Application Studies of Purified Tyrosinase from Isolated Aeromonas sp. SNS with Detailed Characterization and Kinetic Studies

摘要

Tyrosinase is the key enzyme accountable for the byconversion of L-tyrosine to L-DOPA and, further, in melanin. The extracellular tyrosinase from bacterium Aeromonas sp. SNS was purified by ion-exchange chromatography using DEAE cellulose as a matrix.The enzyme was eluted with 0.2M NaCl, showing 20.93 x 10~(-3)U mg~(-1) specific activity with a 12.91 % yield and 17.01 purification fold. PAGE estimated the molecular weight of the purified enzyme at 120 and 150 KDa. The zymography of polyacrylamide gel with 5 mM L-DOPA as a substrate confirmed the protein was tyrosinase. Purified tyrosinase shows maximum activity at pH 7 and 40°C temperature. The screening of different inhibitors shows maximum (100 %) inhibition by 1 mM L-ascorbic acid, followed bykojic acid with 91.57 % inhibition. The Km and Vmax for L-DOPA were found to be 0.488 mM and 1.386 x 10~(-4) U mg~(-1), respectively, while for L-tyrosirie, 0.470 mM Km and 2.428x 10~(-4) U mg~(-1) Vmax were recorded. To retain activity for the maximum time, immobilization was performed using sodium alginate and CuCl2. The immobilized beads of tyrosinase were further used for the production of L-DOPA (2.2 mg L~(-1)) in a packed bead reactor and the degradation of toxic dyes. The purification and application study of immobilized tyrosinase for L-DOPA production and dye degradation confirms its role in therapeutic and industrial applications.