Purification and characterization of cellulolytic enzymes from Aspergillus ustus M35 and trichoderma harzianum M5

A number of methods were applied in an effort to purify the cellulolytic enzymes of Aspergillus ustus M35 and Trichoderma harzianum M5. These mutants were induced by gamma radiation and excreted elevated levels of cellulase and β-glucosidase when grown on different cellulosic substrates. Acetone and ammonium sulfate precipitation, high performance liquid chromatography and ion exchange chromatography were applied. The latter method gave the best results and involved the use of anion exchange resins Accell QMA, Mono Q and Sepharose Q. Mono Q was part of a fast protein liquid chromatography apparatus. The application of Accell QMA revealed the existence of at least two carboxymethylcellulase, two β-glucosidase and one cellobiohydrolase forms in both mutants. Purification of A. ustus M35 with Sepharose Q resulted in the isolation of two β-glucosidase, one carboxymethylcellulase and a mixture of cellulase and β-glucosidase forms. Further purification of the mixture with Mono Q resulted in the isolation of one cellobiohydrolase form. Certain kinetic characteristics of A. ustus M35 purified enzymic forms showed optimum pH values at 3.8, 4.2 and 5.0 for CMCase and β-glucosidase I and II forms respectively. The respective temperature optima were 65, 65 and 45 deg. C. Further purification of cellulolytic systems of both mutants is under investigation. (author). 12 refs, 7 figs, 2 tabs