Thermostable and recyclable Candida antarctica lipase B immobilized on Bacillus subtilis using sortase technology.

UNLABELLED: Bacterial surface display is a transformative strategy for enzyme immobilization, enhancing enzyme stability and reusability for industrial applications. This study presents a novel whole-cell biocatalyst by immobilizing Candida antarctica lipase B (CALB) on Bacillus subtilis cells via a sortase-mediated system. A genetically engineered CALB variant, incorporating a C-terminal cell wall-binding motif, was co-expressed with the sortase enzyme in B. subtilis WASD (ΔwprA ΔsigD) cells, facilitating stable and site-specific anchoring of CALB directly on the bacterial surface without the need for purification or chemical modification. The immobilized CALB exhibited superior performance compared to its free counterpart, displaying optimal activity at 37°C and pH 8.0, with significantly enhanced thermostability. It retained ~50% activity after nine reuse cycles and maintained >50% of its initial activity after 8 weeks of storage. Additionally, surface-displayed CALB efficiently hydrolyzed fish oil, selectively releasing omega-3 polyunsaturated fatty acids (EPA and DHA), demonstrating its potential for sustainable bioprocessing in the food, pharmaceutical, and nutraceutical industries. This work establishes a scalable and environmentally friendly approach for enzyme immobilization. By leveraging B. subtilis as a host, sortase-mediated anchoring provides a robust platform for developing thermostable, reusable, and industrially viable whole-cell biocatalysts.

IMPORTANCE: This work presents a groundbreaking advancement in biocatalysis, addressing critical challenges in enzyme stability, reusability, and sustainability for industrial applications. This work directly addresses the industrial bottlenecks of enzyme instability, high turnover costs, and environmental impact. The sortase-mediated system offers a universal platform for displaying diverse enzymes, enabling tailored biocatalysts for sectors ranging from bioremediation to synthetic biology.