1. Evaluation of chitosan-binding amino acid residues of chitosanase from Paenibacillus fukuinensis
- Author
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Hideo Kusaoke, Danya Isogawa, Kouichi Kuroda, Hisashi Kimoto, Hironobu Morisaka, Shin-ichiro Suye, and Mitsuyoshi Ueda
- Subjects
Models, Molecular ,Glycoside Hydrolases ,Protein Conformation ,Biology ,Applied Microbiology and Biotechnology ,Biochemistry ,Analytical Chemistry ,Chitosan ,Hydrolysis ,chemistry.chemical_compound ,Paenibacillus ,Chitosanase ,Molecular Biology ,chemistry.chemical_classification ,Chitosanase activity ,Organic Chemistry ,Chitosan binding ,General Medicine ,Oligosaccharide ,biology.organism_classification ,Yeast ,Amino Acid Substitution ,chemistry ,Mutation ,Protein Binding ,Biotechnology - 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.
- Published
- 2014
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