Nitrile hydratase-amidase cascade system

Temperature Dependence of the Nitrile Hydratase-Amidase Cascade System 275... 

This was performed for each enzyme independently, feeding the reactor with the appropriate substrate (nitrile for the cascade reaction, amide for the sole amidase). The activation energies of both catalysed reactions were evaluated together with those of the inactivation process that inevitably takes place even under the most suitable operational conditions. In the nitrile hydratase/amidase cascade system nitrile hydratase is the more labile enzyme that imposes process temperature choice. These findings make accessible the complete kinetic expression of the dependence from temperature of reaction rate, allowing accurate prediction on reactor performances for process scale-up. 

A., and Cantarella, M. (2010) Application of continuous stirred membrane reactor to 3-cyanopyridine bioconversion using the nitrile hydratase-amidase cascade system of Microbacterium imperiale CBS 498-74. Enzyme Microb. Technol, 47,... 

A., and Cantarella, M. (2013) Nitrile, amide and temperature effects on amidase-kinetics during acrylonitrile bioconversion by nitrile-hydratase/amidase in-situ cascade system. Bioresour. Technol., 142, 320-328. 

The operational thermal stability of enzymes can be easily evaluated in experiments carried out in a CSMR fed with a saturating substrate concentration, while varying the temperature but maintaining all the other parameters constant. Each enzyme of the cascade system was tested by feeding the CSMR with the appropriate substrate. The kinetic characterization of amidase-catalyzed reactions in runs fed with a nitrile was hampered by the fact that the intracellular enzyme works in cascade with nitrile hydratase. The concentration of amide, produced in situ in the first step, varied with the time and reaction conditions and did not assure the differential conditions needed for an accurate analysis, the amide being completely converted by amidase in some runs. Hence, amidase activity was characterized independently by feeding the reactor with amide as the substrate [35]. 

Our original studies with unprotected P-hydroxynitriles showed that these compounds were hydrolyzed by R. rhodochrous ATCC BAA-870, expressing a benzamide-induced cobalt type nitrile hydratase, to the corresponding amides and acids [11]. The formation of the amide implies a nitrile hydratase and amidase system (although sometimes nitrilases can release partially hydrolyzed substrates as amides [63]). Further studies in our laboratories demonstrated that the system was indeed a nitrile hydratase and amidase cascade reaction functioning via a two... 

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