Biochemical characterization of a novel fibrinolytic enzyme secreted by Hornodermoporus martius LBM 224

ACOSTA, GABRIELA ALEJANDRA; FONSECA, MARIA ISABEL; FARIÑA, JULIA INES; ZAPATA, PEDRO DARIO

Online

Workshop; Tercer Encuentro & Primer Workshop de la Red Argentina de Tecnología Enzimática; 2021

Red Argentina de Tecnología Enzimática

Fibrinolytic enzymes have become very important for the potential treatment of cardiovasculardiseases. Our group has optimized the culture parameters for the production and purification of afibrinolytic enzyme secreted by Hornodermoporus martius LBM 224 with The aim of this work was tocharacterize a novel fibrinolytic enzyme produced by H.martius LBM 224.The fibrinolytic activity was measured using a fibrin degradation assay method. First, 200 µL of 0.72% w/v bovine fibrinogen was placed in a test tube with 50 µL of 50 mM Tris-HCl buffer (pH 7.4) andincubated at 37 °C for 5 min. Then, 50 µL of a 20 U/mL thrombin solution was added and incubatedat 37 °C for 10 min. Fifty µL of enzyme solution were added and the mixture was incubated at 37 °Cfor 1 h, mixing at 20 and 40 min. Finally, 350 µL of 0.2 M trichloroacetic acid (TCA) were added andmixed. The reaction mixture was centrifuged at 15,000 x g for 10 min at 4 °C. Subsequently, a 600-µL aliquot from supernatant was collected and the absorbance at 275 nm was measured. Accordingto this assay, 1 Unit of enzymatic activity, expressed as fibrin degradation unit (FU), is defined as0.01-per-minute increase in absorbance at 275 nm of the reaction solution.To determine the optimal temperature, enzyme activity was measured at different temperatures (20?60 °C) in 50 mM Tris-HCl buffer (pH 7.4). Thermal stability was investigated by measuring theresidual activity after incubating the purified enzyme at different temperatures (20?60 °C) for 1 h in50 mM Tris-HCl buffer (pH 7.4).Optimal pH of the purified enzyme was assayed by measuring fibrinolytic activity at 37 °C in differentpH buffers (pH 3.6?9.0). For testing pH stability, the enzyme was incubated in various pH buffers(pH 3.6?9.0) for 1 h at 37 °C, and the residual enzyme activity was analyzed. Buffers used were:sodium acetate (50 mM, pH 3.0?4.9), sodium phosphate (50 mM, pH 5.0?6.9), Tris-HCl (50 mM, pH7.0?9.0).The effect of various metal ions on the enzyme activity was studied using Zn2+, Ca2+, Cu2+, Co2+,Mg2+, Mn2+, Fe2+, Fe3+, Na+, and K+(at 1 mM final concentration). Inhibition effects were studied usingethylene diamine tetraacetic acid (EDTA) (1?5 mM). Effects of surfactants were studied with Tween80 (1% v/v), Triton X-100 (1% v/v), glycerol (30% v/v), and Sodium Dodecyl Sulfate (SDS) (0.5%w/v). Effect of different reagents on enzyme activity was also studied by incubating each of themalong with the enzyme: β-mercaptoethanol (0.5% v/v), Bovine Serum Albumin (BSA) (1% w/v),gelatin (1% w/v), peptone (1% w/v), acetone (10% v/v), and urea (8 M). Enzyme was preincubatedwith metal ions, surfactants, inhibitor, or reagents in 50 mM Tris-HCl buffer (pH 7.4) at 37 °C for 1 hand then, the residual activities were determined.The optimal temperature for fibrinolytic activity was 40 °C, with a relative fibrinolytic activity at 37 °Cof 88.70 ± 2.69 %. Concerning thermal stability, the fibrinolytic enzyme was highly stable up to 40 °Cretaining >80% of its activity after 1 h. The enzyme activity and thermo-stability gradually declinedabove 45 °C. The fibrinolytic enzyme showed maximal activity at either pH 7.4 or 8.0, withoutstatistical difference (P ≥ 0.05), and showed high stability ranging from pH 6.7?9.0, maintaining >80%of its activity or 1 h.Fibrinolytic activity of the purified enzyme was also influenced by various metal ions. The fibrinolyticactivity was significantly increased in the presence of 1 mM Zn2+, Ca2+, Co2+, Mg2+, Mn2+, Fe2+, andFe3+whileitwasslightlyreducedby Cu2+. Meanwhile, when confronted o EDTA (5 mM), a chelator of divalent cations, it retained 84% of its original activity, thus highlighting the importance of divalentmetals in enzyme stabilization.Regarding the influence of other reagents on enzyme activity, glycerol (85.15 ± 2.04 %) and TritonX-100 (89.57 ± 2.15) exerted a slight inhibition, while gelatin (61.64 ± 1.48 %), β-mercaptoethanol(61.32 ± 1.47 %), SDS (47.27 ± 1.13 %), urea (11.68 ± 0.28 %), and acetone (0 %) showed inhibitoryeffects on enzyme activity. Conversely, peptone (109.46 ± 2.63 %), BSA (119.80 ± 2.87 %), andTween-80 (176.15 ± 4.23 %) enhanced enzyme activity.One of the most important characteristics of the present fibrinolytic enzyme was its high activity andstability in a wide pH range. Furthermore, the purified enzyme showed high enzymatic activity at 37°C and pH 7.4 (physiological conditions), which reveals a great potential for thrombosis treatmentapplication. However, more research will be required in order to analyze the in vivo performance ofthis fibrinolytic enzyme.