Biofísica Química, Cátedra de Química Biológica, Department Química/ ICTA, Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Av. Vélez Sarsfield 1611, X5016CAG, Córdoba, Argentina.
Colloids Surf B Biointerfaces. 2010 Apr 1;76(2):387-96. doi: 10.1016/j.colsurfb.2009.11.005. Epub 2009 Nov 20.
In the present work we applied the sol-gel method to obtain glass lentils entrapping beta-D-galactosidase (beta-Gal) (Ebeta-Gal) within a silicate matrix. The effect of pH, temperature, polarity and salt concentration on the activity of Ebeta-Gal was studied. Apparent kinetic parameters for ortho-nitro-phenyl-beta-D-galactopyranoside hydrolysis catalyzed by Ebeta-Gal (V'max, K'M) were lower compared to the soluble enzyme (Sbeta-Gal), reflecting the solute diffusion restriction imposed by the matrix observed in the time curves, a partial protein inactivation upon encapsulation, and an improvement in the affinity of Ebeta-Gal for the substrate as compared with Sbeta-Gal. At pH<4, Ebeta-Gal stability was higher than that of Sbeta-Gal. Ebeta-Gal could be reused after storage at 4 degrees C for up to 90 days, and retained its activity profile within the range of pH=2-10 and saline concentration 0-400 mM. Pre-incubation at 75 degrees C for 30 min fully inactivated Sbeta-Gal while Ebeta-Gal retained approximately 90% of its activity, even in the reused samples. Encapsulation did not introduce additional impairments to the reaction rate measured in heterogeneous dispersions, beyond those derived from their own particle-crowded environment. This reusable Ebeta-Gal was resistant to typical technological conditions applied in milk processing that would lead to the unfolding and inactivation of Sbeta-Gal. The results are discussed from the biophysical viewpoint.
在本工作中，我们应用溶胶-凝胶法获得玻璃微珠，其内包埋β-D-半乳糖苷酶（β-Gal）（Eβ-Gal）于硅酸盐基质中。研究了 pH、温度、极性和盐浓度对 Eβ-Gal 活性的影响。与可溶性酶（Sβ-Gal）相比，Eβ-Gal 催化对硝基苯-β-D-半乳糖苷水解的表观动力学参数（V'max，K'M）较低，反映了基质对溶质扩散的限制，包埋后部分蛋白质失活，以及 Eβ-Gal 对底物的亲和力提高。在 pH<4 时，Eβ-Gal 的稳定性高于 Sβ-Gal。Eβ-Gal 可在 4°C 下储存长达 90 天，并且在 pH=2-10 和盐浓度 0-400 mM 范围内保持其活性谱。75°C 预孵育 30 分钟可完全使 Sβ-Gal 失活，而 Eβ-Gal 保留了约 90%的活性，即使在重复使用的样品中也是如此。封装在异相分散体中测量的反应速率不会引入除其自身颗粒拥挤环境引起的额外损伤，除了那些之外。这种可重复使用的 Eβ-Gal 能够抵抗典型的在牛奶加工中应用的技术条件，这些条件会导致 Sβ-Gal 的展开和失活。结果从生物物理的角度进行了讨论。
