Biochemical study of alkaline phosphatase in Escherichia coli

Abstract

Abstract The present research aimed to isolate and purify Alkaline phosphatase enzyme from crude protein extract (Lysate supernatant) of Escherichia coli, by using different biotechnologies. To proceed, the following steps were taken: Firstly, The verification of the existence of enzyme in bacteria, the bacteria were diagnosed by using the API 20 stripe that consists of (20) items. the enzyme was isolated to ensure its availability in bacteria within the logarithmic phase and this was done through proliferating them for 18 hours in a nutrient agar. It had been detected that the enzyme was intracellular because of the occurrence of enzyme activity in the lysate supernatant without occurring it in the cell free culture supernatant. Secondly, Enzyme purification, the enzyme had been purified through three stages: precipitation of protein by ammonium sulphate, dialysis and finally, the protein extract was passed through column chromatography by using Sephadex G-100 gel, the estimated enzyme activity after this step was 22.0 in comparison with its activity before the purification processes (crude protein extract). The approximate molecular weight of alkaline phosphatase was 81.000 Dalton estimated by using gel filtration technique.