Leloir glycosyltransferases enabled to flow synthesis: Continuous production of the natural <i>C</i>‐glycoside nothofagin
Hui Liu, Bernd Nidetzky
Abstract
Abstract C ‐glycosyltransferase (CGT) and sucrose synthase (SuSy), each fused to the cationic binding module Z basic2 , were co‐immobilized on anionic carrier (ReliSorb SP400) and assessed for continuous production of the natural C ‐glycoside nothofagin. The overall reaction was 3ʹ‐ C ‐ β ‐glycosylation of the polyphenol phloretin from uridine 5ʹ‐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 3 mg/mg (=3.8 × 10 5 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.