title: 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 (74YGSDVT79sequence) 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 (Vmax, Km, kcatand, 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.
authors.affiliation: School of Biology, College of Science, University of Tehran, Tehran, Iran; Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
authors.forename: Maryam Rezaei
authors.initials: MR
authors.lastname: Arjomand
authors.affiliation: School of Biology, College of Science, University of Tehran, Tehran, Iran; Nano-Biomedicine Center of Excellence, Nanoscience and Nanotechnology Research Center, University of Tehran, Tehran, Iran. Electronic address: mhabibi@ut.ac.ir.
authors.forename: Mehran
authors.initials: M
authors.lastname: Habibi-Rezaei
authors.affiliation: Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran. Electronic address: ahmadian@nigeb.ac.ir.
authors.forename: Gholamreza
authors.initials: G
authors.lastname: Ahmadian
authors.affiliation: Department of Medicinal Chemistry & Drug Design and Development Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Malihe
authors.initials: M
authors.lastname: Hassanzadeh
authors.affiliation: Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
authors.forename: Ali Asghar
authors.initials: AA
authors.lastname: Karkhane
authors.affiliation: School of Biology, College of Science, University of Tehran, Tehran, Iran.
authors.forename: Mandana
authors.initials: M
authors.lastname: Asadifar
authors.affiliation: Department of Medicinal Chemistry & Drug Design and Development Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Massoud
authors.initials: M
authors.lastname: Amanlou
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry*; D001234:Aspergillus niger / Q000737:chemistry* / Q000201:enzymology; D002384:Catalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005632:Fructose / Q000737:chemistry; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D013816:Thermodynamics
chemical_list: D005656:Fungal Proteins; D005632:Fructose; D001224:Aspartic Acid; D006026:Glycoside Hydrolases; C023086:inulinase
keywords: Catalytic performance; Exo-inulinase; Functional thermostability; Site-directed mutagenesis; Ω-Loop