Engineering a reusable carrier-free dual-enzyme Combi-CLEA for pretreatment of high-strength tannery effluent

The development of carrier-free immobilized biocatalysts such as cross-linked enzyme aggregates (CLEAs) offers a robust and economically attractive strategy for enhancing enzyme performance under industrial conditions. Here, an integrated optimization strategy was established to co-immobilize PersiLipase1 and PersiProtease1 into a dual-enzyme Combi-CLEA for pretreatment of high-strength tannery effluent. Preparation parameters were optimized for each enzyme and then harmonized into a single protocol: 30 min PEI treatment, ammonium sulfate precipitation (55% w/v), and glutaraldehyde cross-linking (18% w/v). Kinetic analysis showed that immobilization increased catalytic efficiency (nearly twofold for PersiProtease1 and markedly for PersiLipase1), indicating improved substrate affinity and turnover. The Combi-CLEA system exhibited high robustness, retaining substantial activity at 80°C, across pH 5–9, and during storage relative to free enzymes. FTIR and FESEM confirmed covalent cross-linking and aggregate compaction with reduced α-helix content, increased β-sheet enrichment, and enhanced structural rigidity. In application-relevant assays using a synthetic tannery wastewater matrix, PersiProtease1–Combi-CLEA achieved rapid collagen hydrolysis (∼87% DH within 15 min; ∼10 × higher than the free enzyme), while PersiLipase1–Combi-CLEA hydrolyzed ∼30% of sheep fat whereas the free form showed negligible activity. Reusability tests indicated strong operational stability, with PersiProtease1–Combi-CLEA retaining ∼64% activity after seven cycles; PersiLipase1–Combi-CLEA showed a sharper decline yet consistently outperformed the free enzyme. Overall, this scalable, cost-effective framework yields a high-performance carrier-free dual-enzyme biocatalyst supporting integrated protein/lipid hydrolysis as an engineering pretreatment step for tannery wastewaters.