Extraction, Purification and Characterization of β-Galactosidase from Tomato (Lycopersicom esculentum)

Abstract

β-galactosidase enzyme EC (3.2.1.23), lactase, can be described as an enzyme of glycoside hydrolase which performs the catalyzing of β-galactosidas hydrolysis to monosaccharides by breaking glycosidic bond. The objective of this study was to extraction and purification β-galactosidase from tomato (Lycopersicom esculentum.), ten Different types of extraction were investigated to selection of the best extraction of the enzyme, The Na. phosphate buffer (0.1M and pH6) had given a highest Specific activity of crude enzyme has been 212.27 U/mg. protein. The purification procedures were performed with the use of the precipitation of ammonium sulfate, Ion-exchange and gel filtration chromatographic techniques. ammonium sulfate (70) % saturation has been the best method for precipitation and partially purification of enzyme with a purification fold 1.83 and enzymatic yield 88.31%. This followed by the use of ion exchange chromatography by DEAE sephadex A50 column, the purification times of the enzymatic extract were 2.36, with an enzymatic yield 25.48%. After the final purification step of gel filtration chromatography using SephadexG-100 column, the enzyme has been purified 3.92 fold with 16.33% of enzymatic yield. The optimum enzymatic activity was found at pH (6). The plant extracts tomato (Lycopersicom esculentum) were used to characterize the enzyme in the term of pH, temperature,. The enzyme activity measured by its ability to hydrolyze the substrate 2-nitrophenyl β-D-galactopyranoside (ONPG). The enzyme activity was reached maximum at 45°C and at pH 5.5. The enzyme’s molecular weight has been estimated to 74 KDa by the gel filtration chromatography method, and 73 k D on SDS-PAGE.