Department of Civil and Mechanical Engineering, University of Cassino and of Lazio Meridionale, Via Di Biasio 43, 03043 Cassino (FR), Italy.
Bioresour Technol. 2013 Aug;142:320-8. doi: 10.1016/j.biortech.2013.04.126. Epub 2013 May 9.
In this study the amidase kinetics of an in situ NHase/AMase cascade system was explored as a function of operational parameters such as temperature, substrate concentration and product formation. The results indicated that controlling amidase inactivation, during acrylonitrile bioconversion, makes it possible to recover the intermediate product of the two-step reaction in almost a pure form, without using purified enzyme. It has been demonstrated, in long-term experiments performed in continuous stirred UF-membrane bioreactors, that amidase is kinetically controlled by its proper substrate, depending on the structure, and by acrylonitrile. Using acrylamide, AMase-stability is temperature dependent (5°C, kd=0.008 h(-1); 30°C kd=0.023 h(-1)). Using benzamide, amidase is thermally stable up to 50°C and no substrate inhibition/inactivation occurs. With acrylonitrile, AMase-activity and -stability remain unchanged at concentrations <200 mM but at 200 mM, 35°C, after 70 h process, 90% irreversible inactivation occurs as no AMase-activity on benzamide revives.
在这项研究中，考察了原位 NHase/AMase 级联系统的 amidase 动力学，作为操作参数（如温度、底物浓度和产物形成）的函数。结果表明，在丙烯腈生物转化过程中控制 amidase 的失活，使得有可能几乎以纯态回收两步反应的中间产物，而无需使用纯化酶。在连续搅拌 UF-膜生物反应器中进行的长期实验中已经证明，amidase 动力学受到其适当底物的控制，这取决于结构和丙烯腈。使用丙烯酰胺，AMase 稳定性取决于温度（5°C，kd=0.008 h(-1)；30°C kd=0.023 h(-1)）。使用苯甲酰胺时，amidase 在 50°C 以下热稳定，并且不会发生底物抑制/失活。对于丙烯腈，在浓度 <200 mM 时，AMase 活性和稳定性保持不变，但在 200 mM、35°C 下，70 h 后，由于没有 AMase 活性在苯甲酰胺上恢复，不可逆失活达到 90%。
