Determination of stability and kinetic characterization of lysozyme (isolated from Bacillus subtilis BSN314)

This study investigated the stability and anti-biofilm activity of a lysozyme purified from Bacillus subtilis BSN314. The hypothesis that the enzyme's anti-biofilm effect is concentration- and time-dependent was tested. Lysozyme stability was assessed by measuring its relative enzymatic activity (REA%) under varying conditions. REA% increased with temperature from 20°C to 40°C, with optimal activity observed at 40°C and pH 6.0. The enzyme demonstrated notable thermal stability, retaining 32% of its activity after treatment at 70°C for 30 min. Anti-biofilm activity was evaluated quantitatively and qualitatively against Gram-positive ( Micrococcus luteus ATCC 4698 and Staphylococcus aureus ATCC 25923) and Gram-negative ( Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 13883) biofilm-producing bacteria. Biofilm dispersion was dependent on lysozyme concentration and exposure time, with optimal dispersion achieved using 250 µg/mL for 60 min. Under these conditions, M. luteus exhibited the highest dispersion rate (97%), followed by S. aureus (92%), E. coli (80%), and K. pneumoniae (80%). Scanning electron microscopy (SEM) analysis confirmed the disruptive effect of lysozyme on biofilm structure and bacterial growth. Kinetic characterization using M. luteus as a substrate yielded a Michaelis constant (Km) of 0.12 mg·mL⁻¹ and a maximum reaction velocity (Vmax) of 60.24 mg·mL⁻¹·min⁻¹, indicating high substrate affinity and catalytic efficiency. In conclusion, the lysozyme from B. subtilis BSN314 exhibited significant pH, coupled with potent, concentration- and time-dependent anti-biofilm activity against both Gram-positive and Gram-negative pathogens, highlighting its potential as a natural anti-biofilm agent. • Determination of the pH, temperature, and thermal stability profile of B. subtilis BSN314 lysozyme • Demonstration of concentration- and time-dependent anti-biofilm activity against Gram-positive and Gram-negative bacteria • Quantitative assessment of biofilm dispersion rates, with optimal effect at 250 µg/mL lysozyme for 60 minutes • SEM imaging visually confirming the biofilm-dispersing and bactericidal effects of lysozyme treatment • Kinetic characterization revealing high substrate affinity (low Km) and catalytic efficiency (high Vmax) via Michaelis-Menten analysis