Peptidases from Maclura Pomifera for Preparation of Food Protein Hydrolysates: Purification by Single-Step Chromatography and Characterization of Pomiferin I

Abstract

Our objective was to isolate peptidases from the latex of Maclura pomifera fruits and usethem to hydrolyze food proteins, as well as to purify and characterize the main peptidase.Two partially purified proteolytic extracts were prepared by ethanol (EE) and acetone(AE) precipitation from an aqueous suspension of exuded fruit latex. EE was used tohydrolyze food proteins with a ratio of 0.19 caseinolytic units (Ucas) per mg of substrate.Different values of hydrolysis degree were observed for hydrolysates of egg white, soyprotein isolate, and casein at 180 min (9.3%, 31.1%, and 29.1%, respectively). AE wasemployed to purify a peptidase which exhibited an isoelectric point (pI) of 8.70 andwhose abundance in AE was 28.3%. This enzyme was purified to homogeneity using asingle-step procedure by cation-exchange chromatography, achieving an 8.1-fold purification and a yield of 16.7%. The peptidase was named pomiferin I and showed amolecular mass of 63,177.77 Da. Kinetic constants (KM 0.84 mM, Vmax 27.50 uM s−1,kcat 72.37 s−1, and kcat/KM 86.15 mM−1 s−1) were determined employing N-α-carbobenzoxy-L-alanyl-p-nitrophenyl ester as substrate. Analysis by PMF showed onlypartial homology of pomiferin I with a serine peptidase from a species of the same family.