D-tagatose is a rare natural food sweetener with special health care functions. Currently, the isomerization pathway for D-tagatose production using D-galactose as substrate is limited by the basic thermodynamic equilibrium, which often leads to low substrate conversion rate. However, D-tagatose biosynthesis based on redox pathway can avoid unnecessary reverse reactions by thermodynamically coupling cofactors and has a higher theoretical conversion rate. While, the existing aldose reductase, the key enzyme of the redox pathway, has a wide substrate spectrum and is not specific for substrate D-galactose, which is the bottleneck that restricts the development of this bioprocess. In this study, the xylose reductase from Scheffersomyces stipitis CBS 6054 (SsXR) was used as the template, and 11 homologous sequences were screened from National Center for Biotechnology Information (NCBI) based on amino acid homology analysis, and homology modeling and molecular docking were performed. Based on the binding free energy and the distance of action sites between D-galactose and protein molecule, the depth and matched-degree of substrate in protein pockets, the XR from Spathaspora gorwiae (SgXR) with highest substrate specificity was selected. SsXR and SgXR were expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the two purified enzymes were studied. The substrate specificity of SgXR for D-galactose was 4-times that of SsXR. The Km value and kcat of SgXR for D-galactose were 1/4 and 1.11 times that of SsXR, respectively. Furthermore, by introducing galactitol dehydrogenase RlGDH from Rhizobium legumenosarum, the recombinant strain containing SgXR produced 5.47 g/L D-tagatose from 10 g/L D-galactose within 48 h, which was 1.23 times higher than that of SsXR. The D-tagatose yield from D-galactose was 54.7%, which was higher than most reports based on isomerization pathways. These results laid a foundation for the development of efficient D-tagatose biosynthesis process based on redox pathway.
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