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Directed divergent evolution of thermostable D-tagatose epimerase towards improved activity for two hexose substrates. 2015.

Type of publication Peer-reviewed
Publikationsform Original article (peer-reviewed)
Publication date 2015
Author Bosshart Andreas, Hee C. S., Bechtold Matthias, Schirmer T., Panke Sven,
Project Process intensification with integrated multi-enzyme systems
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Original article (peer-reviewed)

Journal ChemBioChem
Volume (Issue) 16
Page(s) 592 - 601
Title of proceedings ChemBioChem

Abstract

Functional promiscuity of enzymes can often be harnessed as starting point for the directed evolution of novel biocatalysts. Here we describe the divergent morphing of an engineered thermostable variant (Var8) of a promiscuous D tagatose epimerase (DTE) 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 catalytic activity of variant ILS6 is thought to relate to subtle changes in the hydration of the bound substrate. The structures can now be used to select additional sites for the directed evolution of the ketohexose epimerase.
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