Deletion of loop fragment adjacent to active site diminishes the stability and activity of exo-inulinase

Abstract

Inulinases are classified as hydrolases and widely used in the food and medical industries. Here, we report the deletion of a six-membered adjacent active site loop fragment (⁷⁴YGSDVT⁷⁹ sequence) from third Ω-loop of the exo-inulinase containing aspartate residue from Aspergillus niger to study its structural and functional importance. Site-directed mutagenesis was used to create the mutant of the exo-inulinase (Δ6SL). To investigate the stability of the region spanning this loop, MD simulations were performed 80ns for 20-85 residues. Molecular docking was performed to compare the interactions in the active sites of enzymes with fructose as a ligand. Accordingly, the functional thermostability of the exo-inulinase was significantly decreased upon loop fragment deletion. Evaluation of the kinetics parameters (V_max, K_m, k_cat and, k_cat/K_m) and activation energy (E_a) of the catalysis of enzymes indicated the importance of the deleted sequence on the catalytic performance of the enzyme. In conclusion, six-membered adjacent active site loop fragment not only plays a crucial role in the stability of the enzyme, but also it involves in the enzyme catalysis through lowering the activation energy of the catalysis and effective improving the catalytic performance.

Keywords: Catalytic performance; Exo-inulinase; Functional thermostability; Site-directed mutagenesis; Ω-Loop.