Engineering a robust laccase biocatalyst from bacterial lysates via selective hetero-functional immobilization

Abstract

Synthetic dyes are persistent pollutants in industrial effluents due to their aromatic structures and resistance to biodegradation, generating significant environmental concerns. Conventional treatment methods are often inefficient or costly, prompting interest in sustainable biocatalytic alternatives. Laccases, multicopper oxidases that use molecular oxygen as the terminal electron acceptor, are promising candidates for oxidative dye removal. In this study, a laccase-enriched crude extract from Escherichia coli was directly immobilized onto a heterofunctional glyoxyl–iminodiacetic acid (IDA) agarose support, combining selective enrichment and a subsequent stabilization treatment under conditions favoring covalent attachment within the same immobilization strategy. The resulting biocatalyst was optimized regarding protein loading and evaluated for pH and thermal tolerance, operational lifetime, and long-term storage stability. The optimized preparation showed remarkable robustness, remaining active after 221 days of storage at 4 ◦C and retaining 50% of its initial activity after an estimated 912 h of operation. Immobilization significantly enhanced stability compared with the free extract,particularly under acidic and neutral conditions. A 10-fold reactor-scale proof-of-concept was subsequently demonstrated specifically for RBBR, with higher early-stage productivity in the aerated airlift configuration than in the batch system.These results demonstrate that selective hetero-functional immobilization of induced and non-purified bacterial extracts represents an effective strategy for generating stable and reusable oxidative biocatalysts supporting further development toward industrial wastewater treatment applications.