Purification and Partial Characterization of -Galactosidase from Cajanus cajans (Pigeon Pea)
Journal Title: Biotechnology Journal International - Year 2016, Vol 14, Issue 1
Abstract
Aims: This study reported the purification and partial characterization of the enzyme β-galactosidase (EC 3:2: 1: 23) from the seeds of Cajanus cajans. Methodology: The dry seeds of Cajanus cajans were purchased at Shomolu market in Lagos Nigeria. One hundred and fifty gram of the fine powder of the seed was defatted twice in Soxhlet extractor using 300 mL of cold acetone and maintained at room temperature for 4 hours. There after the dark brown supernatant was decanted and filtered on a Buchner glass funnel. The dark brown residue was then treated with additional quantity of cold acetone until no evidence of fat was found on the fine particles. The acetone extract was allowed to evaporate overnight in the open at room temperature. The dry powder was suspended in 0.1 M phosphate buffer pH 7.4 stirred in the cold at 4ºC for 4 hours and centrifuged at 10,000 rpm for 30 minutes at 4ºC and ammonium sulphate was added to the supernatant to 40-60% saturation level. The pellets were collected by centrifugation and dissolved in 50 mL of 0.1 M phosphate buffer pH 7.4 and dialyzed against distilled water for 48 hours with stirring. The concentrated crude extract was loaded into a column (1.7x3.0 cm) of Sephadex G-75 eluted with 5 mL sodium phosphate buffer pH 7.4 and collected in 4 mL fractions. The fractions collected were assayed for β-galactosidase activity. Results: A single band was obtained which indicated the presence of single polypeptide chain; optimum pH was found to be 4.0 while the optimum temperature was 45°C in the active enzyme. Conclusion: Enzymatic electrophoresis and molecular evidence in this study indicated that the purified 4.8 kDa protein is a pigeon pea β-galactosidase.
Authors and Affiliations
Adedoyin Dayo, Ojokuku Sikiru Abiola, O. Okwuraiwe Azuka
Keywords
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- EP ID EP237958
- DOI 10.9734/BBJ/2016/21345
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