M, pH 7.0), adjust the pH of the solution to 7.0 with concentrated ammonia, then add 2.5mg crude threonine deaminase enzyme, 1mg crude leucine dehydrogenase enzyme and 5mg crude formate dehydrogenase enzyme, After reacting on a 160 rpm rotary shaker at 30°C for 24 hours, samples were taken for HPLC analysis. The conversion rate was about 65%, and the ee value was greater than 99%.
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