Biotransformation of the Fluoroquinolone Antibiotic, Levofloxacin, by the Free and Immobilized Secretome of Coriolopsis gallica

Abstract

Antibiotics play a crucial role in human and animal medical healthcare, but widespread use and overuse of antibiotics poses alarming health and environmental issues. Fluoroquinolones constitute a class of antibiotics that has already become ubiquitous in the environment, and their increasing use and high persistence prompt growing concern. Here we investigated a fungal secretome prepared from the white-rot fungus Coriolopsis gallica, which is able to effectively degrade the environmentally persistent fluoroquinolone, levofloxacin. We tested various physical-chemical factors such as concentrations of 1-hydroxybenzotriazol (HBT), of enzyme, and of antibiotic, and pH and temperature of the reaction for biotransformation of the antibiotic. We compared the free with the immobilized Coriolopsis gallica secretome proteins, and analyzed the collective reaction products for residual activity against E. coli (growth inhibition test). We also performed HPLC analysis. The results show that treatment with the free secretome yielded a highest removal efficiency of 50 mg L^-1 levofloxacin in the presence of 2.5 mM HBT, whereas the immobilized secretome was only able to degrade 10 mg L^-1 levofloxacin with the same concentration of mediator, but presenting the advantage of being reusable.

Keywords: Coriolopsis gallica secretome; alginate immobilization; biotransformation; fluoroquinolone; fungal bioremediation; levofloxacin; mediator.

Figure 1

Laccase-like activity of C. gallica produced in M7 medium supplemented with 300 µM CuSO₄. The error bars represent the standard deviation from the mean of the measured parameter across three experimental replicates.

Figure 2

Effect of levofloxacin concentration on its biotransformation using 5 U mL⁻¹ of laccase-like activity containing free (A) and immobilized (B) Coriolopsis gallica secretome after 6 and 24 h of incubation at 30 °C and pH 5. ■ Antimicrobial activity at T0. ■ Residual antimicrobial activity after 6 h of incubation. ■ Residual antimicrobial activity after 24 h of incubation. Chemical controls (antibiotic in the culture medium) were performed for each antibiotic concentration and were selected as the reference (100% of residual antimicrobial activity for LEVO and 0% of residual antimicrobial activity for Cga secretome). The error bars represent the standard deviation from the mean of the measured parameter across three experimental replicates.

Figure 2

Effect of levofloxacin concentration on its biotransformation using 5 U mL⁻¹ of laccase-like activity containing free (A) and immobilized (B) Coriolopsis gallica secretome after 6 and 24 h of incubation at 30 °C and pH 5. ■ Antimicrobial activity at T0. ■ Residual antimicrobial activity after 6 h of incubation. ■ Residual antimicrobial activity after 24 h of incubation. Chemical controls (antibiotic in the culture medium) were performed for each antibiotic concentration and were selected as the reference (100% of residual antimicrobial activity for LEVO and 0% of residual antimicrobial activity for Cga secretome). The error bars represent the standard deviation from the mean of the measured parameter across three experimental replicates.

Figure 3

Effect of HBT concentration on the biotransformation of levofloxacin by (A) free and (B) immobilized secretome after 24 h of incubation at 30 °C and pH 5. ■ Antimicrobial activity at time = 0 h. ■ Residual antimicrobial activity after 6 h of incubation. ■ Residual antimicrobial activity after 24 h of incubation. Chemical controls (antibiotic and the culture medium) were performed for each HBT concentration and were selected as the reference LEVO and Cga secretome (100% of residual antimicrobial activity for LEVO and 0% of residual antimicrobial activity for Cga secretome). The error bars represent the standard deviation from the mean of the measured parameter across three experimental replicates.

Figure 4

Effect of (A) free and (B) immobilized secretome concentration on levofloxacin biotransformation after 24 h of incubation at 30 °C and pH 5. ■ Antimicrobial activity at time = 0 h. ■ Residual antimicrobial activity after 6 h of incubation. ■ Residual antimicrobial activity after 24 h of incubation. Chemical controls (antibiotic and the culture medium) were performed for each secretome concentration and were selected as the reference LEVO and Cga secretome (100% of residual antimicrobial activity for LEVO and 0% of residual antimicrobial activity for Cga secretome). The error bars represent the standard deviation from the mean of the measured parameter across three experimental replicates.

Figure 5

Effect of temperature on levofloxacin biotransformation by (A) free and (B) immobilized secretome after 24 h of incubation at pH = 5. ■ Antimicrobial activity at time = 0 h. ■ Residual antimicrobial activity after 6 h of incubation. ■ Residual antimicrobial activity after 24 h of incubation. Chemical controls (antibiotic and the culture medium) were performed for each temperature value and were selected as the reference LEVO and Cga secretome (100% of residual antimicrobial activity for LEVO and 0% of residual antimicrobial activity for Cga secretome). The error bars represent the standard deviation from the mean of the measured parameter across three experimental replicates.

Figure 6

Effect of pH on levofloxacin biotransformation of by (A) free and (B) immobilized secretome after 24 h of incubation at 30 °C. ■ Antimicrobial activity at time = 0 h. ■ Residual antimicrobial activity after 6 h of incubation. ■ Residual antimicrobial activity after 24 h of incubation. Chemical controls (antibiotic and the culture medium) were performed for each pH value and were selected as the reference LEVO and Cga secretome (100% of residual antimicrobial activity for LEVO and 0% of residual antimicrobial activity for Cga secretome). The error bars represent the standard deviation from the mean of the measured parameter across three experimental replicates.

Figure 6

Effect of pH on levofloxacin biotransformation of by (A) free and (B) immobilized secretome after 24 h of incubation at 30 °C. ■ Antimicrobial activity at time = 0 h. ■ Residual antimicrobial activity after 6 h of incubation. ■ Residual antimicrobial activity after 24 h of incubation. Chemical controls (antibiotic and the culture medium) were performed for each pH value and were selected as the reference LEVO and Cga secretome (100% of residual antimicrobial activity for LEVO and 0% of residual antimicrobial activity for Cga secretome). The error bars represent the standard deviation from the mean of the measured parameter across three experimental replicates.

Figure 7

UV chromatograms (325 nm) of levofloxacin degradation products at 30 °C with tartaric acid buffer (pH 5) after 24 h of incubation [in green, levofloxacin + tartaric acid as controls (n = 3); in blue, Coriolopsis gallica secretome + levofloxacin + BHT 2.5 mM + tartaric acid (pH 5) (n = 3); in red, laccase + levofloxacin + BHT 2.5 mM + tartaric acid buffer (pH 5) (n = 1)].