Tailoring of horseradish peroxidase activity in cationic water-in-oil microemulsions

Horseradish peroxidase (HRP) in cationic water-in-oil (W/O) microemulsions has always been ignored in reverse micellar enzymology, mainly because cationic surfactants are inhibitors of enzyme peroxidase. In the present study, for the first time, we have successfully introduced the cationic W/O microemulsion as an attractive host for efficient HRP activity. To this notion, much improved activity of HRP was observed in the W/O microemulsion of cetyltrimethylammonium bromide (CTAB) with an increase in n-hexanol concentration and W0 ([water]/[surfactant]), presumably due to the increased interfacial area of the microemulsions. In support of our above observation, six surfactants were synthesized with an increased headgroup size where the methyl groups of CTAB were subsequently replaced by the n-propyl and 2-hydroxyethyl groups, respectively, to prepare mono-, di-, and tripropylated/hydroxyethylated n-hexadecylammonium bromide. The peroxidase activity enhanced with headgroup size and also followed an overall trend similar to that found in the case of CTAB. Possibly, the reduced positive charge density at the augmented interfacial area by means of increase, either in headgroup size, cosurfactant concentration, and/or W0, is not capable of inactivating HRP. Also, the larger space at the interface may facilitate easier solubilization of the enzyme and increase the local concentration of enzyme and substrate, leading to the higher activity of HRP. The best activity was obtained with surfactant N-hexadecyl-N,N,N-tripropylammonium bromide, the highest ever found in any cationic W/O microemulsions, being almost 3 times higher than that found in water. Strikingly, this observed highest activity is comparable with that observed in an anionic bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT)-based system, the best W/O microemulsions used for HRP.