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Inulinase is a member of the glycoside hydrolase family 32 (GH32). It catalyzes the randomly hydrolyzation of 2,1-β-D-fructosidic linkages in inulin and plays a role in the production of high-fructose syrup. In this study, detailed roles of the conserved residues W79, F113, M117, R181, C239, and W334 of the exo-inulinase from  Kluyveromyces cicerisporus  CBS4857 (KcINU1) in substrate binding and stabilization were evaluated by  in silico  analysis and site-directed mutagenesis. These residues belong to the conserved WG, FSGSMV, RDP, ECP, and WQY regions of the GH32 and are located around the catalytic pocket of KcINU1. Zymogram assay showed relatively weaker band for F113W and similar band for M117A compared to the wild-type enzyme toward inulin and sucrose, whereas all other variants showed no observable stain on the native polyacrylamide gel electrophoresis. These results were further confirmed with the dinitrosalicylic acid colorimetric method. It showed that the residual activities of F113W toward inulin and sucrose were 33.8 ± 3.3% and 96.2 ± 5.5%, respectively, and that of M117A were 103.8 ± 1.3% and 166.5 ± 12%, respectively. Results from fluorescence spectra indicated that there is a significant conformational change that happened in F113W compared to the wild-type enzyme, while M117A exhibited limited impact although the quenching effect was increased.

The Important Roles Played in Substrate Binding of Aromatic Amino Acids in Exo-Inulinase From Kluyveromyces cicerisporus CBS 4857