title: Nitrile, amide and temperature effects on amidase-kinetics during acrylonitrile bioconversion by nitrile-hydratase/amidase in situ cascade system.
abstract: 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.
authors.affiliation: Department of Civil and Mechanical Engineering, University of Cassino and of Lazio Meridionale, Via Di Biasio 43, 03043 Cassino (FR), Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Cantarella
authors.forename: Alberto
authors.initials: A
authors.lastname: Gallifuoco
authors.forename: Agata
authors.initials: A
authors.lastname: Spera
authors.forename: Maria
authors.initials: M
authors.lastname: Cantarella
mesh_terms: D000577:Amides / Q000378:metabolism*; D000581:Amidohydrolases / Q000378:metabolism*; D019149:Bioreactors; D002851:Chromatography, High Pressure Liquid; D006836:Hydro-Lyases / Q000378:metabolism*; D007700:Kinetics; D009570:Nitriles / Q000378:metabolism*; D013696:Temperature
chemical_list: D000577:Amides; D009570:Nitriles; D000581:Amidohydrolases; C019665:amidase; D006836:Hydro-Lyases; C048831:nitrile hydratase
keywords: Amidase kinetics; Amide inactivation; Nitrile inactivation; Temperature dependence; UF-membrane bioreactor