Purification and characterization of a hydrophobic amino acid -: specific endopeptidase from Halobacterium halobium S9 with potential application in debittering of protein hydrolysates

An extracellular endopeptidase has been isolated and purified by a single step protocol using Bacitracin-NHS (N-hydroxysuccinimide) Sepharose affinity system. Halobacterium halobium S9 endopeptidase has very high salt requirements in the range of 4 M NaCl. Molecular weight as determined by SDS-PAGE and gel filtration analysis was approximately 71 and 45 kDa respectively. N-terminal amino acid sequence analysis shows LVPNDAR as the probable residues. The enzyme has optimal activity at pH 8.7 and at a temperature of 40 degreesC. H. halobium endopeptidase exhibited significant stability under wide variations of pH and temperature. The effect of divalent cations on the enzyme is positive and Ca2+ is the most significant of all the divalent salts tested. Inhibitor studies show inhibition by Ser, His and Asp specific inhibitors. Substrate specificity studies using peptides reveal high specificity for peptides containing Pro in the penultimate position and preference for hydrophobic amino acids in the carboxy terminal side of the peptides. The enzyme exhibits azocasein activity at low salt concentrations as against activities at higher salt concentrations for peptides. The action of endopeptidase on the complex peptide mixture of a tryptic digest of beta-casein resulted in the degradation of hydrophobic peptides. Thus, these characteristics of the purified peptidase make it suitable for an array of industrial applications with special impetus in debittering of meso, cheese and protein hydrolysates in food processing industry. (C) 2002 Published by Elsevier Science Ltd.