Directed Divergent Evolution of a Thermostable D‐Tagatose Epimerase towards Improved Activity for Two Hexose Substrates

Abstract

Abstract:We exploit the functional promiscuity of an engineered thermostable variant of a promiscuous D-tagatose epimerase (DTE Var8) to morph it into two efficient catalysts for the C3 epimerization of D-fructose to D-psicose and of L-sorbose to L-tagatose. Iterative single-site randomization and screening of 48 residues in the first and second shell around the substrate binding site of Var8 yielded 8-sites mutant IDF8 with an 9-fold improved kcat for the epimerization of D-fructose, and the 6-sites mutant ILS6 with an 14-fold improved epimerization of L-sorbose compared to Var8. Structure analysis of IDF8 revealed a charged patch at the entrance of its active site that supposedly facilitates the entry of the polar substrate, whereas the improvement in variant ILS6 is thought to relate to subtle changes in the hydration of the bound substrate. The structures will inform future engineering efforts of these and other isomerizing enzymes for the production of rare.

Production of d‐allulose from d‐glucose by Escherichia coli transformant cells co‐expressing d‐glucose isomerase and d‐psicose 3‐epimerase genes

“…Through random and site‐directed mutagenesis, the double‐site I33L/S213C variant of DPEase from A. tumefaciens was constructed, which showed obvious enhancement in thermostability, including a 7.5 °C increase in optimal temperature, a 29.9‐fold increase in half‐life ( t ½ , 50 °C) and 7.5 °C increase in melting temperature ( T m ) . Recently, by site‐directed mutagenesis, the Y68I/G109P variant of C. bolteae DPEase was obtained, which improved the catalytic efficiency and thermostability with a 17.9% decrease in K m value, a 108 min increase in t ½ (55 °C) and a 2.4 °C increase in T m. Based on the systematic analysis of subunit–subunit interface interactions and the residues around active site development of iterative saturation mutagenesis and high‐efficient screening results in beneficial A. tumefaciens DPEase variants with greatly improved thermostability and substrate specificity, respectively . In addition, the food‐grade expression of DPEase from Clostridium scindens 35704 in Bacillus subtilis based on an antibiotic resistance gene‐independent replicative plasmid was constructed, producing 196 g L −1 d ‐allulose from 700 g L −1 of d ‐fructose …”

Recent advances in d -allulose: Physiological functionalities, applications, and biological production

“…By iterative randomization and screening around the substrate-binding site, the eight-site mutant IDF8 and the six-site mutant ILS6 were achieved. Compared to Var8, IDF8 showed 9-fold improved k cat for the epimerization of D-fructose, while ILS6 14-fold improved k cat for the epimerization of L-sorbose (Bosshart, Hee, Bechtold, Schirmer, & Panke, 2015). All these mutants mentioned above might have a great potential in the industrial production of D-allulose.…”

Through the Looking Glass: Chiral Recognition of Substrates and Products at the Active Sites of Racemases and Epimerases

“…[196] Because of the commercial interesti n this enzyme, at hermostable variant of promiscuous DTE from P. cichorii was engineered into two variants:o ne catalyzing the epimerization of d-fructose/d-psicose (IDF8) and the other cat-alyzing the epimerizationo fl-sorbose/l-tagatose (ILS6). [197] The X-ray crystal structures of IDF8 (PDB 4PFH) and ILS6 (PDB 4PGL) were solved with the substrates bound at the active site. Although crystalso fI DF8 were soakedi nd-fructose, additional electron density was observed at the active site consistent with the presence of both the d-fructose and d-psicose epimers.…”