In this study, sucrase A (SacA) from Bacillus subtilis was successfully displayed on the outer membrane of Escherichia coli via fusion with the AIDA-I autotransporter from E. coli. The pAIDA-sacA plasmid was constructed by fusing sacA with the ctxB signal sequence and the β-barrel domain of aida gene, enabling surface expression under both aerobic and anaerobic conditions. Functional expression of AIDA–SacA was confirmed by the appearance of reducing sugars in enzymatic assays of sucrose hydrolysis and by acid production on phenol red agar. Structural prediction suggested correct localisation of the catalytic domain on the extracellular surface. Enzymatic characterisation revealed that AIDA-SacA exhibits optimal activity at 40 °C and pH 7. The calculated Km for sucrose was 1.18 mM, while the corresponding Vmax was 2.32 U mL⁻¹. Thermal stability assays showed that the enzyme retained over 80% of its activity after 60 min at 45 °C, indicating notable resistance to moderate temperatures. Metal ion assays indicated that K⁺ enhanced enzymatic activity, while Zn²⁺, Cu²⁺, and Mg²⁺ were inhibitory. SDS-PAGE analysis confirmed the expression of the recombinant fusion protein, with a distinct band at approximately 114 kDa corresponding to the expected size. These results demonstrate the feasibility of employing the AIDA-I system for the surface display of SacA in E. coli, providing a functional platform for future applications in whole-cell biocatalysis.

This study aimed to immobilise sucrase A (SacA) from Bacillus subtilis in E. coli using the AIDA-I system for the whole-cell biocatalysis to transform sucrose to lactate. The pAIDA-sacA plasmid, containing the sacA gene, was fused to the signal peptide of the toxin subunit B from Vibrio cholerae (ctxB) and the autotransporter of the aida gene, encoding a connector peptide and the β-barrel domain of the AIDA-I system. This plasmid was employed to transform E. coli strains W3110, WDHFAK, and WDHFAP, which are unable to naturally use sucrose. These strains were anaerobically cultured in batch fermentations using 10 g L⁻¹ sucrose as the sole carbon source. All strains successfully hydrolysed and fermented sucrose, exhibiting a homolactic profile. Among them, WDHFAP/pAIDA-sacA achieved the highest lactic acid titre of 9.84 ± 0.15 g L⁻¹ and a yield of 0.89 ± 0.02 g g⁻¹. Deletion of the mgsA gene in WDHFAP/pAIDA-sacA confirmed that lactic acid production occurred via the methylglyoxal bypass pathway, as lactic acid titres were reduced by over 80%, while ethanol production increased to 4.27 ± 0.26 g L⁻¹. Adaptive laboratory evolution of WDHFAK/pAIDA-sacA was conducted to improve its capacity and fermentation efficiency under elevated sucrose concentrations. The resultant strain, designated as WDHFAK^EV/pAIDA-sacA, consumed up to 65 g L⁻¹ sucrose, achieving 64.61 ± 1.65 g L⁻¹ lactic acid with a yield of 0.99 ± 0.03 g g⁻¹. These findings underscore AIDA-I-mediated SacA immobilisation as a robust strategy for whole-cell biocatalysis, enabling E. coli strains to efficiently ferment sucrose.