Photoregulation of Papain Activity through Anchoring Photochromic Azo Groups to the Enzyme Backbone

The enzyme papain has been chemically modified by 4-carboxyazobenzene (1), 3-carboxyazobenzene, (2), and 2-carboxyazobenzene, (3). The activities of the modified enzymes relative to native papain and the extent of loading by the azobenzene groups have been determined, trans-l-papain exhibits reversible photochromic properties, and upon illumination (l = 320 nm), interconversion to m-l-papain occurs. Further illumination of w-l-papain (l > 400 nm) regenerates trans-l-papain. The biocatalyst irons-l-papain is 2.75-fold more active than cis-l-papain toward hydrolysis of Nabenzoyl- DL-arginine-4-nitroanilide (BAPNA; 4). The difference in activities of the trans/cis photochromic enzyme allows photoregulation of the hydrolytic process. Kinetic analyses reveal that the difference in activities of the two photochromic forms of l-papain originates from poorer binding properties of cis-l-papain toward the substrate, as compared to trans-l-papain. The biocatalyst trans-l-papain is immobilized into Alginate beads, and cyclic photoregulated hydrolysis of is effected.