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

作者：Arjomand Maryam Rezaei; Habibi Rezaei Mehran^*; Ahmadian Gholamreza^*; Hassanzadeh Malihe; Karkhane Ali Asghar; Asadifar Mandana; Amanlou Massoud

来源：International Journal of Biological Macromolecules, 2016, 92: 1234-1241.

DOI：10.1016/j.ijbiomac.2016.08.039

• Exo-inulinase
• Omega-Loop
• Site-directed mutagenesis
• Functional thermostability
• Catalytic performance

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 ((74)YGSDVT(79) sequence) from third Omega-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 (Delta 6SL). 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 (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.

• 出版日期2016-11