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-1-papain exhibits reversible photochromic properties, and upon illumination (lambda = 320 nm), interconversion to cis-1-papain occurs. Further illumination of cis-1-papain (lambda > 400 nm) regenerates trans-1-papain. The biocatalyst trans-1-papain is 2.75-fold more active than cis-1-papain toward hydrolysis of N-alpha-benzoyl-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 1-papain originates from poorer binding properties of cis-1-papain toward the substrate, as compared to trans-1-papain. The biocatalyst trans-1-papain is immobilized into Alginate beads, and cyclic photoregulated hydrolysis of BAPNA is effected.