Kinetics guided engineering of cyclodextrin glycosyltransferase with enhanced intermolecular transglycosylation activity

Cyclodextrin glycosyltransferase (CGTase) catalyzes intermolecular transglycosylation through either disproportionation or cyclization‐coupling pathway. Kinetics analysis reveals that the hesperidin glycosylation process catalyzed by a CGTase variant (M1) is primarily accomplished through the disproportionation pathway. The cyclization‐coupling pathway exhibits a lower reaction rate and competitively consumes glycosyl donor and yield byproducts that impair disproportionation. Under the guidance of reaction kinetics, mutagenesis was targeted at residues in the −3, +1, and +2 subsites, known to control the selectivity between disproportionation and cyclization. A quadruple variant was identified with 2.9 times hesperidin glycosylation activity compared to M1, and 20.3 times compared to the wild‐type. Kinetic analysis reveals a fourfold improvement of k cat / K mA for disproportionation and an 85.5% reduction in k cat / K m for cyclization after mutagenesis. Binding free energy analysis further confirms that the mutagenesis favors the binding of hesperidin, and destabilizes the binding of cyclodextrin.