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Life is fueled by multi-enzymatic tandem processes that display unmatched catalytic efficiencies owing to certain features of the biological reactors such as compartmentalization, nano-confinement, and out-of-equilibrium dynamics. With an attempt to match such natural catalytic systems, herein, we present a chemoenzymatic pH clock mediated transient assembly of a vesicular nanozyme. Distinct confinement of two catalytically discrete units, Histidine groups on the periphery and hemin in the lipid bilayer, results in an efficient hydrolase-peroxidase tandem catalysis in a temporally controlled fashion. The pH clock, constituted by alkaline TRIS (Tris(hydroxymethyl)aminomethane hydrochloride) buffer (promoter) and glucose oxidase (GOx) catalyzed oxidation of glucose, steers the transience in an asymmetric fashion. Alkaline TRIS buffer enhances the pH of the system and triggers the formation of imine linked Supramolecular Peptide Amphiphiles (SPAs) that further assemble into vesicles. On the other hand, oxidation of glucose produces gluconolactone and H

This work reports the significant enhancement in performance of interfacially active enzymes, Chromobacterium viscosum (CV) lipase and horseradish peroxidase (HRP) in mixed reverse micelles of cetyltrimethylammonium bromide (CTAB) and imidazolium-based amphiphiles having varying tail lengths. Lipase activity in these mixed systems was always higher than that in the individual cationic reverse micelles of CTAB or any imidazolium surfactant, highest being observed in the mixed system of CTAB (50 mM) and 6 (1-tetradecyl-3-methyl imidazolium bromide, 40 mM)/water/isooctane/n-hexanol (0.24 M), second-order rate constant, k2=1301+/-5 cm3 g(-1)s(-1), approximately 200% higher compared to that in CTAB and approximately 65% more than the most popular AOT-microemulsion. Activity increased with concentration of imidazolium surfactant and also with its alkyl tail length. To have a more profound view on the structure-activity relationship, CTAB was replaced by cetyltriethylammonium bromide (CTEAB) and cetyltripropylammonium bromide (CTPAB) with subsequent increase in the headgroup size. The generalized influence of these mixed cationic systems on surface-active enzyme was also verified using HRP, where the activity improved approximately 100%. This enhancement in enzyme activity is presumably due to the activating effect of the imidazolium cation in the enzymatic reactions by improving the nucleophilicity of interfacial water in vicinity of enzyme through hydrogen bonding.

The catalytic efficiency of trypsin was estimated in cationic reverse micelles as a function of the concentration of water-pool components and aggregate size to determine their independent influence on enzyme activity. The variation in the aggregate size/water-pool size was achieved by changing both the W 0 (mole ratio of water to surfactant) and the headgroup area of surfactant through introduction of hydroxyethyl groups at the polar head. The local molar concentrations of water present inside the water-pool ([H 2 O] wp ) of different cationic reverse micelles across varying W 0 was estimated using a modified phenyl cation-trapping protocol. The [H 2 O] wp in cationic reverse micelles (surfactant/isooctane/ n -hexanol/water) increases with W 0 and attains the molarity of normal water beyond W 0 = 40 irrespective of the nature of headgroup. Concurrently, the catalytic activity of trypsin compartmentalized within the water-pool increases with the increase in [H 2 O] wp upto an optimal W 0 = 40 in organized solutions of any surfactant. The aggregate size (determined by static light scattering) also increases expectedly with W 0 and noticeably with the area of the surfactant headgroup at similar W 0 . Since the enzyme activity rises both with the increase in water-pool size and [H 2 O] wp , trypsin's efficiency was compared with these two parameters across reverse micelles of varying surfactant headgroup size at similar W 0 to determine their probable independent influence in regulating the enzyme activity. Noticeably, the efficiency of trypsin rises two to ninefold in spite of the [H 2 O] wp being distinctly lower in case of hydroxyethyl group substituted surfactants compared to cetyltrimethylammonium bromide w/o microemulsions at similar W 0 . Thus, the influence of the aggregate size possibly plays an important role alongwith the [H 2 O] wp in modulating the enzyme activity.