Decolorization and purification of crude protease from Rhizopus oryzae by activated charcoal and its electrophoretic analysis

Activated charcoal decolorized and partially purified the protease from a crude extract of solid state fermentation of wheat bran by Rhizopus oryzae. Treatment for 5 min was sufficient. Depending on the initial colour intensity of crude, the charcoal to crude extract ratio could be optimized to achieve 90% decolorization, 85% enzyme recovery, and over a 3-fold purification, even up to 20-fold variation in batch size (from 1 ml to 20 ml crude extract). Decolorization followed the Freundlich and the Langmuir models, the Freundlich constant, n, being 2.74. Partial purification was confirmed by native PAGE and the protease band identified by gelatin-PAGE. SDS-PAGE showed the protease consisted of two sub-units (about 22 and 24 kDa).List of symbols:c_o, initial solute concentration in liquid before adsorption; c^*, equilibrium solute concentration in liquid after adsorption; k, empirical constant for Freundlich adsorption isotherm; U, unit of protease activity; v, volume of solution per unit weight of adsorbent.