Abstract
The potential of 12 fungal strains to produce carboxymethyl cellulase (CMCase) and protease on Eichhornia crassipes (water hyacinth) wastes was investigated under conditions of solid state fermentation. Ulocladium botrytis (Preuss) was selected as the best fungus for the production of both enzymes. The best nitrogen sources for production of CMCase and protease were yeast extract and malt extract, respectively. CMCase and protease were purified by isopropanol (1:1) precipitation and column chromatography on Sephadex G-100 and DEAE-cellulose. Purification fold of 47.34 and 51.78, with 852.11 and 1,469.38 U/mg specific activity was achieved with 40.3 and 56.25% recovery after purification of CMCase and protease, respectively. The purified CMCase expressed its maximal activity at 60 degrees C and pH 5.2, showed good stability in the pH range of 5.2-5.4 and its midpoint of thermal inactivation (Tm) was 60 degrees C after 75 min exposure. The purified protease expressed its maximal activity at 35 degrees C and pH 5.2, showed good stability in the pH range of 5.6-6.0 and its midpoint of thermal inactivation (Tm) was 35 degrees C after 75 min exposure. The best substrate concentration for CMCase was 1.2% (w/v) Na-CMC and for protease, it was 0.8% (w/v) casein. The best enzyme concentration for the two tested enzymes was 0.4 U/ml. Ions of Ca2+, Na+ and K+ showed a stimulatory effect but sodium arsenate and iodoacetate showed an inhibitory effect. Moreover, Ag2+ and Hg2+ inhibited both enzyme activities completely. The purified enzymes from Ulocladium botrytis had molecular weights of 50 and 83 kDa for CMCase, and 47 kDa for protease on SDS-PAGE.