Purification and characterization of membrane-bound <em>Borassus flabellifer</em> L. thermostable peroxidase

Abstract

Generating wealth out of waste is a goal in solid waste management. <em>Borassus flabellifer</em> shells with stone part are discarded as waste after removing its nutritious fruit. An enzyme extracted from the discarded stone part has potential application in molecular diagnosis and it is a wealth. The waste stone part bound <em>Borassus flabellifer</em> peroxidase was purified by salting-out, salting-in and DEAE-Cellulose anion exchange chromatographic technique to apparent homogeneity. Relative molecular weight under denaturating condition was around 40 kDa. The preparation had single isoenzyme as evidenced under nondenaturating condition. It was a glyco- and hemoprotein. It retained 100% activity for 120 h at 70°C. pH optima with benzidine, o-dianisidine, guaiacol and tetramethylbenzidine was around 5.0. Kinetic studies showed it had a higher affinity towards tetramethyl benzidine than other three substrates. The thermodynamic parameter of the enzyme with benzidine, o-dianisidine, guaiacol and tetramethylbenzidine was 35, 1.3, 35 × 10⁶ KJ/mole and 1007 × 10⁶ KJ/mole respectively. It obeyed Ping-Pong kinetics. The fluorescence intensities of the enzyme increased linearly as hydrogen peroxide concentration increased due to exposure of its hydrophobic moiety to the environment. Peel staining with Guaiacol substantiated it as a membrane-bound protein. Peroxidase was inhibited reversibly by various aromatic alcohols and its IC₅₀ values were determined.