Engineering the L-Arabinose Isomerase from Lactobacillus sakei for Whole-Cell Conversion Production of D-Tagatose Under High Substrate Concentration Conditions

D-tagatose, a low-calorie sugar substitute, widely used in the food and pharmaceutical industries, can be produced from the substrate D-galactose using L-arabinose isomerase (L-AI). L-AI genes derived from Lactobacillus fermentum (LFAI), Lactobacillus brevis (LBAI), Lactobacillus plantarum (LPAI), and Lactobacillus sakei (LSAI) for the bioconversion of D-galactose into D-tagatose using whole Escherichia coli (E. coli) cells were investigated. The temperature, time, pH, whole-cell concentration, and metal ion concentration were optimized to enhance catalytic production. The results revealed that at the low substrate concentration of 10 g/L, LFAI, LBAI, LPAI, and LSAI exhibited considerable catalytic activity, with conversion rates of 55.31%, 68.17%, 61.45%, and 58.22%, respectively. In contrast, at the high substrate concentration of 250 g/L, their conversion rates were 19.78%, 18.47%, 20.26%, and 36.24%, respectively, with LSAI demonstrating superior catalytic performance. In order to facilitate the industrial production of D-tagatose, site-directed mutagenesis was performed on LSAI. Among the engineered LSAI variants, the P99H, K142R, and L465R mutants all showed increased catalytic rates for D-tagatose. Specifically, at the high substrate concentration of 250 g/L, the P99H mutant achieved a conversion rate of 45.73%, representing a 32.7% improvement compared to the optimized LSAI. These results indicate that the designed site-directed mutagenesis strategy effectively enhanced whole-cell bioconversion performance of LSAI toward D-galactose.