Purification and partial characterization of a neutral protease from a virulent strain of Bacillus cereus.
The factors involved in the pathogenesis of Bacillus cereus (B. cereus) in non-gastrointestinal diseases are poorly investigated. Some researchers suggest that B. cereus proteases may be involved in these illnesses. The aim of this work was to purify and characterize a protease isolated from a virulent strain of B. cereus to explain its assumptive damaging effect. The enzyme was purified in a four-step procedure involving ammonium sulfate fractionation, acetone precipitation, Bio-Gel filtration and column chromatography on DEAE-cellulose (DE-52 cellulose). The enzyme appeared homogenous using disc electrophoresis. The specific activity of the protease was 72 U/mg of protein. The enzyme was shown to have a relative molecular mass of 29 kDa. The protease was most active at pH 7.0 and 40 degrees C with haemoglobin as the substrate. The enzyme was made completely inactive by ethylenediaminetetraacetic acid (EDTA), beta-mercaptoethanol, dithiothreitol (DTT) and benzamidine (at a concentration of 1 mM) and by diisopropylfluorophosphate (DIPF), L-cysteine, L-histidine, 1,10-phenanthroline (at a concentration of 10 mM). Divalent cations, especially Ca2+ increased enzyme activity. The enzyme hydrolysed haemoglobin, albumin and casein as the substrates. With haemoglobin and albumin as the substrates Michaelis-Menten kinetics was observed. The obtained Km values were 86 +/- 40 microM (SD, n = 3) and 340 +/- 100 microM (SD, n = 3) for haemoglobin and albumin, respectively. The corresponding Vmax values were 1.26 +/- 0.1 (SD, n = 3) and 0.38 +/- 0.07 (SD, n = 3) mumol of tyrosine liberated per min, per ml, and per mg, while those for casein were not determined. It is concluded that this enzyme is a metal-chelator-sensitive, neutral protease damaging haemoglobin and albumin.
Department of Biochemistry, Medical University of Lódź, Poland.
J K
JK
Sierecka
D001409:Bacillus cereus / Q000201:enzymology* / Q000472:pathogenicity; D002412:Cations; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium; D008666:Metalloendopeptidases / Q000032:analysis*; D013379:Substrate Specificity; D013696:Temperature; D014774:Virulence; D015032:Zinc
D002412:Cations; D004791:Enzyme Inhibitors; D008666:Metalloendopeptidases; D008274:Magnesium; D015032:Zinc