Purification and Characterization of Alkaline Phosphatase in Porcine Intestinal Mucosa

The pig intestinal mucosa was fully dissolved in Tris-HCl buffer(pH8.5),degreased with n-butanol,frozen and centrifuged,and lyophilized in vacuum. Phenyl high performance hydrophobic chromatography,DEAE fast flow anion chromatography,Sephacryl S-200 gel chromatography were used to purified and the porcine small intestinal mucosa ALP(alkaline phosphatase)was obtained. Secondary structure and physicochemical properties of the ALP were determined. The results showed that the ALP protein band was separated by sodium lauryl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),and the molecular weight was about 57.0 kDa. The purification factor of ALP was 68.2,and the specific activity factor was 19.1 U/mg. The secondary structure of the protein was determined by circular dichroism spectroscopy,and the results showed that the protein was-helix 3.6%,section-fold 41.8%,section-corner 21.5%,and irregular curl 33.1%. The enzyme catalyzed the substrate disodium nitrophenylphosphate(p-NPP)with an optimal temperature of 30 ℃ and an optimal pH value of 9.5.Metal ions Mg²⁺ and Ca²⁺ could activate ALP,and the inhibition ions were Zn²⁺ and EDTA.