Extracellular acid protease from Rhizopus oryzae: purification and characterization

Abstract

Extracellular aspartate protease from Rhizopus oryzae was purified 91 times with 26% recovery using (NH₄)₂SO₄ fractionation, ion-exchange and size-exclusion chromatographic techniques. The enzyme was found to be monomeric in nature having a molecular mass of 34 kDa. The enzyme acts optimally at 60°C with activation energy of 15.16 kcal/mol and was stable in the temperature range of 30-45°C. The purified enzyme is an acid protease with optimum pH of 5.5 and retained 96% of residual activity between pH 5.5 to 7.5. Ca²⁺ activation (250 times) and varying substrate concentration gave an hyperbolic response. The Lineweaver-Burk plot showed K_m value of 5 mg/ml, when skim milk was used as substrate. The enzyme inhibition of 73 and 93% by pepstatin at 10 and 20 μ M, respectively proved it to be an aspartate protease; however, the additional requirement of histidine residue for enzyme activity has been indicated by differential spectra of diethyl pyrocarbonate treated versus untreated enzyme.