Endoesterolytic activity in NEP proteolytic extract (Natrialba magadii extracellular protease)

Abstract

Natrialba magadii (formerly Natronobacterium) belongs to haloalkaliphilic group, dominate in hyper saline environments (low water activity). The endoesterolytic activity of Natrialba magadii extracellular protease (NEP) was determined by the Silverstein´s method (1974), modified according to optimal conditions of the enzyme. The activity was studied using N-alpha-carbobenzoxy-p-nitrophenyl esters of some amino acids as substrate. Assays were made at 40 ºC in 0.1 M Tris-HCl buffer (pH 8) containing Cys (20 mM), NaCl (1.5 M) and 0.15, 0.3, and 6.25 mM of each substrate in the reaction mixture. P-nitrophenol was followed at 405 nm in a Cintra 6 UV-Visible spectrophotometer. An arbitrary enzyme activity unit (Ucbz) was defined as the amount of protease that released one umol of p-nitrophenolate per min in the assay conditions. For determinate the umol of p-nitrophenolate produced during the reaction, was carried out a standard curve with p-nitrophenol (5-50 uM) in 0.1 M Tris - HCl buffer (pH 8) containing acetonitrile (5%). Natrialba magadii extracellular protease was tested against several N-alpha-carbobenzoxy-p-nitrophenyl esters of different L-amino acids. The preferred substrates of NEP were Gln derivative and in decreasing order those of Asn, Gly, Lys, Tpr. These results showed the high selectivity of NEP by polar amino acids.