Purification, characterization and effect of physico-chemical agents on the stability of ?-galactosidase from betel leaves

Abstract

Context: β-galactosidase are present in a wide variety of organisms including plants, animals and microorganisms. Exploration of this enzyme from plant source will help to address the problems faced in the food and allied industries that look for enzymes with novel properties.  

Objectives: The aim of this study is to explore the purification, characterization and analysis of β-galactosidase from betel leaves.  

Materials and Methods: Aomal Bangla variety of betel leaf (Piper betle Linn.) was collected from betel vine. The column chromatographic method was done at 4°C using conventional method. The protein concentration was determined by UV-spectrophotometer at 280 nm. The activities of β-galactosidase were done by spectrophotometric method. All other reagents used in the study were of analytical grade. Unless specified, all the experimental conditions are maintained at 4°C.  

Results: After extraction of β-galactosidase from betel leaves, the crude enzyme was applied to DEAE-cellulose chromatography with sodium phosphate buffer (pH 7.0). The active fraction from DEAE-column chromatography was dialyzed with buffer and applied to CM-cellulose chromatography. The β-galactosidase activity from CM-cellulose chromatography was loaded to Sephadex G-75 Gel filtration chromatography. In this column, the enzyme β-galactosidase was eluted in a single peak. The homogeneity of purity was checked by disc gel electrophoresis and a single band was obtained. The optimum pH of β-GS-I, β-GS-II and β-GS-III were 3.5, 3.8 and 4.2, respectively. The optimum temperatures of the enzymes were 53, 51 and 56°C, respectively.  

Conclusion: The results obtained in this study suggest that three β-galactosidases namely β-GS-I, β-GS-II and β-GS-III were purified from betel leaves. This is the first report of purification and characterization of β-galactosidase from betel leaves.  

Keywords: Betel leaves; β-galactosidase; MW; characterisation; stability.

DOI: http://dx.doi.org/10.3329/jbs.v18i0.8785

JBS 2010; 18(0): 108-115