Resumo

Proteases are amongst the most studied and important enzymes applied in industrial processes. Prerequisites for their use in detergent industries are high activity and stability in broad ranges of pH and temperature, as well as low sensitivity to commercial detergent components, such as SDS, Tween 80 and oxidizing agents. The thermophilic filamentous fungus Humicola grisea var. thermoidea produces enzymes with excellent thermal stability. This study describes the production of alkaline proteases by Humicola grisea var. thermoidea grown in glucose, as well as some biochemical properties of the crude enzyme and verifies its application in laundry detergent industries. The fungus showed a better protease production when grown for 48 hours, with 0.75% glucose as carbon source, at 40° C and 100 rpm of agitation. Crude H. grisea alkaline protease showed maximal activity at pH 8.5, but relative activities higher than 75% of the maximal were estimated from pH 6.5 to 10.0. H. grisea proteases were stable in a wide range of pH 3-13. Crude alkaline protease exhibited maximal activity in a wide temperature range, from 45 to 60ºC, and about 50% of the maximum was even estimated at 30 and 65ºC. Proteolytic activity was reduced by only 20 and 30% after 1 h incubation of the crude filtrate in aqueous medium at 55 and 60ºC, respectively. Zymogram analysis after SDS-PAGE suggested the presence of at least four proteases in the crude filtrate from H. grisea grown in glucose. Alkaline protease activity was activated by 1.7-fold by Fe+2 , inhibited about 94%, 35% and 35% by 25 mM PMSF, p-chloromercuric benzoic acid and EDTA, respectively, but activated about 1.6-fold by sodium perborate and insensitive to commercial detergents. Further, the proteases showed good stability towards H2O2, SDS, Triton X-100 and Tween 80. Urea at 0,5M slightly activated Humicola proteases. Mixtures of commercial soaps and Humicola proteases improve the washing performance of washing of cotton cloth pieces artificially stained with blood. The good stability of crude H. grisea proteases towards commercial detergents, pH, high temperatures, oxidizing agents and ionic and non-ionic surfactants, allied to its high storage stability qualify this fungus as a new potential mold source of enzymes for use in detergent industry.