Membrane Bound Aminopeptidase B of a Potential Probiotic Pediococcus acidilactici NCDC 252: Purification, Physicochemical and Kinetic Characterization

Abstract

An arginine aminopeptidase (EC 3.4.11.6) called aminopeptidase B was purified to apparent homogeneity from membrane extract of a potential probiotic Pediococcus acidilactici NCDC 252 using successive chromatographies on sephadex G-100 and phenylsepharose CL-4B. Purified enzyme was a heterotrimer with molecular mass of\u2009~\u2009101.36\xa0kDa. Predicted molecular weight of the enzyme from its gene (93.9\xa0kDa) was close to the calculated molecular weight. The enzyme was optimally active at pH 7.5 and 40\xa0°C. It was strongly inhibited by metal chelating agent and thiol protease inhibitors suggesting that enzyme is a metalloprotease with involvement of thiol. The Km and Vmax of enzyme for Arg-4mβNA were calculated to be 26\xa0μM and 19.9\xa0nmol/ml/min respectively. Its 3-D structure was modeled and validated using in-silico approach. In-silico analysis revealed Ser, His, Phe, Tyr and Thr to be present at active site of aminopeptidase B. Docking studies revealed that Arg-4mβNA binds with high affinity to the enzyme followed by Lys-4mβNA. The enzyme also hydrolyzed dipeptide-4mβNA derivatives containing hydrophobic amino acids and diaminocarboxylic acids (Arg, Lys and Asp) at the N-termini but not tripeptides, endopeptidase substrates and -βNA derivatives or peptides with proline and phenyl at their N-termini or C-termini.