title: Evaluation of chitosan-binding amino acid residues of chitosanase from Paenibacillus fukuinensis.
abstract: Chitosan oligosaccharides longer than a hexamer have higher bioactivity than polymer or shorter oligosaccharides, such as the monomer or dimer. In our previous work, we generated Paenibacillus fukuinensis chitosanase-displaying yeast using yeast cell surface displaying system and demonstrated the catalytic base. Here we investigated the specific function of putative four amino acid residues Trp159, Trp228, Tyr311, and Phe406 engaged in substrate binding. Using this system, we generated chitosanase mutants in which the four amino acid residues were substituted with Ala and the chitosanase activity assay and HPLC analysis were performed. Based on these results, we demonstrated that Trp159 and Phe406 were critical for hydrolyzing both polymer and oligosaccharide, and Trp228 and Tyr311 were especially important for binding to oligosaccharide, such as the chitosan-hexamer, not to the chitosan polymer. From the results, we suggested the possibility of the effective strategy for designing useful mutants that produce chitosan oligosaccharides holding higher bioactivity.
authors.affiliation: a Division of Applied Life Sciences , Graduate School of Agriculture, Kyoto University , Kyoto , Japan.
authors.forename: Danya
authors.initials: D
authors.lastname: Isogawa
authors.forename: Hironobu
authors.initials: H
authors.lastname: Morisaka
authors.forename: Kouichi
authors.initials: K
authors.lastname: Kuroda
authors.forename: Hideo
authors.initials: H
authors.lastname: Kusaoke
authors.forename: Hisashi
authors.initials: H
authors.lastname: Kimoto
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Suye
authors.forename: Mitsuyoshi
authors.initials: M
authors.lastname: Ueda
mesh_terms: D019943:Amino Acid Substitution; D048271:Chitosan / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008958:Models, Molecular; D009154:Mutation; D056507:Paenibacillus / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase
keywords: Paenibacillus fukuinensis; chitosanase; substrate binding amino acid; yeast cell surface displaying system