Departamento de Química, Universidad Nacional de Río Cuarto, Agencia Postal # 3. C.P. X5804BYA Río Cuarto, Argentina.
Langmuir. 2013 Jul 2;29(26):8245-54. doi: 10.1021/la401103q. Epub 2013 Jun 20.
The reverse micelle (RM) media are very good as nanoreactors because they can create a unique microenvironment for carrying out a variety of chemical and biochemical reactions. The aim of the present work is to determine the influence of different water-dimethyl sulfoxide (DMSO) mixtures encapsulated in 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/n-heptane RMs on the enzymatic hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide (Bz-Try-pNA) by α-chymotrypsin (α-CT). The reaction was first studied in homogeneous media at different DMSO-water mixture compositions and in DMSO-water/AOT/n-heptane RMs. The hydrolysis rates of Bz-Try-pNA catalyzed by α-CT were determined by UV-vis spectroscopy. The reaction follows the Michaelis-Menten mechanism and the kinetic parameters: kcat, KM, and kcat/KM were evaluated under different conditions. In this homogeneous media, DMSO plays an important role in the solubilization process of the peptide which is almost insoluble in water, but it has a tremendous impact on the inactivation of α-CT. It is shown that the enzyme dissolved in a 20% molar ratio of the DMSO-water mixture does not present enzymatic activity. Dynamic light scattering has been used to assess the formation of DMSO-water/AOT/heptane RMs at different DMSO compositions. The results also show that there is preferential solvation of the AOT RM interface by water molecules. To test the use of these RMs as nanoreactors, the kinetic parameters for the enzymatic reaction in these systems have been evaluated. The parameters were determined at fixed W(S) {W(S) = ([water] + [DMSO])/[AOT] = 20} at different DMSO-water compositions. The results show that the Michaelis-Menten mechanism is valid for α-CT in all the RM systems studied and that the reaction takes place at the RM interface. Surprisingly, it was observed that the enzyme encapsulated by the RMs show catalytic effects with similar kcat/KM values at any DMSO composition investigated, which evidence that DMSO molecules are localized far from the RM interface.
反胶束（RM）介质非常适合作为纳米反应器，因为它们可以为进行各种化学和生化反应创造独特的微环境。本工作的目的是确定不同水-二甲基亚砜（DMSO）混合物在 1,4-双-2-乙基己基琥珀酸酯（AOT）/正庚烷 RM 中包封对α-糜蛋白酶（α-CT）催化 N-苯甲酰-L-酪氨酸对硝基苯胺（Bz-Try-pNA）水解的影响。首先在不同 DMSO-水混合物组成的均相介质中和 DMSO-水/AOT/正庚烷 RM 中研究了该反应。通过紫外-可见光谱法测定 Bz-Try-pNA 的水解速率。反应遵循米氏-门坦机制，在不同条件下评估动力学参数：kcat、KM 和 kcat/KM。在这种均相介质中，DMSO 在溶解几乎不溶于水的肽的过程中起着重要作用，但对α-CT 的失活有巨大影响。结果表明，在 DMSO-水摩尔比为 20%的混合溶液中溶解的酶没有酶活性。动态光散射已用于评估不同 DMSO 组成下 DMSO-水/AOT/正庚烷 RM 的形成。结果还表明，水分子优先溶剂化 AOT RM 界面。为了测试这些 RM 作为纳米反应器的用途，评估了这些体系中酶反应的动力学参数。在固定 W(S) {W(S) = ([water]+[DMSO])/[AOT]=20}的情况下，在不同 DMSO-水组成下测定了参数。结果表明，对于所有研究的 RM 体系中的α-CT，米氏-门坦机制是有效的，反应发生在 RM 界面上。令人惊讶的是，观察到包封在 RM 中的酶在任何研究的 DMSO 组成下都表现出相似的 kcat/KM 值的催化效应，这表明 DMSO 分子远离 RM 界面定位。
