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

Abstract

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 degree C (stable up to 65 degree 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 degree 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.

Thrombolytic Potential of Novel Thiol-Dependent Fibrinolytic Protease from Bacillus cereus RSA1

“…EMB9 [74], and B. circulans M34 [75] produced organic solvent solution-tolerant proteases. Fibrinolytic protease by Bacillus subtilis K42 [76] and Serratia sp. [33] sustained 80-85% of its activity in the presence of organic solvents.…”

Protease production byStaphylococcus epidermidisand its effect onStaphylococcus aureusbiofilms

“…Aprotinin, a serine protease inhibitor (Yin et al, 2010;Park et al, 2012b) (5 mg mL À1 in sterile water, Sigma), was added to the azocasein assay mixture. Similarily, iodoacetamide (100 mM, Sigma) and EDTA (100 mM, Sigma) were added in the azocasein assay as cysteine and metalloprotease inhibitors (Jankiewicz & Bielawski, 2002;Keller et al, 2004;Yin et al, 2010;Hassanein et al, 2011), respectively. The assay consisted of 400 lL azocasein, 500 lL S. epidermidis SN and 300 lL inhibitor.…”

“…-: S. epidermidis was not isolated from that sample. EDTA are used as cysteine and metalloprotease inhibitors, respectively (Jankiewicz & Bielawski, 2002;Keller et al, 2004;Yin et al, 2010;Hassanein et al, 2011). Adding aprotinin and iodoacetamide to the azocasein assay significantly (P < 0.05) reduced the protease activity, indicating that both serine and cysteine proteases are produced by the S. epidermidis isolates.…”

Ability of Microorganisms from Different Ecological Niches to Hydrolyze the Insoluble Proteins

“…Комплексні ферментні препарати з такою специфічністю можуть бути використані в різ них галузях медицини та промисловості. Протеолітичні ферменти з фібринолітичною і коагулазною дією можуть деякою мірою вирішити проблему створення тромболітичних препаратів [9]. Протеолітичні ферменти з колагеназною активністю використовуються у виробництві шкіри, для дезінтеграції сполучної тканини, отримання суспензії клітин або клітинних новоутворень, оскільки при збереженні життєздатності клітини необхідне вибіркове руйнування позаклітинного матриксу без ушкодження поверхні живих клітин [18].…”