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.

菊粉酶是糖苷水解酶家族 32 (GH32) 的成员。它催化菊粉中2,1-β-D-果糖苷键的随机水解，并在高果糖浆的生产中发挥作用。在本研究中，通过计算机分析和定点诱变评估了来自克鲁维酵母 CBS4857 (KcINU1) 的外切菊粉酶的保守残基 W79、F113、M117、R181、C239 和 W334 在底物结合和稳定中的详细作用。这些残基属于 GH32 的保守 WG、FSGSMV、RDP、ECP 和 WQY 区域，位于 KcINU1 催化口袋周围。酶谱分析显示，与针对菊粉和蔗糖的野生型酶相比，F113W 的条带相对较弱，M117A 的条带相似，而所有其他变体在天然聚丙烯酰胺凝胶电泳上均未显示可观察到的染色。通过二硝基水杨酸比色法进一步证实了这些结果。结果表明，F113W对菊粉和蔗糖的残留活性分别为33.8±3.3%和96.2±5.5%，M117A的残留活性分别为103.8±1.3%和166.5±12%。荧光光谱结果表明，与野生型酶相比，F113W 发生了显着的构象变化，而 M117A 尽管猝灭效应有所增强，但影响有限。