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D-tagatose, a low-calorie rare sugar, has significant potential in food, medicine, cosmetics, and other industries owing to its high application value and market potential. In this study, Escherichia coli BL21 was used as the starting strain to express the β-galactosidase (β-Gal) gene-BgaB-derived from Bacillus stearothermophilus and the L-arabinose isomerase (L-AI) gene-araA-derived from Thermus sp., yielding the genetically engineered strains E. coli BL21-pET28a-BgaB and E. coli BL21-pET28a-araA. These strains synthesized D-tagatose using β-Gal and L-AI with a conversion rate of 23.73%. Based on this, we constructed a multienzyme cascade pathway comprising β-Gal, L-AI, glucose isomerase (GI), fructose kinase (FK), D-tagatose-bisphosphate aldolase (GatZ), polyphosphate kinase (PPK), and phosphatase (PGP), further enhancing D-tagatose biosynthesis. This multienzyme approach improved the conversion of the intermediate product D-glucose to D-tagatose by 3.84% compared with the dual-enzyme system. Thus, our study provides a theoretical basis and technical support for the industrial production of D-tagatose., Competing Interests: Declarations. Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors. Competing interests: The authors declare no competing interests and funding., (© 2025. The Author(s).)

D-Mannose and L-ribose are two important monosaccharides, which have attracted public attention recently because of their great application potentials in food, cosmetic and pharmaceutical industries. Sugar isomerases catalyze the sugar isomerization and therefore can be used as the biocatalysts for production of the high-value sugars from inexpensive sugars. L-arabinose isomerase catalyzes the conversion of L-arabinose to L-ribulose, while D-lyxose isomerase catalyzes L-ribulose and D-fructose to L-ribose and D-mannose, respectively. In this paper, a putative D-LI from Bacillus velezensis (BvLI) was identified, characterized and used to produce D-mannose and L-ribose from D-fructose and L-arabinose, respectively. The recombinant BvLI exhibited a maximum activity at 55 °C and pH 6.5, in the presence of 0.1 mM Co 2+ . Approximately 110.75 g/L D-mannose was obtained from 500 g/L D-fructose in 6 h by the recombinant BvLI, and approximately 105 g/L L-ribose was obtained from 500 g/L L-arabinose in 8 h by the successive biocatalysis of L-arabinose isomerase from Bacillus licheniformis (BlAI) and BvLI.

L-arabinose isomerase (L-AI) (EC 5. 3. 1. 4. L-AI) that mediates the isomerization of D-galactose to D-tagatose was isolated from Lactobacillus brevis (MF 465792), and was further purified and characterized. Pure enzyme with molecular weight of 60.1 kDa was successfully obtained after the purification using Native-PAGE gel extraction method, which was a monomer in solution. The L-AI was found to be stable at 45-75 °C, and at pH 7.0-9.0. Its optimum temperature and pH was determined as 65 °C and 7.0, respectively. Besides, we found that Ca 2+ , Cu 2+ , and Ba 2+ ions inhibited the enzyme activity, whereas the enzyme activity was significantly enhanced in the presence of Mg 2+ , Mn 2+ , or Co 2+ ions. The optimum concentration of Mn 2+ and Co 2+ was determined to be 1 mM. Furthermore, we characterized the kinetic parameters for L-AI and determined the K m (129 mM) and the V max (0.045 mM min - 1 ) values. Notably, L. brevisL-AI exhibited a high bioconversion yield of 43% from D-galactose to D-tagatose under the optimal condition, and appeared to be a more efficient catalyst compared with other L-AIs from various organisms.