游离细胞腈水合酶催化丙烯腈水合反应的双稳态反应动力学

Bi-steady state reaction kinetics of nitrile hydratase in free resting cells

腈水合酶是能够催化丙烯腈水合生成丙烯酰胺的一种重要的工业酶。本研究建立了游离细胞腈水合酶催化丙烯腈水合反应的双稳态反应动力学模型,关联了底物浓度、产物浓度和温度等主要因素对反应速率(表观酶活)的影响。在实验研究的基础上,通过麦夸特及全局最优化算法求解了动力学模型。结果表明,游离细胞腈水合酶催化的双稳态反应动力学模型是比较典型的产物抑制型,当产物浓度逐渐增大时,高浓度的产物将抑制腈水合酶的活性。当底物浓度<10g·L-1时,由于底物加入反应体系时产生的局部瞬时高浓度,腈水合酶催化的丙烯腈水合反应的表观反应速率不随底物浓度变化。当底物浓度≥10g·L-1时,底物产物浓度对反应速率具有显著影响。温度对酶活的影响也十分显著,相同底物产物浓度下,28℃时的酶催化水合反应速率是15℃时的3.3倍。

Nitrile hydratase （NHase） is an important industrial enzyme used for acrylamide production from acrylonitrile hydration.The reaction kinetics of NHases in free resting cells of Rhodococcus sp.or Nocardia sp.was presented based on a bi-steady state assumption.Effects of substrate concentration,product concentration and hydration temperature were investigated experimentally and correlated by Levenberg-Marquardt method and global optimal method.The kinetics model showed that reaction would be inhibited when the concentration of product was high.When the mean concentration of substrate was lower than 10 g·L-1,the extrinsic hydration reaction rate showed a changeless base-value with respect to the altered substrate concentration,due to an instantaneous partial high concentration effect of substrate when being added into the reaction system.Effect of hydration temperature to reaction rate was rather significant.The nitrile hydratase activity at 28℃ was 3.3-fold of that at 15℃ under the same concentrations of substrate and product.