Product-oriented chemical surface modification of a levansucrase (SacB) <i>via</i> an ene-type reaction

Abstract

Carbohydrate processing enzymes are sophisticated tools of living systems that have evolved to execute specific reactions on sugars. Here we present for the first time the site-selective chemical modification of exposed tyrosine residues in SacB, a levansucrase from <i>Bacillus megaterium</i> (<i>Bm</i>-LS) for enzyme engineering purposes <i>via</i> an ene-type reaction. <i>Bm</i>-LS is unable to sustain the synthesis of high molecular weight (HMW) levan (a fructose polymer) due to protein-oligosaccharide dissociation events occurring at an early stage during polymer elongation. We switched the catalyst from levan-like oligosaccharide synthesis to the efficient production of a HMW fructan polymer through the covalent addition of a flexible chemical side-chain that fluctuates over the central binding cavity of the enzyme preventing premature oligosaccharide disengagement.