| In this paper, I isolated and purified a kind of alkaline phosphatase (AKP) from Apis cerana cerana Fabricius by n-butanol extraction, ammonium sulfate fractionation and sodium chloride dialyzation. The purified enzyme was gained by means of gel filtration on Sephadex G-150 column. The purification attained to 16. 79 folds and the specific activity was 135. 85 U/mg.Some properties of the enzyme were undertaken. The optimum temperature for the enzyme to catalyze the hydrolysis of p-nitrophenyl phosphate(pNPP) is at 45℃. If the pH value of the assay system was equal to 8. 6, the enzyme would become the most active. The Michaelis-Menten constant(Km) of the hydrolysis pNPP catalyzed by the enzyme is 0. 97 mmol/L.The product HPO42- and the product-analog WO42- Mo042- could competitively inhibit the activity of the enzyme. The survey of effects of metal ions on the AKP activity shows that Na+ K+ and Li+ have not any effect on the enzyme activity, Mg2+ Ca2+ Ba2+ Ni2+ Mn2+ Co2+ and Zn2+ activate the enzyme, Cu2+ Hg2+ Cd2+ and Ag inhibit the enzyme. On the other hand, the AKP is also inhibited by formaldehyde carbinol ethanol and glycol. The AKP is inactivated by urea, and the inactivation can be divided into two kinds according to the concentration of the urea: lower than 3mol/L and higher than 3mol/L.The alkaline phosphatase was selectively modified by DTNB NBS PMSF BrAc acetyl acetone and ME, and changes in the activities ofthe enzyme have been detected. The reaction of alkaline phosphatase with NBS BrAc and ME resulted in a strong inhibition of the enzyme activities which decreased with the increase of modifier concentration. The DTNB PMSF were found without any inhibition effect. The acetyl acetone was found to have little inhibition effect on AKP activity. All of these suggested that Trp and His residues should considered as indispensable functional groups of AKP , and partial disulfide bonds as essential for the function of the enzyme. |
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