Fungal glycosidase for carbohydrate transformations

One hundred and nineteen thermophilic fungi, isolated by enrichment in basal medium containing 0.5 percent locust bean gum (LBG), from 132 soil samples and 22 pure culture were screened for extracellular glycosidase production mainly, mannanase and galactanase, using 1 percent LBG as a substrate. Crude enzyme from the 141 isolates showed mannanase and galactanase activities with the mannanase activity being 12 times as high as that of galactanase. The fungal isolates 24S, identified as Aspergillus fumigatus 24S, produced the highest mannanase activity in the medium containing (g/l) 15 LBG, 1 corn steep solid, 2 K2HPO4, 0.3 MgSO4. 7H2O, 0.3 CaCl2, 1 ml mineral salts and 3 NaNO3. The culture was prepared by inoculation with 2 percent (v/v) of 30 hr seed culture and incubated for 48 hr at 45 deg C with shaking at 200 rpm. The yield of mannanase was 14.7 U/ml giving the specificity of 28.5 U/mg. The Beta-mannanase production was induced by LBG. It has the pH optima of 5-6 whereas the galactosidase component was slightly more heat stable than Beta-mannanase. Purification of the crude enzyme by ion exchange chromatography and gel filtration could not separate mannanase free from Alpha-galactosidase. The multiple enzyme composed of 6 fractions. The fraction, M (F1-1) showed mainly Beta-mannanase activity and the fraction G(F2-1) showed mainly Alpha-galactosidase activity. The M fraction had a pH optimum of 5.6 and the optimum temperature of 50 deg C, similar to the pH and temperature optima obtained from the crude enzyme. The G fraction had the pH and temperature optima of 5.0-5.5 and 60-65 deg C respectively. The Km and V max of enzyme for p-nitrophenyl-alpha - D- galactopyranoside were 0.80 mM and 0.0112 micro mole/min respectively. Thin layer chromatography analysis of the hydrolysis products from LBG before and after purification of Beta-mannanase gave similar results revealing trisaccharide, disaccharide and monosaccharide. There was no significant difference in oligosaccharide synthesis by Alpha-galactosidase at pH 6 and 55 deg C using 40 percent (w/v) galactose as substrate as detected by HPLC. New data for Beta-mannanase and Alpha-galactosidase production from A. fumigatus 24S using locust bean gum as substrate was reported for the first time. The enzyme production can be improved by further selection of strain with higher productivity. The high amount of enzyme production will have an advantage for use in the hydrolysis of galactomannan from agriculture waste to the useful by- products such as tri- and disaccharide. Beta-mannanase and Alpha-galactosidase produced by Aspergillus fumigatus 24S have a specific reaction at high temperature 50 deg C. Purified enzymes were stable at high temperature. This will be an advantage in the hydrolysis process since the chance of getting contaminations from other organisms will be lowered. The amount of enzymes produced is 3X lower than the other organism reported by Araujo and Ward (1990a). Strain improvement by mutation and genetic engineering technique are required. Screening for effective producing strain from new natural resources is nescessary.