Purification and characterization of an acid phosphatase from cell membrane fraction of Lactococcus lactis ssp lactis 303

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 B-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-, Al3+, phenylmethylsulfonyl fluoride, p-chloromercuribenzoic acid and iodoacetic acid and was activated by dithiothreitol and B-mercaptoethanol. Pyrophosphate behaved as a competitive inhibitor for the hydrolysis of p-nitrophenylphosphate, with a K-i of 6.4 mM. Kinetic measurements at pH 5.2 gave a K-m 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. (C) 1999 Canadian Institute of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved.