Chemical engineering of enzymes: altered catalytic activity, predictable selectivity and exceptional stability of the semisynthetic peroxidase seleno-subtilisin

Abstract

The increasing demand for enzymes as highly selective, mild, and environmentally benign catalysts is often limited by the lack of an enzyme with the desired catalytic activity or substrate selectivity and by their instability in biotechnological processes. The previous answers to these problems comprised genetically engineered enzymes and several classes of enzyme mimics. Here we describe the potential of chemical enzyme engineering: native enzymes can be modified by merely chemical means and basic equipment yielding so-called semisynthetic enzymes. Thus, the high substrate selectivity of the enzymatic peptide framework is combined with the catalytic versatility of a synthetic active site. We illustrate the potential of chemically engineered enzymes with the conception of the semisynthetic peroxidase seleno-subtilisin. First, the serine endoprotease subtilisin was crystallized and cross-linked with glutaraldehyde to give cross-linked enzyme crystals which were found to be insoluble in water or organic solvents and highly stable. Second, serine 221 in the active site (Enz-OH) was chemically converted into an oxidized derivative of selenocystein (Enz-SeO2H). As a consequence, the former proteolytic enzyme gained peroxidase activity and catalyzed the selective reduction of hydroperoxides. Due to the identical binding sites of the semisynthetic peroxidase and the protease, the substrate selectivity of seleno-subtilisin was predictable in view of the well-known selectivity of subtilisin.