Immobilization of the hydantoin cleaving enzymes from Arthrobacter aurescens DSM 3747.

The immobilization procedure of the two industrially important hydantoin cleaving enzymes--hydantoinase and L-N-carbamoylase from Arthrobacter aurescens DSM 3747--was optimized. Using different methods (carbodiimide, epoxy activated carriers) it was possible to immobilize the crude hydantoinase from A. aurescens DSM 3747 to supports containing primary amino groups with a yield of up to 60%. Immobilization on more hydrophobic supports such as Eupergit C and C 250 L resulted in lower yields of activity, whereas the total protein coupled remained constant. All attempts to immobilize the crude L-N-carbamoylase resulted in only low activity yields. Therefore, the enzyme was highly purified and used in immobilization experiments. The pure enzyme could easily be obtained in large amounts by cultivation of a recombinant Escherichia coli strain following a three step purification protocol consisting of cell disruption, chromatography on Streamline diethylaminoethyl and Mono Q. The immobilization of the L-N-carbamoylase was optimized with respect to the coupling yield by varying the coupling method as well as the concentrations of protein, carrier and carbodiimide. Using 60 mM of water-soluble carbodiimide, nearly 100% of the enzyme activity and protein could be immobilized to EAH Sepharose 4B.