Abstract
Alkaline phosphatase was purified from Fenneropenaeus merguiensis from Persian Gulf to homogeneity level by using (NH4)2SO4 precipitation, DEAE-cellulose and DEAE-Sephadex anion-exchange chromatography and gel filtration chromatography. The molecular weight of the enzyme was 70kDa, measured by SDS-PAGE. The alkaline phosphatase showed the strongest affinity with p-NPP as the substrate, and the highest catalytic efficiency for hydrolysis of the substrate at pH 11 and 50°C. Activation energy (Ea) for catalysis of alkaline phosphatase was 8.3kcalmol−1K−1, while temperature quotient (Q10) was 3.25. It exhibited Michaelis–Menten kinetics with kcat of 95s−1 and Km of 0.3μM, respectively. Thermodynamic parameters for soluble p-NPP hydrolysis were as follows: ΔH#=7.7kcalmol−1K−1, ΔG#=14.7kcalmol−1K−1, ΔS#=−23.5calmol−1K−1, ΔGE-S#=−0.7 kcal mol−1K−1 and ΔGE-T#=−3.4 kcal mol−1 K−1. Thermodynamic parameters (ΔH*, ΔG*, ΔS*) for irreversible inactivation of alkaline phosphatase at different temperatures (35–50°C) were also determined.
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