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

ACOSTA GA; FONSECA MI; FARIÑA JI; ZAPATA, PD.,

Congreso; Red Argentina de Tecnologia Enzimática; 2021

INTRODUCTION:Fibrinolytic enzymes have become very important for the potential treatment of cardiovasculardiseases. Our group has optimized the culture parameters for the production and purificationof a fibrinolytic enzyme secreted by Hornodermoporus martius LBM 224. The aim of this workwas to characterize a novel fibrinolytic enzyme produced by H. martius LBM 224.MATERIALS AND METHODS: The fibrinolytic activity was measured using a fibrindegradation assay method according to Wang et al. (2011).To determine the optimal temperature, enzyme activity was measured at differenttemperatures (20?60 °C) in 50 mM Tris-HCl buffer (pH 7.4). Thermal stability was investigatedby measuring the residual activity after incubating the purified enzyme at differenttemperatures (20?60 °C) for 1 h in 50 mM Tris-HCl buffer (pH 7.4).Optimal pH of the purified enzyme was assayed by measuring fibrinolytic activity at 37 °C indifferent pH buffers (pH 3.6?9.0). For testing pH stability, the enzyme was incubated in variouspH 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, pH 7.0?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 werestudied using ethylene diamine tetraacetic acid (EDTA) (1?5 mM). Effects of surfactants werestudied with Tween-80 (1% v/v), Triton X-100 (1% v/v), glycerol (30% v/v), and SodiumDodecyl Sulfate (SDS) (0.5% w/v). Effect of different reagents on enzyme activity was alsostudied by incubating each of them along 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 preincubated with metal ions, surfactants, inhibitor, orreagents in 50 mM Tris-HCl buffer (pH 7.4) at 37 °C for 1 h and then, the residual activitieswere determined.RESULTS:The optimal temperature for fibrinolytic activity was 40 °C(Fig. 1a). Concerning thermalstability, the fibrinolytic enzyme was highly stable up to 40 °C retaining >80% of its activityafter 1 h (Fig. 1a). The enzyme activity and thermo-stability gradually declined above 45 °C(Fig. 1b). 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 1HCONCLISIONS: One of the mostimportant characteristics of thepresent fibrinolytic enzyme was itshigh activity and stability in a wide pHrange. Furthermore, the purifiedenzyme showed high enzymaticactivity at 37 °C and pH 7.4(physiological conditions), whichreveals a great potential forthrombosis treatment application.However, more research will berequired in order to analyze the invivo performance of this fibrinolyticenzyme.