CHARACTERIZATION OF A GLUTARALDEHYDE STABILIZED YEAST-CELL BIOCATALYST WITH BETA-GALACTOSIDASE ACTIVITY

A whole cell biocatalyst (Kluyveromyces lactis) consisting in permeabi lized cells stabilized with glutaraldehyde under conditions that prese rve the beta-galactosidase activity was characterized in connection wi th autolytic processes, cell wall effects and sensitivity to shear str ess and exogenous proteases. Permeabilized cells display a typical aut olytic process that was characterized by the accumulation of extracell ular amino-nitrogen due to proteolysis, carbohydrate solubilization an d release of enzyme activities. The stabilization of the cell system w as manifested by the inhibition of the autolytic process. This phenome non was evident by the absence of proteolysis and enzyme solubilizatio n. Cell wall autolysis appears not to be totally blocked by the glutar aldehyde treatment. The beta-galactosidase contained in the stabilized cells was not solubilized when the cell wall was totally digested wit h exogenous lytic enzymes. Thus, a steric effect mediated by the cell wall was not involved in the insolubilization of the enzyme activity. Actually, the cross-linking reaction gave rise to the formation of an insoluble structure in which the intracellular, and partially the cell wall components, were immobilized. The property of this cluster of be ing insoluble is independent of the cell wall integrity. This structur al feature were confirmed by electron microscopy. The formation of the insoluble structure requires the glutaraldehyde reaction to occur in the intracellular environment. When the cell components were dispersed in the reaction mixture, the cross-linking reaction produces soluble high molecular aggregates containing the beta-galactosidase activity. The beta-galactosidase was inactivated when the stabilized cells were agitated with glass beads, indicating that the whole cell biocatalyst was sensitive to shear stress. Treatment of the stabilized cells with exogenous proteases, such as trypsine, both solubilize and inactivate the beta-galactosidase activity.