title: Demonstration of catalytic proton acceptor of chitosanase from Paenibacillus fukuinensis by comprehensive analysis of mutant library.
abstract: Chitosanase from Paenibacillus fukuinensis D2 is an attractive enzyme, and it exhibits both chitosanase and beta-1, 4 glucanase activities. In our previous study, we generated P. fukuinensis chitosanase-displaying yeast cells using a yeast cell surface-displaying system. Chitosanase-displaying yeast can be utilized as a chitosanase cluster without many time-consuming purification steps. In this study, using the system, we have investigated whether Glu302, which is supposed as a putative proton acceptor, is an essential amino acid residue for exhibiting chitosanase activity and analyzed the contribution of mutual interaction between Glu302 and Asn312 to the activity. A mutant library in which Glu302 and Asn312 were comprehensively substituted by the other amino acid residues was constructed on the yeast cell surface. From the results of chitosanase and beta-1, 4 glucanase activity assays, we demonstrated that Glu302 was a proton acceptor for chitosanase activity, and Asn312 also participated in the hydrolysis of chitosan and cellulose.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto, 606-8502, Japan.
authors.forename: Danya
authors.initials: D
authors.lastname: Isogawa
authors.forename: Takeshi
authors.initials: T
authors.lastname: Fukuda
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 / Q000235:genetics; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D048271:Chitosan / Q000378:metabolism; D004252:DNA Mutational Analysis; D005936:Glucans / Q000378:metabolism; D005973:Glutamine / Q000235:genetics / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011522:Protons
chemical_list: D001426:Bacterial Proteins; D005936:Glucans; D011522:Protons; D005973:Glutamine; D001216:Asparagine; D048271:Chitosan; C040088:1,4-glucan; D006026:Glycoside Hydrolases; C019868:chitosanase