β-Galactosidase from Petunia hybrida: characterization and differentiation from E. coli β-galactosidase activity

Abstract

Summary Petunia β-galactosidase was partially purified, characterisized, and differentiated from Petunia β-glucosidase and E. coli β-galactosidase. Partially purified Petunia β-galactosidase preparations show a pH optimum of 3.1 to 4.3, and a temperature optimum of 55 °C. These characteristics allow a differentiation from the heat labile E. coli β-galactosidase with its pH optimum from 7.0 to 7.7. Testing at relevant optima it was possible to detect both activities even in a mixture of crude plant extracts with commercially available E. coli β-galactosidase. Also other properties, like the behavior against substrate analogues and DEAE cellulose chromatography, show a differentiation of plant and bacterial β-galactosidase activities. Polyacrylamide gel disc electrophoresis of the plant preparations gave only one zone of β-galactosidase activity. By isoelectrofocusing on Ampholine PAG plates, however, 4 main and several minor bands of activity showing isoelectric points from 5.5. to 6.65 could be detected. The E. coli β-galactosidase activity with its isoelectric point of 4.6 could be clearly separated. Therefore, isoelectrofocusing seems to be best suited not only to study plant β-galactosidase isoenzymes but also for detecting minor amounts of bacterial β-galactosidase in plant preparations. This is specially important in experiments where one tries to transfer the bacterial gene material for β-galactosidase into plant cells.