Evaluation of chitosan-binding amino acid residues of chitosanase from Paenibacillus fukuinensis
Evaluation of chitosan-binding amino acid residues of chitosanase from Paenibacillus fukuinensis
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DOI：
10.1080/09168451.2014.917263
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发表时间：
2014-07
期刊：
影响因子：
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通讯作者：
Danya Isogawa;Hironobu Morisaka;K. Kuroda;H. Kusaoke;H. Kimoto;S. Suye;M. Ueda
中科院分区：
文献类型：
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作者：
Danya Isogawa;Hironobu Morisaka;K. Kuroda;H. Kusaoke;H. Kimoto;S. Suye;M. Ueda
长于六聚体的壳寡糖具有比聚合物或较短寡糖（例如单体或二聚体）更高的生物活性。在我们之前的工作中，我们利用酵母细胞表面展示系统构建了展示壳聚糖酶的酵母菌，并对其催化基础进行了论证。在这里，我们研究了特定的功能，推定的四个氨基酸残基Trp 159，Trp 228，Tyr 311，和Phe 406从事底物结合。利用该系统，我们产生了壳聚糖酶突变体，其中四个氨基酸残基被替换为丙氨酸和壳聚糖酶活性测定和HPLC分析进行。基于这些结果，我们证明了Trp 159和Phe 406对于水解聚合物和寡糖都是关键的，并且Trp 228和Tyr 311对于结合寡糖（例如壳聚糖-六聚体）而不是结合壳聚糖聚合物特别重要。从结果中，我们提出了有效的策略，设计有用的突变体，产生具有更高的生物活性的壳寡糖的可能性。图解摘要阐明了底物结合裂隙中氨基酸残基对壳寡糖结合的重要性差异。
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. Graphical Abstract We clarified the difference of importance for binding chitosan oligosaccharides among amino acid residues in substrate binding cleft.