Production of Glucoamylase by Marine Endophytic Aspergillus sp. JAN-25 under Optimized Solid-state Fermentation Conditions on Agro Residues

For effective extraction of glucoamylase from the fermented rice bran by endophytic Aspergillus sp. JAN-25 in solid state fermentation, optimized leaching parameters, 1:6 (w/v) of 0.2 M citrate buffer as leaching agent, soaking time with moldy bran 120 min, leaching temperature 45 oC and leaching pH 4.0 at 150 rpm were found to be the optimum leaching parameters that leached the highest yield of glucoamylase (264.5 U gds). Mixed bran of wheat and rice bran (at a ratio of 1:2) as abundant agro-industrial residues in Egypt, stimulated the highest glucoamylase yield as solid substrate in SSF by marine endophytic Aspergillus sp. JAN-25 to 398.52 U gds. Under the optimized extraction and production parameters (2:50 substrate mass to flask volume ratio; IMC 50-60 %, inoculum size 10/ gds; supplementation of bran medium with 0.04 g starch / gds, 0.02 g soybean / gds, 1% CaCl2 and 1% Tween-80 at pH 5.0 and 30 oC for four days the glucoamylase production was markedly increased to 12.13-fold. Following successive purification steps including, (NH4)2SO4 fractionation, gel filtration (sephadex G-200) and DEAE sepharose chromatography, the purified enzyme exhibited 23.34% of the total initial activity and there was a 35.41-fold increase in specific activity (613.30 U/mg) with maximum activity at pH 4.5 5.5 and 50 60 oC. Glucoamylase activity markedly decreased to 41 and 52 % at 50 mM of EDTA and EGTA, respectively demonstrated the ion requirements of this JAN-25 glucoamylase activity (metallo enzyme).