Bacterial Enzyme Serratiopeptidase Shows Promise Against Deadly Biofilms

A bacterial enzyme long sold as a dietary supplement may be far more interesting than its wellness-market reputation suggests. In a study published in the journal 3 Biotech, researchers at JAIN (Deemed-to-be University) in Bangalore purified and characterized serratiopeptidase from the bacterium Serratia marcescens SP6 and demonstrated that the enzyme can weaken the protective biofilms formed by two of medicine’s most troublesome pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. The work, led by Sejal Kumar and Sourav Bhattacharya, provides one of the most detailed biochemical portraits of this enzyme to date and positions it as a candidate adjunct therapy for biofilm-associated infections that routinely defeat conventional antibiotics.

Biofilms are structured communities of bacteria encased in a self-produced matrix of proteins, extracellular DNA, and polysaccharides. Within these fortresses, pathogens become dramatically more tolerant of antibiotics and immune attack, which is why biofilms complicate the treatment of cystic fibrosis lungs, chronic wounds, medical implants, and catheter-associated infections. Pseudomonas aeruginosa and Staphylococcus aureus are flagship biofilm builders, and both appear on global lists of antibiotic-resistant priority pathogens. An enzyme that can chew through the proteinaceous scaffolding of a biofilm, or disrupt the physiology of the cells inside it, therefore represents a genuinely different therapeutic strategy: rather than killing bacteria outright, it dismantles the architecture that makes them so hard to eradicate.

Serratiopeptidase, sometimes marketed as serrapeptase, is an extracellular protease naturally secreted by Serratia marcescens. It has been used for decades, particularly in Japan and India, as an anti-inflammatory and anti-edema agent, and earlier studies have hinted that it can affect Pseudomonas physiology and biofilm integrity. What has been missing is a rigorous, quantitative characterization of a purified preparation, since crude enzyme mixtures make it impossible to attribute activity to a single protein. The new study addressed this gap with a four-step purification pipeline: precipitation, dialysis, ion-exchange chromatography, and gel filtration chromatography, which together yielded a 15.91-fold increase in purity, sufficient for reliable biochemical and kinetic analysis.

With the purified enzyme in hand, the team determined its molecular weight to be approximately 47 kilodaltons, consistent with the serralysin-like metalloproteases produced by Serratia species. The enzyme displayed broad substrate specificity but showed its highest affinity toward casein, a milk protein commonly used as a benchmark substrate in protease assays. Activity peaked at pH 7 and 40 degrees Celsius, close to physiological conditions, and the enzyme proved remarkably durable under these optima, retaining 89.2 percent and 82.4 percent of its activity, respectively, after 60 minutes of incubation. That combination of neutral pH preference and moderate temperature optimum is exactly what one would want in a therapeutic protein intended to function inside the human body.

The kinetic analysis revealed an enzyme of impressive catalytic power. The Michaelis constant, Km, was 0.034 millimolar, equivalent to 842 micrograms per milliliter, indicating tight substrate binding, while the maximum velocity, Vmax, reached 738.2 units per milliliter. The turnover number, kcat, came in at 55.3 per second, meaning each enzyme molecule processes more than 55 substrate molecules every second. Dividing kcat by Km yields a catalytic efficiency of 1.64 × 10⁶ per molar per second, a figure that places this serratiopeptidase firmly in the category of highly efficient catalysts. For a protein being considered as a drug candidate, such numbers matter: an efficient enzyme can deliver biological effects at lower doses, reducing cost and potential side effects.

Equally informative was the enzyme’s response to chemical modulators. Metal ions including zinc, cobalt, barium, and manganese enhanced activity, as did the non-ionic detergent Tween-20 and the reducing agent beta-mercaptoethanol. Conversely, EDTA, a chelator that strips away metal cofactors, and PMSF, a classic serine protease inhibitor, both shut the enzyme down. This dual sensitivity is diagnostic: it confirms that the SP6 enzyme is a serine-metalloprotease, a hybrid classification consistent with the serralysin family, in which a catalytic serine operates within a metal-dependent active site. Knowing this helps predict how the enzyme will behave in complex biological environments and informs strategies for stabilizing it in formulation.

