Cloning and expression of the gene for thermostable pullulanase from Clostridium thermohydrosulfuricum in Escherichia coli

Using a pUC19-based genomic library of the anaerobic thermophilic bacterium C. thermohydrosulfuricum a DNA fragment that confers pullulanase activity to E. coli cells has been identified. Subcloning and restriction mapping procedures was carried out and the primary structure of the 5'-region of the pullulanase gene (pul) was determined. The pul enzyme was shown to be a protein with molecular weight of approximately 60,000. It was found that both pullulanase and glucoamylase activities resides in pullulanase. The intracellular distribution of pullulanase was studied. An E. coli strain that produces large amounts of thermostable pullulanase has been constructed.