β-Galactosidases of suspension-cultured Medicago sativa cells growing on lactose

Abstract

During the growth of suspension-cultured Medicago sativa cells, β-galactosidase activities were investigated, using the standard substrate O-nitrophenyl- β-galactoside (ONPG), with respect to the lactose uptake and residual sugar levels in the cells and in the broth. The pH profiles showing two optima at pH 4 and pH 7 in crude extracts of cells, strongly suggested the occurrence of two β-galactosidase activities. Only one optimum pH was detected in the broth at about pH 4.4. The β-galactosidase activity at pH 7 was found to be located inside the cells while that observed at pH 4 was bound to the cell wall. At the onset of the active growth phase an increase in the β-galactosidase activity occurred in the medium, probably due to a release of the cell wall linked enzyme. The separation of the intracellular β-galactosidases by gel filtration on a Sephadex G-200 column was achieved and the pH dependances of the enzymes were confirmed. The two enzymes were found to be efficient on lactose hydrolysis.