Downstream Processing of Nucleoside‐Diphospho‐Sugars from Sucrose Synthase Reaction Mixtures at Decreased Solvent Consumption

Abstract

Nucleoside-diphospho-sugars (NDPsugars)a re highly demanded as specialty chemicals and as substrates for enzymatic glycosylations.Biocatalysis is efficient for their synthesis;h owever, downstreamp rocessing (DSP) is ab ottleneck of the overall production. We describe twos calable DSP routes for recovery of NDP-glucose (NDP-glc) from sucrose synthase (SuSy) reaction mixtures. In both routes product precipitation with ethanol replaceds ize exclusion chromatography( SEC) which,d ue to high solvent consumption, constitutes the main limiting step of current DSP protocols.W ea lso show that selective phosphomonoester hydrolysisb ya lkaline phosphatase,c haracterized in that it did not degrade the phosphodiester linkage in NDP-glc, was au seful alternative to anion-exchange chromatography (AEC) for capture andi nitial purification of the product.B ased on comprehensive route optimization, we show uridine 5'-diphosphate glucose (UDPglc) recovery at the ! 0.5 gs cale in ay ield of about 80% andi napurityo f8 6% (phosphatase route) and 95% (AEC route).S olvent savings of up to 38-fold comparedt ot he conventional AEC-SEC route were achieved. Therei saclear demand for both new routes,a sr equirements on DSP regarding purity and costs vary with the field of application. Both DSP routes seem to be applicablet oo ther NDP-sugars and different methods of their synthesis.

Unlocking the Potential of Leloir Glycosyltransferases for Applied Biocatalysis: Efficient Synthesis of Uridine 5′‐Diphosphate‐Glucose by Sucrose Synthase

“…Besides the (bio)catalytic conversion, product isolation is a recurring bottleneck in cost‐efficient preparation of NDP‐sugars. To eliminate this restriction we recently established downstream procedures which afforded straightforward isolation of UDP‐glucose from SuSy conversions at gram‐scale …”

Determinants of the Nucleotide Specificity in the Carbohydrate Epimerase Family 1

“…(product precipitation with ethanol). [ 7 ] After reaction optimization, the nucleotide concentration of GDP‐Glc and CDP‐Glc were adapted to 30 and 5 m m , respectively. Moreover, an additional yeast treatment was performed to remove sucrose and fructose that was still present after ethanol precipitation.…”

“…[ 5,6 ] The vast majority of these biocatalysts are only active on nucleotide‐activated sugars, which generally have a high cost and limited availability, owing to their synthesis from the precursors sugar‐1‐phosphate and NTP by nucleotidyltransferases. [ 7,8 ] However, UDP‐Glc forms an attractive exception since it can easily be produced from sucrose as cheap and abundant resource, by exploiting the reversible reaction catalyzed by sucrose synthase (SuSy). [ 9,10 ]…”

Integrated process design for biocatalytic synthesis by a Leloir Glycosyltransferase: UDP‐glucose production with sucrose synthase

“…Chromatography‐free DSP of UDP‐glc was performed according to Lemmerer et al (). Biomass was roughly removed by vacuum filtration through cellulose filter paper discs (Macherey‐Nagel, MN 619 EH, particle retention 2–26 μm, thickness 0.17 mm, diameter 150 mm, 1 per 100 mL suspension).…”

“…We have recently developed a convenient chromatography‐free DSP of NDP‐sugars from SuSy reaction mixtures (Lemmerer et al, ). The DSP included calf intestinal alkaline phosphatase (CIAP) treatment for selective hydrolysis of the phosphomonoesters (UMP, UDP) present in the reaction mixture (Fig.…”