Improving the thermostability and modulating the inulin profile of inulosucrase through rational glycine-to-proline substitution
Thanapon Charoenwongpaiboon, Nawapat Sommanat, Karan Wangpaiboon, Manatsanan Puangpathanachai, Piamsook Pongsawasdi, Rath Pichyangkura
Abstract
121 inulosucrase (LrInu) on stability and inulin profile were investigated through glycine-to-proline substitutions. Molecular dynamics (MD) simulations were employed to discover the flexible glycine residues, and eight glycine residues, including Gly217, Gly298, Gly330, Gly416, Gly450, Gly624, Gly627, Gly629, were selected for site-directed mutagenesis. The results demonstrated significant changes in both thermostability and inulin profiles of the variants. Particularly, the G624P and G627P variants showed reduced production of long-chain oligosaccharides compared to the WT. This can be ascribed to the increased rigidity of the active site, which is crucial for the induction-fit mechanism. Overall, this study provides valuable insights into the role of flexible glycine residues in the activity, stability, and inulin synthesis of LrInu.