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| Purification and Some Characterization of Phosphatase f rom Grass Carp (Ctenopharyngodon idellus) |
| MA Jin-hu,XUE Fu-lai,WANG Yi-fan,LIN Ya-qiu,SHA Ya-juan,WU Hong,HE Qing-hua |
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Abstract The properties of alkaline phosphatase (AKP) were studied in hepatopancreas of grass carp Ctenopharyngodon idellus .In our study ,the isolation and purification of AKP were conducted by some processes of Tris-HCl(pH 7 .5)extraction ,n-butanol degreasing ,ammonium sulfate fractional precipitati-on and gel filtration (Sephadex G -150) ,and the kinetic properties of AKP and the effects of metal ions on the enzyme activity were studied .The molecular weight of the protein was about 54 ku by SDS-PAGE , with 7 .3 fold purification and specific activity of 949 .8U/mg .Kinetic assay showed that the optimum pH of the enzyme in catalyzed nitrobenzene disodium phosphate (PNPP) hydrolysis reaction was 10 .33 ,the optimum temperature was 38 ℃ ,the Michaelis constant (Km) value was 9 .03 mmol /L and the maximum reaction rate (Vmax) was 1 .5μmol /(L · min) .The findings showed that Li+ ,Na+ ,K+ had no effects on the activity of AKP ,and Ba2+ ,Ga2+ ,Mg2+and Mn2+ led to activation of the enzyme activity ,but EDTA , Cu2+ and Zn2+ inhibited the enzyme activity .Together ,we successfully isolated and purified the AKP from hepatopancreas of the grass carp and stuied the properties of AKP .Our findings might provide some refer-ence for the understanding of the function of AKP in the lipid metabolism of fish .
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Received: 25 August 2014
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