In the present work an automatic methodology, based on sequential injection analysis, (SIA) for the kinetic evaluation of α-chymotrypsin (α-CT) activity in cetyltrimethylammonium bromide (CTAB)/ionic liquid (IL) mixed micelles, was developed. The α-CT-catalysed hydrolysis of N-succinyl-l-phenylalanine-p-nitroanilide (SPpNA) was monitored by following the change in absorbance at 410nm brought about by the formation of p-nitroaniline (pNA). The influence of parameters such as reagents concentration, flow rate, as well as reaction coil length, on the sensitivity and performance of the SIA system were studied and the optimum reaction conditions were subsequently selected. The effect of CTAB/IL micelles on the catalytic constant Kcat and apparent Michaelis-Menten constant (KM) for SPpNA hydrolysis was then studied. The kinetic assays evidence that CTAB/ILs mixed micellar systems can induce α-CT superactivity. In order to perform a critical evaluation of the obtained results, CMC and average micellar size of CTAB/hmim[Cl], CTAB/bmim[Cl], CTAB/bmpyr[Cl] and CTAB/bmpy[Cl] mixed micelles were evaluated by fluorescence and dynamic light scattering, respectively. The SIA methodology showed to be an interesting tool for evaluation of α-CT activity in mixed micelles as it proved to be robust and exhibited good repeatability in all the assay conditions leading also to a reduction of the consumption of solvents as well as of effluent production.
Keywords: Ionic liquids; Kinetic assays; Mixed micelles; SIA; α-Chymotrypsin.
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