Repetitive Batch Mode Facilitates Enzymatic Synthesis of the Nucleotide Sugars UDP-Gal, UDP-GlcNAc, and UDP-GalNAc on a Multi-Gram Scale

Abstract

The availability of nucleotide sugars is considered as bottleneck for Leloir-glycosyltransferases mediated glycan synthesis. A breakthrough for the synthesis of nucleotide sugars is the development of salvage pathway like enzyme cascades with high product yields from affordable monosaccharide substrates. In this regard, the authors aim at high enzyme productivities of these cascades by a repetitive batch approach. The authors report here for the first time that the exceptional high enzyme cascade stability facilitates the synthesis of Uridine-5'-diphospho-α-d-galactose (UDP-Gal), Uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc), and Uridine-5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) in a multi-gram scale by repetitive batch mode. The authors obtained 12.8 g UDP-Gal through a high mass based total turnover number (TTN_mass ) of 494 [g_product /g_enzyme ] and space-time-yield (STY) of 10.7 [g/L*h]. Synthesis of UDP-GlcNAc in repetitive batch mode gave 11.9 g product with a TTN_mass of 522 [g_product /g_enzyme ] and a STY of 9.9 [g/L*h]. Furthermore, the scale-up to a 200 mL scale using a pressure operated concentrator was demonstrated for a UDP-GalNAc producing enzyme cascade resulting in an exceptional high STY of 19.4 [g/L*h] and 23.3 g product. In conclusion, the authors demonstrate that repetitive batch mode is a versatile strategy for the multi-gram scale synthesis of nucleotide sugars by stable enzyme cascades.

Keywords: biocatalysis; glycobiotechnology; multi-enzyme cascade; nucleotide sugar; repetitive batch.