Screening glycosynthase libraries with a fluoride chemosensor assay independently of enzyme specificity: identification of a transitional hydrolase to synthase mutant

The Biochemical Journal
Eduardo AndrésAntoni Planas

Abstract

Glycosynthases have become efficient tools for the enzymatic synthesis of oligosaccharides, glycoconjugates and polysaccharides. Enzyme-directed evolution approaches are applied to improve the performance of current glycosynthases and engineer specificity for non-natural substrates. However, simple and general screening methods are required since most of the reported assays are specific for each particular enzyme. In the present paper, we report a general screening assay that is independent of enzyme specificity, and implemented in an HTS (high-throughput screening) format for the screening of cell extracts in directed evolution experiments. Fluoride ion is a general by-product released in all glycosynthase reactions with glycosyl fluoride donors. The new assay is based on the use of a specific chemical sensor (a silyl ether of a fluorogenic methylumbelliferone) to transduce fluoride concentration into a fluorescence signal. As a proof-of-concept, it has been applied to a nucleophile saturation mutant library of Bacillus licheniformis 1,3-1,4-β-glucanase. Beyond the expected mutations at the glutamic acid (catalytic) nucleophile, other variants have been shown to acquire glycosynthase activity. Surprisingly, an aspartic acid fo...Continue Reading

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Citations

May 14, 2016·ACS Chemical Biology·Phillip M Danby, Stephen G Withers
Sep 5, 2017·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Marc R Hayes, Jörg Pietruszka
Dec 10, 2019·Chemical Society Reviews·Ulrich MarkelUlrich Schwaneberg
Mar 31, 2021·Biotechnology Advances·Zuzana MészárosKristýna Slámová
Nov 12, 2020·Current Opinion in Biotechnology·Chandra Kanth BandiShishir Ps Chundawat
Sep 1, 2021·Biotechnology Advances·Zuzana MészárosKristýna Slámová

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