Demonstration of catalytic proton acceptor of chitosanase from Paenibacillus fukuinensis by comprehensive analysis of mutant library

• Published in: Applied microbiology and biotechnology 2009-11, Vol.85 (1), p.95-104
• Main Authors: Isogawa, Danya, Fukuda, Takeshi, Kuroda, Kouichi, Kusaoke, Hideo, Kimoto, Hisashi, Suye, Shin-ichiro, Ueda, Mitsuyoshi
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution - genetics; Amino acids; Asparagine - genetics; Asparagine - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biological and medical sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Cellulase; Cellulose; Chitosan - metabolism; Crustaceans; DNA Mutational Analysis; E coli; Enzymes; Fundamental and applied biological sciences. Psychology; Glucans - metabolism; Glutamine - genetics; Glutamine - metabolism; Glycoside Hydrolases - genetics; Glycoside Hydrolases - metabolism; Libraries; Life Sciences; Microbial Genetics and Genomics; Microbiology; Models, Molecular; Mutagenesis, Site-Directed; Paenibacillus - enzymology; Paenibacillus - genetics; Paenibacillus fukuinensis; Plasmids; Protein Structure, Tertiary; Protons; Studies; Yeast; Yeasts
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-009-2041-5

Chitosanase from Paenibacillus fukuinensis D2 is an attractive enzyme, and it exhibits both chitosanase and β-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 β-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.