Universidad Nacional de Córdoba, Facultad de Ciencias Exactas, Físicas y Naturales, Departamento de Química, Cátedra de Química Biológica, Av. Vélez Sársfield 1611, 5016 Córdoba, Argentina; CONICET-Universidad Nacional de Córdoba, Instituto de Investigaciones Biológicas y Tecnológicas (IIByT), Córdoba, Argentina.
Universidad Nacional de Córdoba, Facultad de Ciencias Exactas, Físicas y Naturales, Departamento de Química, Cátedra de Química Biológica, Av. Vélez Sársfield 1611, 5016 Córdoba, Argentina.
Biochem Biophys Res Commun. 2019 Jan 1;508(1):270-274. doi: 10.1016/j.bbrc.2018.11.077. Epub 2018 Nov 26.
The effect on protein conformation and thermal stability was studied for β-Galactosidase (β-Gal) encapsulated in the nanopores of a silicate matrix (E). Circular dichroism spectra showed that, compared with the enzyme in buffer (S), E exhibited a higher content of α-helix structure. Heating E up to 75 °C caused a decrease in the content of β-sheet structure and additional augments on E components attributed to helical content, instead of the generalized loss of the ellipticity signal observed with S. Steady state fluorescence spectroscopy analysis evidenced an E structure less compact and more accessible to solvent and also less stable against temperature increase. While for S the denaturation midpoint (Tm) was 59 °C, for Eit was 48 °C. The enzymatic activity assays at increasing temperatures showed that in both conditions, the enzyme lost most of its hydrolytic activity against ONPG at temperatures above 65 °C and E did it even at lower T values. Concluding, confinement in silica nanopores induced conformational changes on the tertiary/cuaternary structure of E leading to the loss of thermal stability and enzymatic activity.
研究了β-半乳糖苷酶（β-Gal）包埋在硅酸盐基质（E）的纳米孔中对其蛋白质构象和热稳定性的影响。圆二色光谱显示，与缓冲液中的酶（S）相比，E 表现出更高含量的α-螺旋结构。将 E 加热至 75°C 会导致β-折叠结构含量降低，并且 E 组分的额外增加归因于螺旋含量的增加，而不是与 S 观察到的椭圆率信号的普遍损失。稳态荧光光谱分析表明，E 的结构不太紧凑，对溶剂更具可及性，并且对温度升高的稳定性也较低。虽然 S 的变性中点（Tm）为 59°C，但 E 的 Tm 为 48°C。在升高温度下进行的酶活性测定表明，在两种情况下，酶在高于 65°C 的温度下对 ONPG 的水解活性丧失了大部分，而 E 在更低的 T 值下就已经丧失了。综上所述，在二氧化硅纳米孔中的限制导致 E 的三级/四级结构发生构象变化，从而导致热稳定性和酶活性丧失。
