Enzymatic Hydrolysis of N‑Benzoyl‑L‑Tyrosine p‑Nitroanilide by α‑Chymotrypsin in DMSO-Water/AOT/n‑Heptane Reverse Micelles. A Unique Interfacial Effect on the Enzymatic Activity

FERNANDO MOYANO ; EVANGELINA SETIEN; JUANA J. SILBER; N. MARIANO CORREA

LANGMUIR

AMER CHEMICAL SOC

Lugar: Washington; Año: 2012 vol. 29 p. 8245 - 8254

0743-7463

ABSTRACT: The reverse micelle (RM) media are very good as nanoreactors because they can create a uniquemicroenvironment for carrying out a variety of chemical and biochemical reactions. The aim of the present work is todetermine 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 andin DMSO-water/AOT/n-heptane RMs. The hydrolysis rates of Bz-Try-pNA catalyzed by α-CT were determined by UV−visspectroscopy. The reaction follows the Michaelis-Menten mechanism and the kinetic parameters: kcat, KM, and kcat/KM wereevaluated under different conditions. In this homogeneous media, DMSO plays an important role in the solubilization process ofthe peptide which is almost insoluble in water, but it has a tremendous impact on the inactivation of α-CT. It is shown that theenzyme dissolved in a 20% molar ratio of the DMSO-water mixture does not present enzymatic activity. Dynamic light scatteringhas been used to assess the formation of DMSO-water/AOT/heptane RMs at different DMSO compositions. The results alsoshow that there is preferential solvation of the AOT RM interface by water molecules. To test the use of these RMs asnanoreactors, the kinetic parameters for the enzymatic reaction in these systems have been evaluated. The parameters weredetermined at fixed WS {WS = ([water] + [DMSO])/[AOT] = 20} at different DMSO-water compositions. The results showthat the Michaelis−Menten mechanism is valid for α-CT in all the RM systems studied and that the reaction takes place at theRM interface. Surprisingly, it was observed that the enzyme encapsulated by the RMs show catalytic effects with similar kcat/KMvalues at any DMSO composition investigated, which evidence that DMSO molecules are localized far from the RM interface.