Immunolocalization of beta-glucosidase immobilized within polysulphone capillary membrane and evaluation of its activity in situ

A new combined method is reported to localize the sites of enzyme immobilization and to determine its catalytic activity on a polymeric capillary membrane reactor. The useful new method resulted from the merging of the classic in situ enzyme activity assay and western blot technique whose both results are easily detectable either at low than at high magnification in light microscopy. ?-Glucosidase from olive fruit was selected as enzyme model because of its suitable relevance in the industrial processing of foods, in biotechnology and in pharmaceuticals and for its activity against the synthetic substrate 5-brome-4-chloro-3-indolyl-?-D-glucopyranosyde which develops an insoluble dyed product. The enzyme was physically immobilized within 30 kDa cut-off capillary polysulphone membranes and results obtained by means of a polyclonal antibody against ?-glucosidase and the synthetic substrate clearly showed a coherent localization of the immobilization enzyme sites and its activity.