Production of cellulases by an isolated fungus and its mutant strain.

Bioconversion, particularly enzymatic hydrolysis of cellulosic materials to useful products has great potential and is being actively investigated [l-31. Biotechnological utilization of cellulose requires a very efficient cellulolytic enzyme system active on native cellulose. Cellulases are nulticomponent enzyme systems in which the synergistic action of endoglucanase, exoglucanase and Betaglucosidase is necessary to promote effective hydrolysis. The present study describes the production of cellulases by one fungus isolated from decomposed wood and of a mutant strain obtained by ultraviolet irradiation. A mesophilic fungal strain which exhibited reasonable amounts of cellulose hydrolysing activity when compared to Trichoderma reesei QM 9414, was isolated from decomposed wood and used throughout these experiments. The fungal strain (F.3) was maintained in PDA slopes at 4OC. Fresh spores were used for mutagenesis experiments. They were suspended in sterile distilled water and subjected to UV irradiation to give about 1% survival rate. After the treatment, the cells were cultured on agar plates containing (5.0 9/11 cellulose microcrystalline, (50 ng/l) bengal rose, (1.0 ml/l) triton X-100 and minerals and incubated at 30 C. One colony (F. M38), which presented a clear circle with a large radius around it was chosen for enzyme production studies. Extracellular cellulase production was carried out under shaking conditions in media contaning carbon source, (1.0 ml/l) Tween 80, and minerals. The carbon sources used were eiher (5.09/1) cellulose microcrystalline (Merck) or (10 9/11. of babassu flour. Filter paper degrading activity (FPA) was estimated according to the procedures of Mandels & a. (4). Carboxymethyl cellulose hydrolysing activity (CMCase) was determined by the increase in reducing sugar after 60 minutes of reaction of a mixture of (0.5 ml) extracellular extract and (1.0 m1) of (1%) carboxymethylcellulose at pH 5.0 in acetate buffer, incubated at 50'C. Microcrystalline cellulose degrading activity was carried out in a similar way as for CMCase, using (1%) microcrystalline cellulose. One unit of enzyme activity was defined as the number of micromoles of reducing sugars released by one millilitre of enzyme per minute. Reducing sugar expressed as glucose was determined by the DNS (3,5-dinitrosalicylic acid) method [ 5 1 . Beta-glucosidase activity ( 8 gluc. ) vas determined according to Jafelice & d. [ 6 1 , using o-nitropheno1-BDglucopiranoside (ONPG) as substrate. Table 1 shows the results for cellulase activities in the medium where the carbon source was microcrystalline cellulose and Table 2 shovs the results for babassu flour.