微生物法生产丙烯酰胺的研究(Ⅱ)——腈水合酶催化反应动力学与失活动力学

Study on Production of Acrylamide by Microbial Method (Ⅱ) ——Enzyme catalytic kinetics and de-active dynamics of nitrile hydratase

在以丙烯腈为原料 ,微生物转化生产丙烯酰胺的过程中 ,酶催化反应是过程的关键。为了了解酶催化的动力学 ,本研究以自由细胞的酶为催化剂 ,进行了腈水合酶的反应动力学和失活动力学的研究。首先研究了菌体浓度、温度、pH值、丙烯腈浓度、丙烯酰胺浓度等对腈水合酶催化反应速度的影响。结果表明 ,在这些因素中 ,温度和丙烯酰胺浓度是最主要的影响因素。 2 8℃时酶活为 5 6 5 9u mL(菌液 ) ,在 5℃时的反应速率仅为 2 8℃时的11 72 % ,相应的表观酶活为 6 6 3u mL(菌液 )。而在丙烯酰胺 45 %浓度条件下的酶活大约只有丙烯酰胺 5 %浓度下的酶活的 1 2。经过对不同温度下的反应速度的研究 ,得到腈水合酶水合反应的活化能为 6 5 5 7kJ·mol- 1 。本文进一步研究了自由细胞状态下 ,菌体浓度、pH值、温度、丙烯腈浓度、丙烯酰胺浓度对腈水合酶失活的影响 ,得到了失活动力学。结果表明 ,在这些因素中 ,对酶失活影响的最主要因素还是温度和丙烯酰胺浓度。尤其当丙烯酰胺浓度到达 35 %时 ,酶活下降得很快 ,在 5 5h后 ,酶活几乎为零。而在丙烯酰胺浓度为 10 %的情况下 ,5 5h的酶活仍然还存在约 5 0 %。试验结果还表明 ,丙烯腈对酶的稳定性的影响很小。经过数据处理 ,得到的 2 8℃的酶失活速率常数为 5℃下的 2 1

The hydration reaction by microbial method is the crisis of the procedure of acrylamide production from acrylonitrile. This research studied the enzyme catalytic kinetics and de\|active kinetics of nitrile hydratase in the type of free cell. Firstly, the effects of the concentration of cells、the temperature、pH value、the concentration of acrylonitrile and the concentration of acryla mide on the activity of nitrile hydratase was investigated. The result is that the temperature and the concentration of acrylamide are the most important among these factors. The activity of the nitrile hydratase was 5659u/mL (broth) at 28℃; the counterpart was only 663u/mL (broth)at 5℃. And the activity of NHase in solution of 45% acrylamide was just about half of that in solution of 5% acrylamide. After study on the relation of temperature and the reaction speed, It was found that the activation energy of the hydration of NHase was 65.57kJ·mol -1 . This paper studied the effects of concentration of cells、temperature、pH value、concentrations of acrylonitrile and acrylamide on the deactivation of Nhase, as well as the related enzyme de active kinetics. The result also showed that the temperature and the concentration of acrylamide are the most important among these factors. In solution of 35% acrylamide, the residual activity was about 0% of the original value after 55h; but in solution of 10% acrylamide, after the same period of time, the residual activity was 50% of the original one. It was also found that the concentration of acrylonitrile had little effect on the stability of NHase. The coefficient of deactivation at 28℃ was 21.77 times of the one at 5℃. Correlating the temperature and the coefficient of deactivation, the activation energy of the de active reaction was found to be 92.28 kJ·mol -1 .