Production of a thermostable pullulanase by a Thermus sp.

A moderate thermophile that produces a large amount of extracellular pullulanase was isolated from soil. The isolate (AMD-33), that grew at 37 to 74°C with an optimum at 65°C, was identified as a Thermus sp. strain. Maximal enzyme production was attained after 3 days shaking cultivation at 60°C on a medium composed of 1% pullulan, 2% gelatin, 0.1% K2HPO4, 0.03% MgSO4·7H2O and 0.25% CaCO3.Pullulanase synthesis was enhanced by pullulan, soluble starch and dextrin as well as maltose but not at all by glucose. The enzyme, which was most active at pH 5.5-5.7 and 70°C, was stabilized by Ca2+, and the optimum temperature for activity shifted to 75°C in the presence of 3mM CaCl2.