Demonstration of catalytic proton acceptor of chitosanase from Paenibacillus fukuinensis by comprehensive analysis of mutant library.
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.
Isogawa
Danya
D
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto, 606-8502, Japan.
Fukuda
Takeshi
T
Kuroda
Kouichi
K
Kusaoke
Hideo
H
Kimoto
Hisashi
H
Suye
Shin-ichiro
S
Ueda
Mitsuyoshi
M
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
D001426:Bacterial Proteins; D005936:Glucans; D011522:Protons; D005973:Glutamine; D001216:Asparagine; D048271:Chitosan; C040088:1,4-glucan; D006026:Glycoside Hydrolases; C019868:chitosanase