A NOVEL ALPHA-AMYLASE-PULLULANASE ENZYME, A DNA SEQUENCE CODING THE SAME, A RECOMBINANT DNA MOLECULE AND A RECOMBINANT CLONE EXPRESSING THE ENZYME

摘要

A gene from Clostridium thermohydrosulphuricum DSM 3783 encoding a thermostable alpha-amylase-pullulanase enzyme was cloned encoding in Escherichia coli as a 7 kb insert using a lambda-vector. For subcloning pUC18 and pUC19 plasmids were used. Using a C. thermohydrosulphuricum promoter at least ten different sizes of soluble intracellular thermostable, mostly degenerated polypeptides having both alpha-amylase and pullulanase activities and Mr in the range 165 000 to 100 000 were produced. The temperature optimum of these polypeptides was (80-85 DEG C), some 5 DEG C lower than the temperature optimum of the extracellular enzyme from the native host, but the heat stability was the same as that of the native enzyme. The enzymes can be used alone or in combination with a glucogenic or a maltogenic enzyme for effective hydrolysis of starch and pullulan.