Purification of Protease II from Escherichia coli by Affinity Chromatography and Separation of Two Enzyme Species from Cells Harvested at Late Log Phase

Abstract

N-Acetyl-D-arginine linked to an agarose matrix has been used to purify protease I1 from Escherichia coli by affinity chromatography. The specific adsorption of protease I1 to this absorbent was achieved in 220 mM potassium phosphate buffer pH 7.6, and the enzyme was eluted with L-arginine.Enzyme preparations from cells harvested at late log phase have been resolved into two molecular species which differ in specific activity, kinetic constants and carbohydrate content. Both species appeared homogeneous by electrophoresis in conventional buffers and also in the presence of sodium dodecyl sulfate. Only one enzyme species was obtained by the same procedure using bacteria harvested at the middle of exponential growth.We have previously demonstrated the presence of an enzyme with trypsin-like activity in crude extracts of Escherichia coli [l]. This enzyme, designated as protease 11, has been recently purified by a conventional method and some of its properties were investigated [2]. It hydrolyzes the synthetic substrates of trypsin and is inhibited by common inhibitors such as diisopropylphosphofluoridate, tosyl lysine chloromethyl ketone and amidines. Nevertheless it is unaffected by peptidic trypsin inhibitors. Protease I1 is also competitively inhibited by two enantiomeric forms of N-substituted derivatives of arginine and by free L-arginine. Taking advantage of the particularity that a number of neutral proteolytic enzymes are capable of binding but not hydrolyzing, significantly, the enantiomeric substrate analog [3,4], we have developed a method of purification of protease I1 by affinity chromatography on N-acetyl-D-arginine coupled to an agarose matrix. The procedure has enabled us to prepare the homogeneous enzyme in relatively simple steps with a final recovery greater than previously obtained by the conventional method : 30 -37 instead of 15 -25 %. Moreover two enzyme forms has been isolated from cell extracts from bacteria harvested at late log phase.

The prolyl oligopeptidase family

“…Oligopeptidase B has been isolated from several sources [80], including E. coli [68,81], T. cruzi [82] and T. brucei [71,83]. It has been demonstrated by osmotic shock that the E. coli enzyme is a cytosolic protein [68].…”

Determination of Immunoreactive Trypsin, Pancreatic Elastase and Chymotrypsin in Extracts of Human Feces and Ileostomy Drainage

“…Enzymatic techniques lack complete specificity and particle-bound enzymes may show altered kinetics versus substrates as compared to the free enzyme. Furthermore, bacterial enzymes might contribute to the activity measured [16][17][18]. Specific immuno logic techniques circumvent several of these problems.…”

The Study of Escherichia coli Proteases. Intracellular Serine Protease of E. coli - an Analogue of Bacillus Proteases

“…The scarcity of biochemical data on the purified E. coli enzymes is caused by difficulties in their isolation, the extremely low concentration within the cells an'd the absence of specific affinity adsorbents suitable for the simultaneous isolation of several proteases. ' Chymotrypsin-like' protease I (Pacaud & Uriel, 1971 ;Pacaud et al, 1976) and 'trypsin-like' protease I1 (Pacaud & Richaud, 1975;Pacaud, 1976) are the only endopeptidases of E. coli to have been purified and studied in some detail.…”