Evaluation of the cellulase system produced by three strains of Clostridium thermocellum on cellobiose and Avicel

Abstract

Clostridiwn thennocellwn, a thermophilic anaerobe, produces an extracellular cellulase system capable of extensively degrading crystalline cellulose, in the presence of Ca2+ and D l T [l]. However, the cellulase system of this bacterium is present in the form of a complex, termed cellulosome with a molecular mass of -2x106 12).Using three strains (YS, LQRI and NCIB 10682) of C.thennocellwn, we have demonstrated that this bacterium produces extracellular cellulosomes when grown on cellobiose and Avicel [3]. In the present paper, cellulosomes produced on cellobiose and Avicel by the above three strains were compared on the basis of in vitro Avicel solubilization, dissociation pattern and the cellulase and xylanase activities of various subunits.All three strains were cultured on cellobiose and Avicel and the cellulase system produced was isolated as described [3]. The cellulase system produced on both carbon sources by these strains solubilized Avicel linearly up to 48 h ( Fig.1). However, the rate and extent of Avicel solubilization by the cellulase system of all three strains of C.thennocellwn obtained on cellobiose (1.6 -1.8% h-1 and 70 -80%) was marginally lower than that produced on Avicel (2.1 -2.8% h-1 and 95 -98%). Furthermore, the cellulase system produced by strain NCIB 10682 on Avicel showed the highest rate and extent of Avicel solubilization, followed by YS and LORI while the rate and extent of Avicel solubilization by the enzyme system produced on cellobiose by all three strains were comparable. Electron microscopic observation of the above mentioned strains, during the growth on cellobiose and Avicel also revealed the presence of cell surface cellulosomes. Thus, biochemical and morphological studies confirmed the production of fully active cellulosomes on cellobiose and Avicel by these strains of C.rhennocellwn.Purified cellulosomes of all three strains from cellobiose and Avicel cultures were dissociated at pH 5.0 and at 25°C for 30 min and separated by SDS-PAGE. The pattern of dissociation showed overall similarity between the cellulosomes from all three strains produced on both carbon sources. However, the S, subunit of cellulosomes from cellobiose culture showed a diffused protein pattern while that from Avicel culture migrated as a sharp protein band. Also, based on the intensity of protein bands, S, and S, were found to be the major subunits in cellulosomes from cellobiose culture whereas S, and S,, appeared to be the most prominent subunits in cellulosomes from Avicel culture.In order to identify whether the subunits of cellulosomes from cellobiose and Avicel cultures show the same type of enzyme activity, the cellulosomes were dissociated as described above and separated by SDS-PAGE. Proteins eluted from the SDS-gel slices were assayed for endoglucanase, exoglucanase and xylanase activities. Cellulosomes produced on both carbon sources contained one major PNPCase and one major CMCase activity peaks corresponding to S, and S,, subunits, respectively. In addition, two minor PNPCase and several ...