Purification and Characterization of Alkaline Phosphatase from Haliotis diversicolor

An alkaline phosphatase was purified from Haliotis diversicolor by following procedures:Tris-HCl buffer (pH 7.5) extraction,n-butanol disposal,ammonium sulfate precipitation,ion-exchange chromatography on DE-32 cellulose,then by gel filtration through SephadexG-150 following again ion-exchange chromatography on DE-32.The final preparation was homogeneous on PAGE and SDS-PAGE.The specific activity of the enzyme was 1 226 U/mg.The optimum pH is 10.08,and the optimum temperature is 48℃ for the hydrolysis of pNPP.Mchaelis-Menten contant (K_m) is 0.8 mmol/L and the maximun velocity (V_m) is 20.1 mmol/L·min~(-1) at pH 10.0 and 37℃.The enzyme is stable in the range of pH from 7 to 11 and in temperature below 55℃. A survey of effects of metal ions on the enzyme showed that the positive monovalent metal ions (Li~+,Na~+ and K~+) have no effect on the enzyme;positive bivalent metal ions (Mg~(2+),Ca~(2+),Ba~(2+),Mn~(2+) and Co~(2+)) activate the enzyme,Cu~(2+)、Cd~(2+)、Zn~(2+)、Hg~(2+)、Ag~+、Bi~(2+) and Pb~(2+) inhibit the enzyme.The inhibition effect of Hg~(2+) is strongest.When the concentration of Hg~(2+)come to 0.2 mmol/L,the ALP is complete inactivation.The result indicates that different metal ions have different effect on the ALP.The study of ALP from Haliotis diversicolor may give some help to Haliotis diversicolor’s culture.