Development of Tagaturonate 3-Epimerase into Tagatose 4-Epimerase with a Biocatalytic Route from Fructose to Tagatose

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.

Potential of Prebiotic D-Tagatose for Prevention of Oral Disease

“…This result appears to be well supported by previous findings indicating that chewing gum containing D-tagatose reduced the number of aerobic and anaerobic bacteria in saliva (Nagamine et al, 2020). D-tagatose is an extremely rare monosaccharide in nature, though can be produced not only chemically, but also biologically using enzymes such as L-arabinose isomerase and Dtagatose 4-epimerase (Kim, 2004;Oh, 2007;Shin et al, 2020). Therefore, the origin of D-tagatose detected in saliva is unknown, though it cannot be denied that it may be derived from a metabolite of oral bacteria.…”

Non-Native Site-Selective Enzyme Catalysis

“…D-Tagatose is a lowcalorie alternative to more abundant natural sugars but is rarely present in nature, making scalable isolation a challenge, and natural enzymes capable of epimerizing D-fructose to Dtagatose have proven elusive. 133 An enzyme that catalyzes C-3 epimerization of a related substrate 135 was engineered to achieve this goal using several rounds of structure-guided sitedirected mutagenesis (Scheme 20B). The final variant 5V was capable of producing D-tagatose at 200 g/L titers and had reduced activity on the native D-tagaturonate substrate compared to the wild-type variant, indicating a significant modification of the substrate scope.…”

Non-Native Site-Selective Enzyme Catalysis

“…In the case of C-3 epimerization, 132 these species catalyze formation of a cis-enediolate, and subsequent protonation from the opposite face of the enolate by a second aspartic acid residue leads to epimizeration. C-4 epimerization 133 proceeds via a retro-aldol reaction of the keto-sugar substrate involving a M 2+ ion and a glutamic acid residue in the epimerase active site. Rotation of the resulting C-4 aldehyde presents the opposite face of the carbonyl to the intermediate M 2+ -bound enolate so that the aldol reaction gives the opposite stereoisomer at C-4.…”

“…D-tagatose is a low-calorie alternative to more abundant natural sugars but is rarely present in nature, making scalable isolation a challenge, and natural enzymes capable of epimerizing D-fructose to D-tagatose have proven elusive. 133 An enzyme that catalyzes C-3 epimerization of a related substrate, 135 was engineered to achieve this goal using several rounds of structure-buided site-directed mutagenesis (Scheme 20B). The final variant 5V was capable of capable of producing D-tagatose at 200 g/L titers and had reduced activity on the native D-tagaturonate substrate compared to the wild-type variant, indicating a significant modification of substrate scope.…”