Going full circle: dynamic covalent enzyme immobilisation via visually trackable boronate esters

Enzyme immobilisation on solid supports enables biocatalyst recycling but generates significant waste due to single-use resins that are discarded when enzyme activity declines. Here we report a reversible immobilisation strategy based on boronate ester formation between alizarin-functionalised enzymes and boronic acid-modified supports. Alizarin-methyliminodiacetic acid (alizarin-IDA) serves dual roles as both a pH-responsive binding handle and a visual reporter, enabling real-time colourimetric tracking of enzyme loading (red solution → orange resin), immobilisation completeness, and pH-triggered release. A universal labelling protocol was established and successfully applied to four structurally diverse enzymes retaining 77-95% of native activity. All alizarin-labelled enzymes achieved >90% immobilisation yield on different supports, were extensively reusable, and could be removed by acidic treatment with full regeneration of the supports. The load-use-cleave sequence was repeated five times without loss of binding capacity, enabling more than 50 catalytic cycles per support across multiple enzyme lifecycles (5 regeneration cycles × 10+ reactions each) with identical performance.