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

Abstract

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.