ISOLATION, PURIFICATION AND CHARACTERIZATION CELLULASE FROM Geotrichum candidum ISOLATED FROM LONGHORN BEETLES (Cerambycidae latreille)

Abstract

The ceaseless quest for economical and efficacious cellulase- an enzyme that hydrolyzes cellulose, has led to the exploration of diverse environments like the gut of insects for cellulase production. The aim of this study is to isolate, purify and characterize cellulose-degrading enzyme from Geotrichum candidum isolated from the gut of longhorned beetle. After the G. candidum was screened for cellulase-degrading ability, the enzyme produced thereafter was subjected to a-three-step purification. However, the physicochemical properties, such as, effect of temperature, pH, metal ions, inhibitors and organic solvents on the activity and stability of the enzyme were determined; while molecular weight and kinetic parameters were also assessed. Two activity peaks designated as cellulase A (CA) and cellulase B (CB) were observed in the ion exchange and loaded separately into Sephadex G-100. A final purification fold of 0.86 and 1.86 with recovery of 0.18 % and 0.44 % were achieved for A and B respectively. Molecular weights of 48.5 KDa and 36.5 KDa were recorded for Cellulase A and B respectively. The optimum pH 5.0 was observed for both purified CA and CB, and both were also stable at acidic pH 4.0. Optimum temperature of 60 oC for CA and dual optima temperature 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 cellulases activity, while Zn2+, Cu2+, Mn2+, K+ and Na+ reduced the cellulase activity. Similarly, urea, ascorbic acid and EDTA also inhibited the activity of CA and CB, whileboth showed remarkable stability of two (2) hours in the presence of some hydrophilic (n-Butanol and Ethanol) as well as hydrophobic (Toluene, n-Hexane and Benzene) organic solvents employed in this study. However, the Km and Vmax values were 3.86 mM and 0.3159 mg/ml/min, 4.12 mM and 0.223 mg/ml/min for CA and CB respectively, using carboxymethyl (CMC) cellulose as substrate.The remarkable and unique physicochemical properties of cellulases from G. candidum could be exploited in industrial and biotechnological applications.