Okayama Prefectural Technology Center for Agriculture, Forestry and Fisheries, Research Institute for Biological Sciences, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan.
Biochimie. 2012 Dec;94(12):2783-90. doi: 10.1016/j.biochi.2012.09.012. Epub 2012 Sep 23.
Mannanase is an important enzyme involved in the degradation of mannan, production of bioactive oligosaccharides, and biobleaching of kraft pulp. Mannanase must be thermostable for use in industrial applications. In a previous study, we found that the thermal stability of mannanase from Streptomyces thermolilacinus (StMan) and Thermobifida fusca (TfMan) is enhanced by calcium. Here, we investigated the relationship between the three-dimensional structure and primary sequence to identify the putative calcium-binding site. The results of site-directed mutagenesis experiments indicated that Asp-285, Glu-286, and Asp-287 of StMan (StDEDAAAdC) and Asp-264, Glu-265, and Asp-266 of TfMan (TfDEDAAAdC) were the key residues for calcium binding affinity. Isothermal titration calorimetry revealed that the catalytic domain of StMan and TfMan (StMandC and TfMandC, respectively) bound calcium with a K(a) of 3.02 × 10(4) M(-1) and 1.52 × 10(4) M(-1), respectively, both with stoichiometry consistent with one calcium-binding site per molecule of enzyme. Non-calcium-binding mutants (StDEDAAAdC and TfDEDAAAdC) did not show any calorimetric change. From the primary structure alignment of several mannanases, the calcium-binding site was found to be highly conserved in GH5 bacterial mannanases. This is the first study indicating enhanced thermal stability of GH5 bacterial mannanases by calcium binding.
甘露聚糖酶是一种重要的酶，参与甘露聚糖的降解、生物活性寡糖的生产和硫酸盐浆的生物漂白。甘露聚糖酶必须具有热稳定性才能用于工业应用。在之前的研究中，我们发现嗜热链霉菌（StMan）和嗜热纤维单胞菌（TfMan）的甘露聚糖酶的热稳定性通过钙得到增强。在这里，我们研究了三维结构和一级序列之间的关系，以确定可能的钙结合位点。定点突变实验的结果表明，StMan 的 Asp-285、Glu-286 和 Asp-287（StDEDAAAdC）和 TfMan 的 Asp-264、Glu-265 和 Asp-266（TfDEDAAAdC）是钙结合亲和力的关键残基。等温滴定量热法表明，StMan 和 TfMan 的催化结构域（StMandC 和 TfMandC）分别以 3.02×10^4 M^(-1)和 1.52×10^4 M^(-1)的 K(a)值结合钙，其化学计量学与每个酶分子结合一个钙结合位点一致。非钙结合突变体（StDEDAAAdC 和 TfDEDAAAdC）没有显示任何量热变化。从几种甘露聚糖酶的一级结构比对中发现，钙结合位点在 GH5 细菌甘露聚糖酶中高度保守。这是首次表明 GH5 细菌甘露聚糖酶通过钙结合增强热稳定性的研究。
