Purification and characterization of an intracellular aminopeptidase from a wil...

• Author(s): Macedo, Angela C. ; Tavares, Tânia G. ; Malcata, F. Xavier
• Date: 2003
• Persistent ID: http://hdl.handle.net/10400.14/6824
• Origin: Veritati - Repositório Institucional da Universidade Católica Portuguesa
• Subject(s): Enzyme; Isolation; Activity; Lactic acid bacteria; Dairy food
• Document Type: Article
• Language: English

Description

An intracellular hydrolase able to cleave Image -lysine-p-nitroanilide was purified from Lactobacillus plantarum strain ESB5004 via two steps of precipitation with ammonium sulfate (at 30 and 50% (w/v)), followed by hydrophobic interaction and ion-exchange chromatographies. The aminopeptidase was purified up to 11-fold, with a final yield of ca. 1%. Its native molecular weight is ca. 70 kDa, and it is apparently composed of two subunits, the molecular weight of which is 34 kDa. The enzyme was assayed using a wide variety of p-nitroanilide (pNA) derivatives as substrates: it hydrolyzed preferentially pNA adducts of hydrophobic and basic amino acid residues; no hydrolysis was in particular observed of Glu-pNA, Gly-pNA or Pro-pNA. The enzyme activity was removed by the metal-chelating agent EDTA, thus suggesting that it is a metallo-enzyme; however, the EDTA-inhibited enzyme was reactivated in the presence of Co2+. Optimal aminopeptidase activity was obtained at 28 °C (pH 7.0) and pH 6.5 (37 °C). The enzyme was inhibited by 10 mM CaCl2 or MgCl2.