Purification and characterization of an arylamine N-acetyltransferase from Lactobacillus acidophilus.

N-acetyltransferase from Lactobacillus acidophilus was purified by ultrafiltration, DEAE-Sephacel, gel filtration chromatography on Sephadex G-100, and DEAE-5pw on high performance liquid chromatography, as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% (w/v) slab gel. The purified enzyme was thermostable at 37 degrees C for 1 h with a half-life of 32 min at 37 degrees C, and displayed optimum activity at 37 degrees C and pH 7.0. The K(m) and Vmax values for 2-aminofluorene were 0.842 mM and 2.406 nmol/min/mg protein, respectively. Among a series of divalent cations and salts, Zn2+, Ca2+, Fe2+, Mg2+, and Cu2+ were demonstrated to be the most potent inhibitors. The enzyme had a molecular mass of 44.9 kD. The three chemical modification agents, iodoacetamide, phenylglyoxal, and diethylpyrocarbonate, all exhibited dose-, time-, and temperature-dependent inhibition effects. Preincubation of purified N-acetyltransferase with acetyl coenzyme A (AcCoA) provided significant protection against the inhibition of iodoacetamide and diethylpyrocarbonate, but only partial protection against the inhibition of phenylglyoxal. These results indicate that cysteine, histidine, and arginine residues are essential for this bacterial activity, and the first two are likely to reside on the AcCoA binding site, but the arginine residue may be located close to the AcCoA binding site. This report is the first demonstration of acetyl CoA:arylamine N-acetyltransferase in L. acidophilus.