Analysis of Endoglucanase Activity

A recently discovered thermophilic isolate, Geobacillus sp. R7, was shown to produce a thermostable cellulase with a high hydrolytic potential when grown on extrusion-pretreated agricultural residues such corn stover and prairie cord grass. At 70°C and 15–20% solids, the thermostable cellulase was able to partially liquefy solid biomass only after 36 h of hydrolysis time. The hydrolytic capabilities of Geobacillus sp. R7 cellulase were comparable to those of a commercial cellulase. Fermentation of the enzymatic hydrolyzates with Saccharomyces cerevisiae ATCC 24860 produced ethanol yields of 0.45–0.50 g ethanol/g glucose with more than 99% glucose utilization. It was further demonstrated that Geobacillus sp. R7 can ferment the lignocellulosic substrates to ethanol in a single step that could facilitate the development of a consolidated bioprocessing as an alternative approach for bioethanol production with outstanding potential for cost reductions.

The focus of this study was on the solid state fermentation (SSF) of cellulase enzymes produced by Aspergillus niger SB-2 utilizing lignocellulosic agricultural waste as carbon and energy source. Optimization of the SSF media and parameters resulted in a 32% increase in the cellulase activity. Maximum enzyme production of 1,325±7.1 IU/g dry fermented substrate was observed on wheat bran and rice bran supplemented with malt dextrin and soybean meal at pH 6 and 300C after incubation for 120 h. The cellulase activities presented here appear to be among the highest reported in literature for A. niger to date. The A. niger SB-2 cellulase was partially purified and characterized. Zymogram analysis of the sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed two bands of cellulase activity with molecular weights of 30 and 45 kDa. To the best of our knowledge, a 45 kDa cellulase from A. niger has not been previously described in literature. The enzyme was active in a broad pH (4-7) and temperature (30-550C) range with a pH optimum of 6 and a temperature optimum of 450C. At 50 and 600C, the cellulase half life was 12.4 and 4.1 h, respectively. Dithiothreitol, iodoacetamide and Mg+2 acted as activators of cellulase activity. Kinetics studies indicated that the substrate specificity of A. niger SB-2 cellulase was 18% higher on insoluble cellulose than on soluble cellulose. Therefore, the cellulase complex of A. niger SB-2 would be useful in bioprocessing applications where efficient saccharification of lignocellulosic biomass is required.