1. Discovery of a Thermostable Tagatose 4-Epimerase Powered by Structure- and Sequence-Based Protein Clustering.
- Author
-
Chen J, Ni D, Zhu Y, Xu W, Moussa TAA, Zhang W, and Mu W
- Subjects
- Kinetics, Archaeal Proteins genetics, Archaeal Proteins chemistry, Archaeal Proteins metabolism, Fructose chemistry, Fructose metabolism, Carbohydrate Epimerases genetics, Carbohydrate Epimerases chemistry, Carbohydrate Epimerases metabolism, Hydrogen-Ion Concentration, Substrate Specificity, Hot Temperature, Amino Acid Sequence, Racemases and Epimerases genetics, Racemases and Epimerases chemistry, Racemases and Epimerases metabolism, Hexoses chemistry, Hexoses metabolism, Enzyme Stability, Molecular Docking Simulation
- Abstract
d-Tagatose is a highly promising functional sweetener known for its various physiological functions. In this study, a novel tagatose 4-epimerase from Thermoprotei archaeon (Thar-T4Ease), with the ability to convert d-fructose to d-tagatose, was discovered through a combination of structure similarity search and sequence-based protein clustering. The recombinant Thar-T4Ease exhibited optimal activity at pH 8.5 and 85 °C, in the presence of 1 mM Ni
2+ . Its kcat and kcat / Km values toward d-fructose were measured to be 248.5 min-1 and 2.117 mM-1 ·min-1 , respectively. Notably, Thar-T4Ease exhibited remarkable thermostability, with a t1/2 value of 198 h at 80 °C. Moreover, it achieved a conversion ratio of 18.9% using 100 g/L d-fructose as the substrate. Finally, based on sequence and structure analysis, crucial residues for the catalytic activity of Thar-T4Ease were identified by molecular docking and site-directed mutagenesis. This research expands the repertoire of enzymes with C4-epimerization activity and opens up new possibilities for the cost-effective production of d-tagatose from d-fructose.- Published
- 2024
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