Nucleoside-diphospho-sugars (NDP-sugars) are highly demanded as specialty chemicals and as substrates for enzymatic glycosylations. Biocatalysis is efficient for their synthesis; however, downstream processing (DSP) is a bottleneck of the overall production. We describe two scalable DSP routes for recovery of NDP-glucose (NDP-glc) from sucrose synthase (SuSy) reaction mixtures. In both routes product precipitation with ethanol replaced size exclusion chromatography (SEC) which, due to high solvent consumption, constitutes the main limiting step of current DSP protocols. We also show that selective phosphomonoester hydrolysis by alkaline phosphatase, characterized in that it did not degrade the phosphodiester linkage in NDP-glc, was a useful alternative to anion-exchange chromatography (AEC) for capture and initial purification of the product. Based on comprehensive route optimization, we show uridine 5′-diphosphate glucose (UDP-glc) recovery at the ≥0.5 g scale in a yield of about 80% and in a purity of 86% (phosphatase route) and 95% (AEC route). Solvent savings of up to 38-fold compared to the conventional AEC-SEC route were achieved. There is a clear demand for both new routes, as requirements on DSP regarding purity and costs vary with the field of application. Both DSP routes seem to be applicable to other NDP-sugars and different methods of their synthesis.
Fields of Expertise
- Human- & Biotechnology