Enabling Stable Recycling of L-Arabinose Isomerase Through Whole-Cell Immobilization for Efficient and Cost-Effective D-Tagatose Production.

D-tagatose is a functional sweetener with glucose-regulating and prebiotic properties, but its bioproduction from D-galactose faces many limitations, particularly the high production costs. In particular, the current biosynthesis of D-tagatose suffers from thermal instability and the substrate selectivity issues of L-arabinose isomerase (L-AI) required to convert D-galactose into D-tagatose. In this study, recombinant BW25113/pQE-80L- expressing double mutant L-AI was immobilized to enhance its stability and reusability. The optimal conditions for whole-cell catalysis were 60 °C, pH 6.5, 5 mM Mn, and 20 h, with a yield of 55.2 g/L of D-tagatose. Immobilization with 3% sodium alginate and 2% CaCl retained 90% of the production efficiency displayed by free cells. Notably, the immobilized cells exhibited enhanced heat resistance (60-70 °C) and operational stability, retaining 76% activity after five cycles. The D-tagatose production was further increased to 129.43 g/L by increasing the substrate concentration to 250 g/L. Compared to free cells, immobilized cells retained 83.6% of the initial yield up to 10 batches. This study presents a cost-effective and sustainable method for the production of D-tagatose using optimized whole-cell catalysis through immobilization, which paves the way to solve industrial challenges such as thermal instability and low substrate efficiency.