Deletion of Loop Fragment Adjacent to Active Site Diminishes the Stability and Activity of Exo-Inulinase Publisher Pubmed



Arjomand MR^{1, 3} ; Habibirezaei M^{1, 2} ; Ahmadian G³ ; Hassanzadeh M⁴ ; Karkhane AA³ ; Asadifar M¹ ; Amanlou M⁴
Source: International Journal of Biological Macromolecules Published:2016

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 80 ns 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 (Vmax, Km, kcat and, kcat/Km) and activation energy (Ea) 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. © 2016