Mycosis fungoides presenting as annular erythema.

UNLABELLED: This study aimed to purify and biochemically characterize serratiopeptidase from Serratia marcescens SP6 and evaluate its anti-biofilm efficacy against clinically relevant biofilm-forming pathogens. Serratiopeptidase derived from S. marcescens SP6 underwent a four-step purification process: precipitation, dialysis, ion-exchange, and gel filtration chromatography resulting in a 15.91-fold increase in purity, essential for reliably characterizing its biochemical and kinetic properties. The enzyme's molecular weight was determined to be approximately 47 kDa. It demonstrated broad substrate specificity, showing the highest affinity toward casein. Optimal enzymatic activity was recorded at pH 7 and 40 °C, with 89.2% and 82.4% of its activity retained under these conditions for 60 min. Kinetic analysis revealed a K m of 0.034 mM (842 µg/mL) and a V max of 738.2 U/mL, with a turnover number (k cat ) of 55.3 s⁻¹ and catalytic efficiency (k cat /K m) of 1.64 × 10⁶ M⁻¹s⁻¹, indicating high catalytic efficiency. The activity of serratiopeptidase was modulated by various metal ions and reagents, with Zn2+, Co2+, Ba2+, Mn2+, Tween-20, and β-mercaptoethanol acting as activators, while EDTA and PMSF inhibited activity, confirming its classification as a serine-metalloprotease. The enzyme exhibited stability at 4 °C, retaining 61.31% of its activity after 60 days of storage and exhibited resistance to degradation by trypsin and serum, with in vitro half-lives of 4 and 5 h, respectively. Notably, serratiopeptidase displayed anti-biofilm activity against Pseudomonas aeruginosa MTCC 2453 and Staphylococcus aureus MTCC 1430, achieving maximum biofilm inhibition of 41.65% and 21.87%, respectively, at 200 µg/mL with corresponding IC50 values (defined relative to the normalized response range) of 56.92 ± 8 µg/mL and 124.6 ± 8 µg/mL, respectively, corresponding to 50% of the observed response range rather than 50% absolute biofilm inhibition. These results highlight the enzyme's potential role in biofilm inhibition and suggest its possible use as an adjunct therapeutic agent alongside conventional antimicrobials to combat biofilm-associated infections.

SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at https://doi.org/10.1007/s13205-026-05063-9.