Discovery of a Thermostable Tagatose 4-Epimerase Powered by Structure- and Sequence-Based Protein Clustering
Jiajun Chen, Dawei Ni, Yingying Zhu, Wei Xu, Tarek A. A. Moussa, Wenli Zhang, Wanmeng Mu
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 k cat and k cat / K m 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 t 1/2 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.