Leloir glycosyltransferases enabled to flow synthesis: Continuous production of the natural C-glycoside nothofagin.

C-glycosyltransferase (CGT) and sucrose synthase (SuSy), each fused to the cationic binding module Z , were co-immobilized on anionic carrier (ReliSorb SP400) and assessed for continuous production of the natural C-glycoside nothofagin. The overall reaction was 3'-C-β-glycosylation of the polyphenol phloretin from uridine 5'-diphosphate (UDP)-glucose that was released in situ from sucrose and UDP. Using solid catalyst optimized for total (∼28 mg/g) as well as relative protein loading (CGT/SuSy = ∼1) and assembled into a packed bed (1 ml), we demonstrate flow synthesis of nothofagin (up to 52 mg/ml; 120 mM) from phloretin (≥95% conversion) solubilized by inclusion complexation in hydroxypropyl β-cyclodextrin. About 1.8 g nothofagin (90 ml; 12-26 mg/ml) were produced continuously over 90 reactor cycles (2.3 h/cycle) with a space-time yield of approximately 11 mg/(ml h) and a total enzyme turnover number of up to 2.9 × 10  mg/mg (=3.8 × 10  mol/mol). The co-immobilized enzymes exhibited useful effectiveness (∼40% of the enzymes in solution), with limitations on the conversion rate arising partly from external liquid-solid mass transfer of UDP under packed-bed flow conditions. The operational half-life of the catalyst (∼200 h; 30°C) was governed by the binding stability of the glycosyltransferases (≤35% loss of activity) on the solid carrier. Collectively, the current study shows integrated process technology for flow synthesis with co-immobilized sugar nucleotide-dependent glycosyltransferases, using efficient glycosylation from sucrose via the internally recycled UDP-glucose. This provides a basis from engineering science to promote glycosyltransferase applications for natural product glycosides and oligosaccharides.