IMPROVED PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF EXTRACELLULAR AMYLOPULLULANASE FROM THERMOANAEROBACTER-ETHANOLICUS 39E

A maltose-limited chemostat culture was used to investigate the expres sion and excretion of amylopullulanase by Thermoanaerobacter ethanolic us 39E (formerly Clostridium thermohydrosulfuricum 39E). In maltose-li mited continuous culture, amylopullulanase was produced and secreted a t tenfold higher levels than in batch culture. The extracellular amylo pullulanase was purified to homogeneity by using an inhibitor-linked a ffinity column matrix. The purified amylopullulanase had a specific ac tivity of 480 units (U)/mg protein for pullulanase and 175 U/mg protei n for alpha-amylase. beta-Cyclodextrin inhibited both alpha-amylase an d pullulanase activities, with a substrate inhibition constant (K(i)) of 0.065 mg/ml. Amylopullulanase had a relative molecular mass (M(r)) of 140000 using sodium dodecyl sulphatepolyacrylamide gel electrophore sis (SDS-PAGE) analysis and an M(r) of 133000 using gel-filtration chr omatography. The N-terminal sequence of the enzyme was Glu-Thr-Asp-Thr -Ala-Pro-Ala. The purified enzyme displayed Michaelis constant (K(m)) values of 0.35 mg/ml for pullulan and 1.00 mg/ml for amylose. The enzy me had an isoelectric point (pl) of 4.0, and displayed an optimum pH f or stability and activity of 6.2 and 5.5, respectively. The enzyme was stable up to 85-degrees-C in the presence of Ca2+, and had a half-lif e of 40 min at 90-degrees-C (pH 6.2). Ca2+ was required for thermal st ability, but not for activity. Amylose, glycogen, and amylopectin were degraded to maltose, maltotriose, and maltotetraose, whereas only mal totriose was formed from pullulan.