Evidence for beta-galactosidase catalyzed hydrolysis of paranitrophenyl-beta-D-galactopyranoside anchored in cyclodextrins.

The absorption maximum of p-nitrophenol upon mixing with molar equivalents of alpha-cyclodextrin (alpha-CD) or hydroxypropyl-beta-cyclodextrin (HPB) showed nearly 5 nm shift to the longer wavelength region, indicative of complex formation, while beta-cyclodextrin (beta-CD), gamma-cyclodextrin (gamma-CD) and hydroxyethyl-beta-cyclodextrin (HEB) produced only a marginal shift of about 1-2 nm, suggestive of a weaker interaction. It has been shown by circular dichroism spectral studies that the aglycon part of p-nitrophenyl-beta-D-glycoside (PNPG) is also encapsulated by alpha-cyclodextrin. The encapsulated form of PNPG could be hydrolyzed by beta-galactosidase, the temperature and pH-optima for hydrolysis of anchored substrates being essentially identical to that of free substrate. However, small but consistent increase in Km values were obtained for alpha-cyclodextrin-, HEB- and HPB-anchored substrates. The kcat values also registered an increase for the HEB- and HPB-anchored substrates. However, there was no increase in kinetic efficiency (kcat/Km) of enzyme. The inhibition noted at higher concentrations of HEB- and gamma-cyclodextrin-anchored PNPG but not with o-nitrophenyl-beta-D-galactoside (ONPG)-cyclodextrin mixture suggests that PNPG-cyclodextrin complexes were responsible for the inhibition. Taken together, these results suggest that the enzyme catalyses the hydrolysis of anchored substrates.

Soybean lipoxygenase catalysed oxygenation of unsaturated fatty acid encapsulated in cyclodextrin.

The linoleic or arachidonic acid entrapped in cyclodextrin (alpha, beta or gamma) serves as an excellent substrate for soybean lipoxygenase-1 catalysis. At pH 9.0 the Km values for the beta-cyclodextrin encapsulated arachidonic acid, referred herein as encapsulated substrate, and the Tween-20 dispersed substrate were 7.7 microM and 7.5 microM, respectively. However, the Vmax values for alpha- and beta-cyclodextrin solubilized substrates were lower in comparison with the Tween-20 dispersed substrate. Interestingly, the pH-activity profile for the enzyme towards cyclodextrin encapsulated arachidonic acid showed optimum around 7.5, while that towards Tween-20 dispersion showed the expected broad optimum in the alkaline range (8.5-10.0). The activity with encapsulated substrate at pH 7.5 was at least 5-fold higher than that obtained with Tween-20 dispersed substrate at the corresponding pH. Similar results were obtained using linoleic acid. The second order rate constant, Kcat/Km, for the encapsulated substrate was an order of magnitude higher when compared to the Tween-20 dispersed substrate. The plot of v obtained at pH 9.0, against S gave hyperbolic curves for both the encapsulated as well as the Tween-20 dispersed substrates, whereas at pH 7.5, the curve for cyclodextrin encapsulated arachidonic acid appeared initially concave and then at higher concentrations of the substrate sigmoidal. The positional specificity of soybean lipoxygenase remained unaltered, however.