Production of a Recombinant Fibrinolytic Protease from an Isolate of Serratia marcescens from the Amazon Basin

Abstract

Intravenous fibrinolytic agents are essential for the treatment of cardiovascular diseases, acting through plasminogen activation to dissolve thrombi. However, current therapies are often limited by low fibrin specificity, high production costs, and side effects such as bleeding and allergic reactions. In this study, we describe a novel fibrinolytic protease, rSM519, derived from Serratia marcescens CBAM 519 and recombinantly expressed in Escherichia coli. The enzyme was purified via affinity chromatography and exhibited a molecular mass of 56 kDa. Biochemical assays revealed that rSM519 is a metalloprotease with optimal activity at pH 9.0 and 37 °C, significantly enhanced by Mn²⁺ ions. Unlike conventional agents such as tissue plasminogen activator or streptokinase, rSM519 acts directly on fibrin without requiring plasminogen activation. It efficiently degraded the α and β chains of fibrinogen, mimicking plasmin-like behavior, and showed no hemolytic activity. These features position rSM519 as a promising thrombolytic candidate with potential advantages over existing therapies, including lower production costs, reduced side effects, and direct fibrin-targeting activity.

1

Secreted proteins of Serratia marcescens CBAM 519 after 24 h of growth in medium containing gelatin (protease induction). After TCA precipitation and resuspension in IEF solution, the total protein content was measured using a Nanodrop (280 nm) and applied onto 15% SDS-PAGE (5, 10, 15, and 20 μg, as indicated above the lanes). Coomassie R 250 stain was used for visualization. The most abundant band (∼56 kDa) was excised and analyzed by mass spectrometry.

2

Comparison of SM519 with other Serratia proteases. The amino acid sequence of the fibrinolytic protease from S. marcescens CBAM 519 was compared with proteases P07268 from S. marcescens ATCC 21074/E-15 (accession number: 617), D4E064 from S. odorifera (accession number: 2647522), and A0A240AD01 from S. ficaria (accession number: 61651). Identical residues are shaded in dark gray (*), conserved residues are shaded in medium gray (:), and light gray indicates semiconservative substitutions (.). Yellow-highlighted characters denote amino acid residues of the enzyme’s active site. Zinc and calcium-binding domains (glycine-rich repeat) are indicated. Numbers written on both sides of the lines indicate the positions of amino acids. Putative residues of the pro-peptide and mature protease are also indicated.

3

Cloning, expression, and purification of rSM519. (A) Protein extract from induced (IND) or noninduced (NI) cultures harboring pET28a-sm519 resolved by 12% SDS-PAGE, analyzing total protein (Tot), insoluble (in), and soluble (sol) fractions after lysis and clarification. (C) Proteins resolved by 12% SDS-PAGE, displaying the sample applied to the Ni²⁺-IMAC column (solubilized inclusion-body fraction in 4 M urea), nonbound proteins (flow), and elution fractions with buffer B at 10%. (B) Fibrin zymography before and (D) after purification. (E) Fibrin plate assay of the purified sample obtained from fraction 1.

4

(A) The fibrinogenolytic activity of rSM519 was analyzed in 12% SDS-PAGE stained with Coomassie Brilliant Blue R-250 after specified time intervals of enzyme and fibrinogen coincubation. Line C: control (fibrinogen), 1 to 120: incubation time (min). Bands corresponding to the α, β, and γ chains are shown. (B) The fibrinogenolytic activity was calculated for each chain over time using densitometry and analysis with the ImageJ software. (C) Additionally, the percentage of each chain relative to the total was also determined throughout the kinetic assay.

5

Effect of temperature on the activity of rSM519. () Optimal temperature: the maximum detected activity exhibited by the enzyme was considered 100% relative activity. () Enzyme stability: the effect of temperature on enzyme stability was measured after 1 h of incubation at different temperatures and expressed as a percentage of residual activity. Assays were conducted under optimal enzyme pH conditions (pH 9.0). Values represent the mean ± SD (n = 3) from three independent experiments.

6

Effect of pH on the activity of rSM519. () Optimal pH: the maximum activity exhibited by the enzyme was considered 100% relative activity. () Enzyme stability: the effect of pH on enzyme stability was measured after 24 h of incubation and expressed as a percentage of residual activity. Buffers used: citrate buffer (pH 4–6), Tris–HCl buffer (pH 7–8), and sodium carbonate-bicarbonate buffer (pH 9). All buffer concentrations were 0.1 M. Assays were conducted at 25 °C. Values represent the mean ± SD (n = 3) from three independent experiments.

7

Effect of inhibitors on the fibrinolytic activity of rSM519. The effect of inhibitors on enzyme activity was measured after 1 h of incubation at 37 °C. The reaction was conducted at optimal pH 9.0. The inhibition level was expressed as a percentage of remaining activity compared to the control activity without an inhibitor. Values represent the mean ± SD of experiments (n = 3).

8

In vitro hemolysis assay of S. marcescens CBAM 519. The assay was performed on blood agar plates and incubated at 37 °C for 3 days. Native enzyme (NE) and recombinant enzyme (RE) were tested. The protein concentration in each well was 0.25 mg/mL (n = 3).