Isolation of a putative virulence agent, cytotoxic serine-elastase,from a newly isolated Pseudomonas aeruginosa ZuhP13

A 48 kDa ZuhP13 elastase from P. aeruginosa isolated from a urine sample was successfully purified to 8.8-fold and 39%recovery by DEAE-Sepharose CL-6B and Sephadex G-100 chromatography. Its ideal reaction values were pH 7.5 and40C. It showed stability at pH 6–9 for 1 h and up to 60C for 30 min with midpoint temperature (Tm) at 61.3Cand isoelectric value (pI) at 5.6±0.2. Its Km and catalytic efficiency (Kcat/Km) for the substrate azocasein were 1.3 mg/mLand 4.629107 M-1s-1, respectively. On contrary to most P. aeruginosa proteases, Zn2?, EDTA, 2,20-bipyridine ando-phenanthroline showed slight inhibition upon its activity, while, the elastase inhibitors (elastatinal and elastase inhibitorII) and the serine protease inhibitors (TLCK, PMSF, SBTI, and aprotinin) markedly decreased the enzymatic activity. Takentogether, we suggest that ZuhP13 is a serine elastase-type. Interestingly, the tested enzyme showed both hemolytic andhemorrhagic activities in vivo. Furthermore, it induced nuclear lysis yielding hyperchromatism within leaky and malformedhepatocytes, suggesting ZuhP13 elastase as a high molecular weight potential pathological agent.

Fibrinolysis and anticoagulant potential of a metallo protease produced by Bacillus subtilis K42.

In this study, a potent fibrinolytic enzyme-producing bacterium was isolated from soybean flour and identified as Bacillus subtilis K42 and assayed in vitro for its thrombolytic potential. The molecular weight of the purified enzyme was 20.5 kDa and purification increased its specific activity 390-fold with a recovery of 14%. Maximal activity was attained at a temperature of 40°C (stable up to 65°C) and pH of 9.4 (range: 6.5–10.5). The enzyme retained up to 80% of its original activity after pre-incubation for a month at 4°C with organic solvents such as diethyl ether (DE), toluene (TO), acetonitrile (AN), butanol (BU), ethyl acetate (EA), ethanol (ET), acetone (AC), methanol (ME), isopropanol (IP), diisopropyl fluorophosphate (DFP), tosyl-lysyl-chloromethylketose (TLCK), tosyl-phenylalanyl chloromethylketose (TPCK), phenylmethylsulfonylfluoride (PMSF) and soybean trypsin inhibitor (SBTI). Aprotinin had little effect on this activity. The presence of ethylene diaminetetraacetic acid (EDTA), a metal-chelating agent and two metallo protease inhibitors, 2,2′-bipyridine and o-phenanthroline, repressed the enzymatic activity significantly. This, however, could be restored by adding Co2+ to the medium. The clotting time of human blood serum in the presence of this enzyme reached a relative PTT of 241.7% with a 3.4-fold increase, suggesting that this enzyme could be an effective antithrombotic agent.