Characterization of <i>Lactiplantibacillus plantarum</i> WCFS1 L‑arabinose isomerase and its application in D‑tagatose production

D‑Tagatose is a low‑calorie rare sugar of growing interest due to its favourable nutritional and physiological properties. Biocatalytic isomerization of D‑galactose using L‑arabinose isomerase (L‑AI) represents a sustainable production route; however, enzyme performance varies significantly among microbial sources. In this study, the L‑AI from the food‑grade strain Lactiplantibacillus plantarum WCFS1 was heterologously expressed as two variants carrying poly‑histidine tags at either the C‑ or N‑terminus. Both enzymes catalysed the isomerization of L‑arabinose to L‑ribulose and D‑galactose to D‑tagatose, but displayed distinct kinetic and functional behaviours depending on tag position. C‑His‑L‑AI showed superior catalytic performance towards D‑galactose, with higher Vmax and catalytic efficiency, while N‑His‑L‑AI exhibited enhanced substrate affinity (lower Km) and turnover (kcat) for the natural substrate L-arabinose. Optimal activity for both variants occurred at pH 7.0 and 45–50 °C, and Mn2+ served as the most effective cofactor. Process optimization using C‑His‑L‑AI demonstrated predictable equilibrium‑limited behaviour, enabling up to 24.2% D‑tagatose formation under mild conditions. Substrate specificity analysis indicated activity primarily restricted to L‑arabinose and D‑galactose. Overall, these findings position L. plantarum WCFS1 L‑AI as a competitive food‑grade biocatalyst for D‑tagatose synthesis and highlight the impact of His‑tag placement on biocatalytic performance.