High-resolution structural characterization of a heterogeneous biocatalyst using solid-state NMR
High-resolution structural characterization of a heterogeneous biocatalyst using solid-state NMR
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DOI：
10.1021/acs.jpcc.6b11575
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发表时间：
2016-12
影响因子：
3.7
通讯作者：
Sabu Varghese;P. Halling;D. Häussinger;S. Wimperis
中科院分区：
文献类型：
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作者：
Sabu Varghese;P. Halling;D. Häussinger;S. Wimperis
采用固态魔角旋转 (MAS) NMR 光谱研究共价固定在环氧官能化二氧化硅上的均匀 15N 和选择性（15N 亮氨酸）富集状态的人碳酸酐酶 II (hCA II) 的结构细节。与溶液中的游离酶相比，固定化的 hCA II 保留了 71% 的比酶活性。基于一维和二维 1H、13C、15N 和 29Si MAS NMR 谱，可以从二氧化硅载体、共价连接体和固定化酶获得化学位移分配。除了来自修饰支持物的信号外，固定化酶中芳香族和羰基信号的出现也证实了酶在环氧二氧化硅上的成功共价固定。最值得注意的是，我们的 MAS NMR 结果表明，hCA II 在环氧二氧化硅上的共价固定不会显着影响蛋白质的结构完整性。
Solid-state magic-angle spinning (MAS) NMR spectroscopy was employed to investigate structural detail in the enzyme human carbonic anhydrase II (hCA II) in uniformly 15N and selectively (15N leucine) enriched states, covalently immobilized on epoxy-functionalized silica. The immobilized hCA II retained 71% of its specific enzymatic activity when compared to the free enzyme in solution. On the basis of the one- and two-dimensional 1H, 13C, 15N, and 29Si MAS NMR spectra, chemical shift assignments could be obtained from the silica support, covalent linker, and immobilized enzyme. The successful covalent immobilization of the enzyme on epoxy–silica was confirmed by the appearance of signals from the aromatic and carbonyl groups in the immobilized enzyme in addition to signals from the modified support. Most notably, our MAS NMR results suggest that the covalent immobilization of the hCA II on epoxy–silica does not significantly affect the structural integrity of the protein.