Isogawa Danya, Morisaka Hironobu, Kuroda Kouichi, Kusaoke Hideo, Kimoto Hisashi, Suye Shin-Ichiro, Ueda Mitsuyoshi
a Division of Applied Life Sciences , Graduate School of Agriculture, Kyoto University , Kyoto , Japan.
Biosci Biotechnol Biochem. 2014;78(7):1177-82. doi: 10.1080/09168451.2014.917263. Epub 2014 Jun 17.
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.
比六聚体更长的壳寡糖比聚合物或更短的寡糖（如单体或二聚体）具有更高的生物活性。在我们之前的工作中，我们使用酵母细胞表面展示系统构建了展示福冈芽孢杆菌壳聚糖酶的酵母，并阐明了催化碱基。在此，我们研究了假定参与底物结合的四个氨基酸残基Trp159、Trp228、Tyr311和Phe406的具体功能。利用该系统，我们构建了四个氨基酸残基被丙氨酸取代的壳聚糖酶突变体，并进行了壳聚糖酶活性测定和高效液相色谱分析。基于这些结果，我们证明Trp159和Phe406对于水解聚合物和寡糖都至关重要，而Trp228和Tyr311对于结合寡糖（如壳聚糖六聚体）而非壳聚糖聚合物尤为重要。根据这些结果，我们提出了设计有用突变体的有效策略的可能性，这些突变体可产生具有更高生物活性的壳寡糖。
