Effect of C-terminal truncation on enzyme properties of recombinant amylopullulanase from Thermoanaerobacter pseudoethanolicus

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 . Kinetic analyses indicated that the overall catalytic efficiency, (cat)/ (m), of TetApuQ818 was 8-32% decreased for the pullulan and the soluble starch substrate, respectively. Changes to the substrate affinity, (m), and the turnover rate, (cat), were decreased significantly in both enzymatic activities of TetApuQ818. TetApuQ818 exhibited less thermostability than TetApuM955 when the temperature was raised above 85A degrees 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.