PURIFICATION AND SOME PROPERTIES OF AN ALKALINE PULLULANASE FROM ALKALOPHILIC BACILLUS SP KSM-1876

A novel alkaline pullulanase (pullulan 6-glucanohydrolase, EC 3.2.1.41) was purified to homogeneity from the culture filtrate of the alkalophilic Bacillus sp. KSM-1876. The pullulanase had an optimum pH for activity of around 10.0-10.5, which is the highest pH for optimum activity of any pullulanases reported to date. The optimum temperature at pH 10 was around 50-degrees-C. The enzyme had a molecular mass of 120 kDa and an isoelectric point of pH 5.2. The enzyme acted specifically on pullulan to generate maltotriose as the major end product; the K(m) for pullulan was 1.8 mg/ml. N-Bromosuccinimide abolished the enzymatic activity, and pullulan protected the enzyme from inactivation by this tryptophan-specific oxidant, suggesting that a tryptophan residue(s) is involved in the mechanism of action of the pullulanase from Bacillus sp. KSM-1876.