Cloning and expression of clostridium thermocellum exoglucanase gene in escherichia coli

Abstract

Three different genes; endo-glucanase gene, exo-glucanase gene and $\beta$ glucosidase gene, are involved in the enzymatic decomposition of cellulose molecules to glucose molecules. The two genes encoding endo-glucanase and $\beta$ glucosidase have been cloned in our laboratory remaining the gene for exoglucanase unclosed. In this research, cloning of the exo-glucanase gene was attempted. Chromosomes of Clostridium thermocellum, a cellulolytic organism, were digested with EcoRI and a genomic library was constructed in Escherichia coli JM 83 using pUC 19 as a vector. A gene located in a 2.9kbp EcRl fragment was inserted in the vector plasmid and the newly constructed plasmid was named pKM29. Escherichia coli strains transformed by pKM29 produced enzyme showing strong activity toward p-nitrophenyl-$\beta$ -D-cellobioside (pNPC) and filter paper with little activity toward p-nitrophenyl-$\beta$ -d-glucopyranoside (pNPG). The enzyme hydrolyzed CM-cellulose with negatively Congo Red tests as well as showed exo type mode by viscometric analysis. From these characteristics we have concluded that the enzyme produced by the cloned gene is exo-glucanase. The exo-glucanase produced by Escherichia coli JM 83 (pKM29) showed optimal at pH 5.5 and was stable at temperature up to $75\,^\circ\!C$. The exo-glucanase activity was enhanced by the presence of endo-glucanase and the synergism between the two enzymes was confirmed.