RESUMO
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.
Assuntos
Quitosana/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Paenibacillus/enzimologia , Substituição de Aminoácidos , Glicosídeo Hidrolases/genética , Hidrólise , Modelos Moleculares , Mutação , Ligação Proteica , Conformação ProteicaRESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Análise Mutacional de DNA , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Paenibacillus/enzimologia , Prótons , Substituição de Aminoácidos/genética , Asparagina/genética , Asparagina/metabolismo , Quitosana/metabolismo , Glucanos/metabolismo , Glutamina/genética , Glutamina/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Paenibacillus/genética , Estrutura Terciária de ProteínaRESUMO
To produce chitoorigosaccharides using chitosan, we attempted to construct Paenibacillus fukuinensis chitosanase-displaying yeast cells as a whole-cell biocatalyst through yeast cell-surface engineering. The localization of the chitosanase on the yeast cell surface was confirmed by immunofluorescence labeling of cells. The chitosanase activity of the constructed yeast was investigated by halo assay and the dinitrosalicylic acid method.