Optimization, Isolation, Purification, and Characterization of a Thermally Stable, Acidophilic Cellulase from Aspergillus awamori AFE1 for Industrial and Biotechnological Applications

Abstract

The ceaseless quest for economical cellulase, an enzyme that hydrolyzes cellulose, has led to exploring diverse environments, such as insect guts. In this study, we report the optimization of cellulase production and isolation, purification, and characterization of cellulose-degrading enzymes from Aspergillus awamori AFE1. Aspergillus awamori AFE1 was screened for its cellulase-degrading ability, and molecular and phylogenetic analyses of the isolate were performed. Two activity peaks were observed during ion exchange chromatography. A final purification fold of 0.86 and 1.86 with a recovery of 0.18% and 0.44% were achieved for cellulase A and B, respectively; molecular weight of 48.5 KDa and 36.5 KDa for A and B, respectively. The optimum pH of 5.0 was observed for both purified cellulases, and both were stable at an acidic pH of 4.0. An optimum temperature of 60 oC for CA and dual optimum temperatures of 60 and 70 oC were obtained for CB, while both were stable at 30 oC with 63 and 61% residual activity after 2 h, respectively. Fe2+ stimulated both cellulase activity, whereas Zn2+, Cu2+, Mn2+, K+, and Na+ inhibited cellulase activity. Similarly, urea, ascorbic acid, and EDTA inhibited the enzyme. The enzymes were stable in the presence of some organic solvents. The Km and Vmax values were found to be 3.86 mM and 0.3159 mg/ml/min, 4.12 mM, and 0.223 mg/ml/min for the enzyme. The remarkable and unique physicochemical properties of cellulases from Aspergillus awamori AFE1 could be exploited for industrial and biotechnological applications.