Enhanced Stabilization of Nitrile Hydratase Enzyme From Rhodococcus Sp. DAP 96253 and Rhodococcus

Treatment of industrial wastewaters contaminated with toxic and hazardous organics can be a costly process. In the case of acrylonitrile production, due to highly volatile and toxic nature of the contaminant organics, production wastewaters are currently disposed by deepwell injection without treatment. Under the terms granting deepwell injection of the waste, alternative treatments must be investigated, and an effective treatment identified. Cells of two Gram-positive bacteria, Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 were evaluated for their potential as biocatalysts for detoxification of acrylonitrile production wastewaters. Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 when multiply induced, are capable of utilizing the hazardous nitrile and amide components present in the wastewater as sole carbon and/or nitrogen sources, employing a 2-step enzymatic system involving nitrile hydratase (NHase) and amidase enzymes. There is a significant potential for overproduction of NHase upon multiple induction. However, high-level multiple induction required the presence of highly toxic nitriles and/or amides in the growth medium. Asparagine and glutamine were identified as potent inducers with overexpression at 40% of total soluble cellular protein as NHase. In native form (either cell free enzymes or whole cells) the desired NHase is very labile. In order to develop a practical catalyst to detoxify acrylonitrile production wastewaters, it is necessary to significantly improve and enhance the stability of NHase. Stabilization of desired NHase activity was achieved over a broad range of thermal and pH conditions using simultaneous immobilization and chemical stabilization. Previously where 100% of NHase activity was lost in 24 hours in the non-stabilized cells, retention of 20% of initial activity was retained over 260 days when maintained at 50-55 C, and for over 570 days for selected catalyst formulations maintained at proposed temperature of the biodetoxification process. In addition, NHase and amidase enzymes from Rhodococcus sp. DAP 96253 were purified. Cell free NHase was characterized for its substrate range and effect of common enzyme inhibitors and was compared to available information for NHase from other organisms. As a result of this research a practical alternative to the deepwell injection of acrylonitrile production wastewaters is closer to reality