This proposal describes a comprehensive program aimed at developing new methods for the production of speciality biochemicals and advanced biomaterials. These materials will be formed by reversing the normal hydrolytic action of two proteases. These proteases will be modified to alter their selectivity and to enhance their stability under reaction conditions. Trypsin or chymotrypsin specificity will be altered by several techniques; site-directed mutagenesis, screening for mutants which produce enzymes with the ability to act on the substrates of interest, refolding partially denatured enzyme around novel substrates and a new approach which will permit the insertion of synthetic amino acids at the active site of the enzyme. Cathepsin C will be modified by a refolding procedure, and immobilized by a variety of techniques. Methods for enhancing the reversal of the hydrolytic reaction for dipeptide and oligomer synthesis with non-natural amino acids will be developed. These include the use of non-aqueous solvents (both water-miscible and immiscible) to increase substrate solubility and the use of directed methods for enzyme immobilization to enhance enzyme stability under reaction conditions. Changes in enzyme conformation induced by non-aqueous solvents and by immobilization will be probed by several techniques, including EPR.
