Purification and characterization of an acid phosphatase from cell membrane fraction of Lactococcus lactis ssp lactis 303
1998
Akuzawa, R. | Fox, P.F.
An acid phosphatase was purified from the cell membrane fraction of Lactococcus lactis ssp. lactis 303 by anion exchange chromatography on DEAE-cellulose, hydrophobic interaction chromatography on Phenyl Sepharose and gel filtration on Sephacryl S-200 HR and Sepharose 4B-200. Gel filtration on Sepharose 4B-200 showed that activity was confined to a single peak eluted at the void volume of the column. The molecular mass of the enzyme was estimated to be 430 kDa by gel filtration equilibrated with 20 mM Tris-HCl buffer, pH 7.2, containing SDS and beta-mercaptoethanol. Activity was optimal at pH 5.2 and 50 degrees C. The enzyme was relatively heat-stable; 23% of activity remained after heating at 70 degrees C for 30 min. The enzyme was stable in 6 M urea after standing for 18 h at 4 degrees C, 83% of the original activity remained when assayed in the present of 3 M urea. The enzyme was inhibited by F-, Al(3)+, phenylmethylsulfonyl fluoride, p-chloromercuribenzoic acid and iodoacetic acid and was activated by dithiothreitol and beta-mercaptoethanol. Pyrophosphate behaved as a competitive inhibitor for the hydrolysis of p-nitrophenylphosphate, with a Ki of 6.4 mM. Kinetic measurements at pH 5.2 gave a Km for p-nitrophenylphosphate of 0.1 mM. The enzyme released inorganic phosphate from a mixture of casein phosphopeptides, mainly alpha s1-casein (fragment 59-79) and beta-casein (fragment 1-25), at a rate of 0.0071 mmol min-1 mg-1 but did not dephosphorylate sodium caseinate.
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