The functional and structural significance of the C-terminal region of Thermoanaerobacter ethanolicus 39E amylopullulanase (TetApu) was explored using C-terminal truncation mutagenesis. Comparative studies between the engineered full-length (TetApuM955) and its truncated mutant (TetApuR855) included initial rate kinetics, fluorescence and CD spectrometric properties, substrate-binding and hydrolysis abilities, thermostability, and thermodenaturation kinetics. Kinetic analyses revealed that the overall catalytic efficiency, k (cat)/K (m), was slightly decreased for the truncated enzymes toward the soluble starch or pullulan substrate. Changes to the substrate affinity, K (m), and turnover rate, k (cat), varied in different directions for both types of substrates between TetApuM955 and TetApuR855. TetApuR855 exhibited a higher thermostability than TetApuM955, and retained similar substrate-binding ability and hydrolyzing efficiency against the raw starch substrate as TetApuM955 did. Fluorescence spectroscopy indicated that TetApuR855 retained an active folding conformation similar to TetApuM955. A CD-melting unfolding study was able to distinguish between TetApuM955 and TetApuR855 by the higher apparent transition temperature in TetApuR855. These results indicate that up to 100 amino acid residues, including most of the C-terminal fibronectin typeIII (FnIII) motif of TetApuM955, could be further removed without causing a seriously aberrant change in structure and a dramatic decrease in soluble starch and pullulan hydrolysis.

Chitinases have been classified into family 18 and 19 glycohydrolases based on their hydrolytic mechanisms and the amino acid sequence similarity of their catalytic domains [1]. The catalytic domains of family 18 chitinases are characterized as having (b ⁄ a) 8 barrel folds [2][3][4][5][6], whereas those of family 19 chitinases have a high a-helical content and a structure similar to those found in chitosanases and lysozymes [7,8] The functional and structural significance of the C-terminal region of Bacillus licheniformis chitinase was explored using C-terminal truncation mutagenesis. Comparative studies between full-length and truncated mutant molecules included initial rate kinetics, fluorescence and CD spectrometric properties, substrate binding and hydrolysis abilities, thermostability, and thermodenaturation kinetics. Kinetic analyses revealed that the overall catalytic efficiency, k cat ⁄ K m , was slightly increased for the truncated enzymes toward the soluble 4-methylumbelliferyl-N-N¢-diacetyl chitobiose or 4-methylumbelliferyl-N-N¢¢-N¢¢¢-triacetyl chitotriose or insoluble a-chitin substrate. By contrast, changes to substrate affinity, K m , and turnover rate, k cat , varied considerably for both types of chitin substrates between the full-length and truncated enzymes. Both truncated enzymes exhibited significantly higher thermostabilities than the full-length enzyme. The truncated mutants retained similar substrate-binding specificities and abilities against the insoluble substrate but only had approximately 75% of the hydrolyzing efficiency of the full-length chitinase molecule. Fluorescence spectroscopy indicated that both C-terminal deletion mutants retained an active folding conformation similar to the full-length enzyme. However, a CD melting unfolding study was able to distinguish between the full-length and truncated mutant molecules by the two phases of apparent transition temperatures in the mutants. These results indicate that up to 145 amino acid residues, including the putative C-terminal chitin-binding region and the fibronectin (III) motif of B. licheniformis chitinase, could be removed without causing a seriously aberrant change in structure and a dramatic decrease in insoluble chitin hydrolysis. The results of the present study provide evidence demonstrating that the binding and hydrolyzing of insoluble chitin substrate for B. licheniformis chitinase was not dependent solely on the putative C-terminal chitin-binding region and the fibronectin (III) motif.

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.