Characterisation of β-galactosidase isozymes of ripening peppers

Abstract

Activity of β-galactosidase (EC 3.2.1.23) and isozymes of the New Mexican type pepper were assayed in ripening fruit and characterised in order to further understand the possible role this enzyme plays in fruit ripening. Total β-galactosidase activity increased in New Mexican type peppers (NM chile) as they ripened. To a lesser extent, xylosidase and β-glucosidase increased from the mature green stage to the turning stage of ripening and then decreased in the red ripe stage. Cation exchange chromatography indicated two prominent β-galactosidase peaks depending on stage of ripeness. In mature NM green chile, one large anionic, and in NM red chile, one large cationic peak was found. Isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) separated one isozyme from mature green NM chile (pI 4.6) and an additional β-galactosidase isozyme as NM chile turned from green to red (pI 8.0). The cationic β-galactosidase isozyme from red NM chile extract had an apparent native-molecular weight of 51 kDa. The optimum pH for β-galactosidase activity in both red and green NM chile was between 3 and 4.5. Stable enzyme activity was observed from 25 to 50°C. β-Galactosidase from red NM chile had a K m of 0.87 mM and V max of 12.3 nKat, while the values for green NM chile β-galactosidase were 2.5 mM and 8.3 nKat. The heavy metal ions Ag 2+ and Hg 2+ inhibited β-galactosidase activity by 100% and 90%, respectively.