Oral administration is generally preferred over injections. However, most peptide-, protein-, and nucleic acid-based therapeutics are not delivered via this route due to their inability to overcome the gastrointestinal barriers presented by enzymatic digestion, the mucus layer, and the epithelium. While significant formulation efforts target digestion and epithelial permeability, the intestinal mucus barrier which is primarily composed of mucins, often remains neglected. We previously reported the use of bacterial mucin-specific proteases (mucinases), such as the zinc-dependent secreted protease of C1 esterase inhibitor (StcE), to transiently degrade mucus and enhance macromolecule diffusion. Although co-administration with a permeation enhancer did improve oral bioavailability of a model peptide drug in dogs, the inter-individual variability was high. Studies investigating the rheological effect of StcE using porcine small-intestinal mucus recapitulated this marked inter-individual variability in StcE's mucolytic efficacy. In this study we investigated the origin of this variability. We assessed enzyme integrity and activity following mucus exposure. Mass spectrometry, polyacrylamide gel electrophoresis, and in vitro activity assays confirmed that StcE remained structurally intact and functional. Thereby excluding rapid degradation or stable inhibition as main causes of inactivation. Inductively coupled plasma mass spectrometry was used to quantify total, and non-protein bound zinc in mucus. Although total zinc content was around 100 nmol/g in most samples, the non-protein-bound fraction was much lower, consistently below 10 nmol/g. Suggesting that zinc limitation could be a factor limiting StcE activity in some samples. Indeed, zinc supplementation improved mucolytic efficiency. These findings support the potential of StcE, in combination with zinc supplementation, to degrade the mucus barrier and motivate further investigation of StcE as a potential strategy to overcome the mucus barrier and improve the oral delivery of complex therapeutics.