Expression of clostridium thermocellum exo-β-1,4-glucanase gene in escherichia coli

Abstract

Exo-$\beta$ -1, 4-glucanase was previously cloned from C. thermocellum in our laboratory. For overproduction, This gene was subcloned into the expression vector pKK223-3 which contains tac promoter. The plasmid pKM29 containing exo-$\beta$ -1, 4-glucanase was digested partially with EcoRI and ligated with EcoRI digested vector. Those ligated mixtures were used to transform E. coli JM103. From the transformants, the desired transformant was isolated and plasmids were purified. It was named pTK29. The tac promoter may be controlled with IPTG. When the culture of E. coli (pTK29) was grown in LB broth to $O.D_{600}$ of 0.5, IPTG was added. The production of exo-$\beta$ -1, 4-glucanase was enhanced and maximum specific activity of this enzyme on induced state was 7 times higher than that of on uninduced state. The molecular weight of exo-$\beta$ -1, 4-glucanase was about 66kD. In the cell, Produced polypeptide processed to smaller, at least, four types which still retain activity.