Purification and characterization of a thermostable pullulanase fromThermoactinomyces thalpophilus

Thermoactinomyces thalpophilus No. 15 produced an extracellular pullulanase in an aerobic fermentation with soluble starch, salts, and complex nitrogen sources. Acetone fractionation, ion-exchange chromatography, and gel filtration purified the enzyme from cell-free broth 16-fold to an electrophoretically homogeneous state (specific activity, 1352 U/mg protein; yield, 4%). The purified enzyme (estimated MW 79 000) was optimally active at pH 7.0 and 70°C and retained 90% relative activity at 80°C (30 min) in the absence of substrate. The enzyme was activated by Co2+, inhibited by Hg2+, and exhibited enhanced stability in the presence of Ca2+. The enzyme hydrolyzed pullulan (Km 0.32%, w/v) forming maltotriose, and hydrolyzed amylopectin (Km 0.36%, w/v), amylopectin beta-limit dextrin (Km 0.45%, w/v) and glycogen beta-limit dextrin (Km 1.11%, w/v) forming maltotriose and maltose.