| A novel pullulanase, which hydrolyzes both $alpha$-1,6 bonds in pullulan and $alpha$-1,4 bonds in amylose, was characterized from Clostridium thermohydrosulfuricum 39E, a thermophilic, anaerobic bacterium. Conditions were optimized for overproduction and secretion of the enzyme by using a maltose limited chemostat culture. The enzyme was purified by using affinity chromatography on an inhibitor-linked matrix, and the biochemical properties determined. Activity staining of PAGE gels, and inhibition kinetics using dual alternate substrates showed that both $alpha$-1,4 and $alpha$-1,6 glucosidic bond cleavage resided on the same enzyme. This amylopullulanase had higher affinity for pullulan than amylose. Both $alpha$-amylase and pullulanase activities were inhibited by $beta$-cyclodextrin, a known inhibitor for both pullulanases and $alpha$-amylases. Amylose, glycogen, and amylopectin were hydrolyzed by the enzyme to maltose, maltotriose, and maltotetraose. $sp{13}$C NMR spectroscopy showed that the enzyme was capable of hydrolyzing both $alpha$-1,6 and $alpha$-1,4 bonds in glycogen. The gene encoding amylopullulanase was identified in a 6.1 kbp chromosomal DNA fragment and cloned into Escherichia coli and subcloned into Bacillus subtilis. The cloned enzyme was processed to the periplasmic space of E. coli, while in B. subtilis it was extracellular. The cloned enzyme from E. coli expressed both $alpha$-amylase and pullulanase activities and maintained thermostability and thermophilicity. The 4.4 kbp amylopullulanase (apu) gene was sequenced. Nested deletion mutants and fusion proteins constructed from the gene allowed the identification of a 2.9 kbp segment in the middle of the coding region, that encoded a M$sb{rm r}$ 100,000 protein which maintained both $alpha$-amylase and pullulanase activities and thermostability, indicating that a single active site was probably involved in both $alpha$-1,6 and $alpha$-1,4 hydrolytic activities. Chemical modification of the enzyme with group specific reagents enabled the putative identification of either aspartate or glutamate, to be involved in catalysis. Site directed mutagenesis determined that Asp$sb{597}$, Glu$sb{626}$, and Asp$sb{703}$ were involved in catalysis, with each amino acid substitution resulting in loss of both $alpha$-1,4 and $alpha$-1,6 glucosidic bond cleavage. |
