Reactivity of trypsin in reverse micelles: pH-effects on the W0 versus enzyme activity profiles.

pH-Dependence of hydrolytic activity of trypsin has been studied in cationic reverse micellar system of cetyltrimethylammonium bromide (CTAB) in (50% v/v) chloroform/isooctane using a positively charged substrate N(alpha)-benzoyl-L-arginine ethyl ester (BAEE). The pH of the medium was varied from 4.0 to 8.5 with addition of 0.025 M citrate-phosphate buffer containing 1 mM CaCl2. Optimum pH for maximum enzyme activity, pH(opt) in reverse micelles is found to be similar to that observed in bulk aqueous solution (8.0-8.5). However, changes in activity of trypsin (k(cat)) as a function of water content W0 (W0 = [H2O]/[CTAB]) in reverse micelles are found to be pH dependent. At low pH (4.0) and low water content (W0 = 5) the enzyme is more active in reverse micelles than in bulk aqueous solution by a factor of 2. This 'superactivity' is lost at higher W0 values and the k(cat) in reverse micelles is found to be similar to that observed in aqueous bulk. At pH 5, the enzyme activity is found to be independent of W0 while at pH 6.0-6.5 the enzyme activity is low at W0 5 and increases with water content to a constant value which is still 50% lower than that in aqueous buffer. Above pH 7, the W0-activity profile becomes distinctly bell shaped with W0 optimum around 10-15. The enzyme activity at optimum W0 is close to that observed in aqueous bulk.