Perhaps the most clinically relevant findings concern stability and resistance to degradation. The enzyme retained 61.31 percent of its activity after 60 days of storage at 4 degrees Celsius, a refrigerated shelf life that simplifies handling and distribution. More strikingly, it resisted digestion by trypsin and by human serum, with in vitro half-lives of 4 and 5 hours, respectively. Therapeutic proteins typically face rapid destruction by circulating proteases and serum components, so a bacterial enzyme that survives hours in serum-like conditions is unusual and valuable. The authors note that extending the half-lives of therapeutic proteins is a major focus of drug development, and intrinsic resistance to proteolysis gives this enzyme a head start.

The anti-biofilm experiments delivered the study’s headline result. Against Pseudomonas aeruginosa MTCC 2453, serratiopeptidase achieved a maximum biofilm inhibition of 41.65 percent at a concentration of 200 micrograms per milliliter, with an IC50 of 56.92 plus or minus 8 micrograms per milliliter. Against Staphylococcus aureus MTCC 1430, the maximum inhibition was 21.87 percent at the same dose, with an IC50 of 124.6 plus or minus 8 micrograms per milliliter. The authors are careful to define these IC50 values relative to the normalized response range, meaning they correspond to 50 percent of the observed inhibitory effect rather than absolute halving of biofilm mass, a methodological honesty that strengthens the credibility of the data. The stronger effect against Pseudomonas aligns with earlier reports that serratiopeptidase alters the physiology of Pseudomonas isolates from cystic fibrosis patients and disrupts Pseudomonas biofilms and functional amyloids.

What makes these numbers exciting is not that the enzyme obliterates biofilms on its own, but that partial disruption is often enough to restore the vulnerability of embedded bacteria. Anti-biofilm agents are increasingly viewed as adjuncts: by loosening or thinning the matrix, they allow antibiotics and immune cells to reach cells that would otherwise remain sheltered. A 41.65 percent reduction in Pseudomonas biofilm formation, achieved by a single enzyme at a modest concentration, could meaningfully amplify the efficacy of co-administered antimicrobials. The enzyme’s activity at neutral pH and 40 degrees Celsius, its serum stability, and its resistance to trypsin all support the plausibility of such combination approaches.

Significant hurdles remain before serratiopeptidase reaches the clinic as an anti-biofilm drug. The current work used a partially purified preparation, and further purification, scale-up of production, formulation studies, toxicity testing, and ultimately animal and clinical trials will be required. The enzyme’s origin from Serratia marcescens, an opportunistic pathogen itself, raises production and safety questions that recombinant expression systems, which have already been used to produce serratiopeptidase in cell-free platforms, could help resolve. Nevertheless, by systematically documenting the enzyme’s molecular weight, kinetics, inhibitor profile, storage stability, serum resistance, and anti-biofilm potency, Kumar and Bhattacharya have transformed a supplement-shelf enzyme into a well-characterized candidate for rational development. As antibiotic resistance continues to erode the effectiveness of conventional drugs, enzymes that dismantle bacterial fortresses rather than merely poisoning the bacteria inside them may prove to be one of the most important weapons in the next generation of anti-infective therapy.

Subject of Research: Biochemical characterization and anti-biofilm activity of serratiopeptidase from Serratia marcescens SP6

Article Title: Elucidation of the biochemical properties of a partially purified Serratia marcescens SP6 serratiopeptidase with anti-biofilm efficacy

Article References: Kumar, S., & Bhattacharya, S. (2026). Elucidation of the biochemical properties of a partially purified Serratia marcescens SP6 serratiopeptidase with anti-biofilm efficacy. 3 Biotech, 16(10), Article 433. https://doi.org/10.1007/s13205-026-05063-9

Image Credits: AI Generated

DOI: 10.1007/s13205-026-05063-9

Keywords: serratiopeptidase, Serratia marcescens, biofilm, Pseudomonas aeruginosa, Staphylococcus aureus, protease, enzyme kinetics, antibiotic resistance, metalloprotease, anti-biofilm therapy, protein purification, therapeutic enzymes