Demonstration of lactase activity in culture medium of melon cells

Abstract

Lactase activity was detected in a culture medium of the cell suspension culture of watermelon (Citrullus vulgaris L.). A simple, rapid and reproducible procedure for identification of extracellular lactase is described using callus cultures of seedlings from the tested plant, hairy roots of 2.5 days old seedlings of watermelon germinating on agar plates as well as cell suspension cultures derived from callus cultures. For the determination of intracellular activities of lactase, 6-bromo-2-naphthyl-β-D-galactopyranoside and p-nitrophenyl-β-D-galactopyranoside were used as synthetic substrates. The extracellular lactase activity was determined by evaluating the day-zone in agar medium. The enzyme from watermelon callus cultures and seedling roots, cultivated on agar plates supplemented with 6-bromo-2-naphthyl-2-bromo-β-D-galactopyranoside, hydrolyzed this substrate releasing 6-bromo-naphthyl. By simultaneous coupling with hexazonium p-rosaniline or Fast Blue BB the corresponding azo dye was formed. The parallel extracellular and intracellular activities were determined in cell suspension cultures derived from callus cultures. The results show a 43.8% intracellular and 54.2% extracellular distribution of lactase activity. The described agar plate method enables a rapid, simple and specific detection of plant processes of extracellular lactase.