Development of Tagaturonate 3-Epimerase into Tagatose 4-Epimerase with a Biocatalytic Route from Fructose to Tagatose
Kyung‐Chul Shin, Tae‐Eui Lee, Min‐Ju Seo, Dae Wook Kim, Lin‐Woo Kang, Deok‐Kun Oh
Abstract
d-Tagatose, a low-calorie functional sweetener, is biotransformed from lactose via galactose. The discovery of an enzyme with 4-epimerization activity toward d-fructose to produce d-tagatose has been a major goal in the sugar industry. However, no enzymes used for the direct production of d-tagatose from d-fructose have been identified until now. Here, we identified a tagaturonate 3-epimerase from Thermotoga petrophila with latent 4-epimerization activity toward d-fructose. We developed this enzyme into tagatose 4-epimerase by increasing the 4-epimerization activity toward d-fructose using structure-based engineering, including favorable interaction analysis and active-site modifications, and random mutagenesis, including saturation mutagenesis and DNA shuffling after error-prone PCR. The developed tagatose 4-epimerase, which showed 184-fold higher 4-epimerization activity toward d-fructose than that of tagaturonate 3-epimerase, produced 213 g/L d-tagatose using d-fructose in 2 h. Our results suggest that the tagatose 4-epimerase is an alternative enzyme for d-tagatose production via a different biocatalytic route.