Influence of initial glucose concentration nitrile hydratase production in Brevibacterium imperialis CBS 498-74

The growth of Brevibacterium imperialis CBS 498-74 (new classification, Microbacterium imperiale), with glucose, acrylonitrile, acrylamide and methacrylamide as the C-source, was studied in a shake flask at 28 °C for culture periods of up to 150 h. The optimum initial glucose concentration for nitrile hydratase (NHase) production (131 U ml−1 broth) was 5 g l−1. Higher concentrations were found to depress the volumetric enzyme production. Acrylonitrile, acrylamide and methacrylamide cannot be used as the sole C-source. At all tested growth conditions, the highest NHase productivity (U ml−1 broth h−1) was reached after 24 h of incubation. Specific activities (U mg−1 DWC) in the cell were found to be: 31 with 5 g l−1 glucose, 43 with 5 g l−1 glucose plus 20 mM acrylonitrile, 47 with 5 g l−1 glucose plus 20 mM acrylamide. The addition of methacrylamide was found to be detrimental under all tested concentrations. Yield coefficient increased progressively with initial glucose concentration until 3.5 g l−1 and then decreased. Maintenance energy requirement was continuously increasing function of the initial glucose concentration. NHase activity in the whole cell suspension was tested following the biotransformation of acrylonitrile (50 mM) into acrylamide at 20 °C in 50 mM sodium phosphate buffer, pH 7.0. The differently induced NHase had very close KM (from 9.35 to 9.80 mM). The enzyme in cells grown using glucose as the sole C-source had a Vmax of 41.86 μmol min−1 mg−1 DWC, whereas the acrylonitrile and acrylamide induced NHase had a Vmax of 51.04 and 56.19 μmol min−1 mg−1 DWC, respectively. The measured activation energy, 28.6 KJ mol−1, indicated a partial control by mass transport through the cell wall.