Purification and characterization of an acid phosphatase from Lactobacillus plantarum DPC2739

An acid phosphatase was partially purified from a cell-free extract of Lactobacillus plantarum DPC2739 by a combination of anion-exchange chromatography on DEAE-Sephacel, hydrophobic interaction chromatography on Phenyl Sepharose, gel permeation chromatography on Sephacryl S200 and high performance anion-exchange chromatography on MonoQ. The native enzyme (~110 kDa) was tetrameric with a subunit molecular mass of ~27 kDa. The enzyme was heat-stable, retaining ~60% of its activity after heating for 30 min at 70°C. It was optimally active in the pH range 3.5-5.0 and at 40°C. The enzyme was strongly inhibited by 0.5 mM sodium fluoride, and hexametaphosphate and by 5 mM orthophosphate, tripolyphosphate and pyrophosphate. It was insensitive to metal chelators (ethylenediaminetetraacetic acid and o-phenanthroline), ascorbic acid, sulphydryl blocking agents (e.g., N-ethylmaleimide), phenylmethylsulphonyl fluoride and divalent metal ions at 5 mM concentration. The enzyme appeared to be a non-specific phosphomonoesterase and hydrolysed a number of phosphate esters. The amino acid sequence of the first 20 residues was determined and showed some homology with mammalian, yeast and Escherichia coli acid phosphatases, phosphoglycerate mutases and phosphoglycerokinases with a common motif Arg-His-Gly. Copyright (C) 1999 Elsevier Science Ltd.