Abstract:The smallest and enzymatically active molecule, TetApuQ818, was localized within the C-terminal Q818 amino acid residue after serial C-terminal truncation analysis of the recombinant amylopullulanase molecule (TetApuM955) from Thermoanaerobacter pseudoethanolicus. Kinetic analyses indicated that the overall catalytic efficiency, k cat/K m, of TetApuQ818 was 8–32% decreased for the pullulan and the soluble starch substrate, respectively. Changes to the substrate affinity, K m, and the turnover rate, k cat, were decreased significantly in both enzymatic activities of TetApuQ818. TetApuQ818 exhibited less thermostability than TetApuM955 when the temperature was raised above 85°C, but it had similar substrate-binding ability and hydrolysis products toward various substrates as TetApuM955 did. Both enzymes showed similar spectroscopies of fluorescence and circular dichroism, suggesting the active folding conformation was maintained after this C-terminal Q818 deletion. This study suggested that the binding ability of insoluble starch by TetApuM955 did not rely on the putative C-terminal carbohydrate binding module family 20 (CBM20) and two FnIII regions of TetApu, though the integrity of the AamyC module of TetApuQ818 was required for the enzyme activity.