Alkaline Phosphatase Purification from Bacillus megaterium. Hussin, H. 1a , Hsu, W.C. 2b Nebenfuhr, M. 3b and Papantoniou, C. 4b a Department of Biology, Faculty Science, University Technology Malaysia, Skudai, 81310, Johor, Malaysia. b Department of Biological Science, University of Essex, Wivenhoe Park, Colchester, C04 3SQ, United Kingdom. { 1a husza@bio.fs.utm.my} Abstract The aim of this study was to isolate and purify an alkaline phosphatase (ALPs) from Bacillus megaterium. The purified enzyme was established by gel filtration chromatography and high performance liquid chromatography (HPLC). Then, followed by non-denaturing gel electrophoresis to separate the ALPs from described purification technique. The purified enzyme had the following properties: ALPs concentration obtained from enzyme assay culture supernatant was 13.453µg and selected purification techniques of gel filtration and HPLC were both 19.74µg and 23.52µg respectively. The enzyme’s activity and specific activity from culture supernatant were both 0.493 U/ml and 36.3 U/mg respectively. Keyword : Bacillus megaterium, alkaline phosphatase, HPLC, non-denaturing gel electrophoresis, gel filtration 1.0 Introduction Alkaline phosphatase (ALPs) orthophosphoric- monoester phosphohydrolase classified as phosphomonoesterases (E.C. 3.1.3.1) are a group of membrane-bound glycoprotein that limited to the monoesterase activity catalysed through the formation of a phophoseryl intermediate [1]. ALPs however vary in sizes, metal requirements and substrate specificities [2]. Phosphatase is known to be essential in most microorganisms to release phosphate from organic compound when inorganic phosphate is limited [2]. Therefore, the mechanism has practically used by most molecular biologists to removed 5’ and 3’ monophosphatase (dephosphorylation) from nucleic acid through ALPs Escherichia coli [3]. In addition, alkaline phosphatase from calf intestine has proven to be useful in enzyme-immunoassays (EIA) [4]. Since then, studies have been made extensively to find new source with significant properties for commercial purpose. As recently, species of Bacillus megaterium has revealed to meet this requirement, as alkaline phosphatase found within them to be heat stable [5]. Therefore, the aim of this study was to isolate and purify an alkaline phosphatase (ALPs) from Bacillus megaterium. The purified enzyme is established by gel filtration chromatography and high performance liquid chromatography (HPLC), followed by non- denaturing gel electrophoresis. The purified enzyme should obtained higher concentration and enzyme activity accordingly to the following properties: ALPs concentration obtained from enzyme assay culture supernatant was 13.453µg and selected purification techniques of gel filtration and HPLC were both 19.74µg and 23.52µg respectively. The enzyme’s activity and specific activity from culture supernatant were both 0.493 U/ml and 36.3 U/mg respectively. Purified ALPs from gel filtration and HPLC were compared to one another for their activities. Those purified enzyme concentrations were calculated based on Bradford method and Standard Bovine Serum Albumin (BSA) curve. While, the ALPs activity and its specific activity were measured by p-nitrophenyl phosphate (p-NPP) substrate. 2.0 Materials and Methods 2.1 Sampling and bacterial strains Bacillus megaterium obtained from Department of Biological Science, University of Essex’s storage is cultured overnight and grown in 30°C. 2.2 Aseptic technique The organism was sub-cultured aseptically from the stock culture in nutrient broth. The cultures were then incubated for 60h at 200 rev min -1 . Identification was done by using gram and spore staining based on methods described [6]. 183