Enzyme Immobilisation on Cellulose via Bifunctional Reactive Dyes: A Simple Route to Textile-Based Biocatalysts

The durable enzyme functionalisation of cellulosic fibres is often limited by enzyme deactivation under alkaline processing and insufficient wash resistance. Here, a cold pad-batch (CPB)-compatible route integrates reactive dyeing and lysozyme anchoring by using the commercial bifunctional dye C.I. Reactive Red 195 (MCT/VS) as an interfacial mediator to build a cellulose-dye-lysozyme ternary layer. The dye is first fixed on cotton, and residual electrophilic motifs are proposed to facilitate subsequent coupling with nucleophilic residues in lysozyme. A nine-run, four-factor/three-level orthogonal design was used to identify a practical processing window; under the selected condition, an ELISA-equivalent releasable lysozyme level of 53.8 U/L was achieved together with moderate colour strength (K/S = 6.5). The treated fabrics exhibited 96.5% inhibition against Escherichia coli and >99.9% against Staphylococcus aureus and retained antibacterial functionality after five ISO 105-C06 laundering cycles. Textile-relevant properties were preserved, including colour fastness (Grade 4-5), tensile strength retention (~86%), and capillary wicking close to pristine cotton. This dye-mediated strategy offers a practical route to wash-resistant bioactive interfaces on cellulose fibres and is extendable to regenerated cellulose and wood-pulp-derived cellulosics.