A study in UF-membrane reactor on activity and stability of nitrile hydratase from Microbacterium imperiale CBS 498-74 resting cells for propionamide production

Abstract

The bioconversion of propionitrile to propionamide was catalysed by nitrile hydratase (NHase) using resting cells of Microbacterium imperiale CBS 498-74 (formerly, Brevibacterium imperiale). This microorganism, cultivated in a shake flask, at 28°C, presented a specific NHase activity of 34.4UmgDCW−1 (dry cell weight). The kinetic parameters, Km and Vmax, tested in 50mM sodium phosphate buffer, pH 7.0, in the propionitrile bioconversion was evaluated in batch reactor at 10°C and resulted 21.6mM and 11.04μmolmin−1mgDCW−1, respectively. The measured apparent activation energy, 25.54kJmol−1, indicated a partial control by mass transport, more likely through the cell wall.UF-membrane reactors were used for kinetic characterisation of the NHase catalysed reaction. The time dependence of enzyme deactivation on reaction temperature (from 5 to 25°C), on substrate concentrations (from 100 to 800mM), and on resting cell loading (from 1.5 to 200μgDCWml−1) indicated: lower diffusional control (Ea=37.73kJmol−1); and NHase irreversible damage caused by high substrate concentration. Finally, it is noteworthy that in an integral reactor continuously operating for 30h, at 10°C, 100% conversion of propionitrile (200mM) was attained using 200μgDCWml−1 of resting cells, with a maximum volumetric productivity of 0.5gl−1h−1.