Cell-free multi-enzyme synthesis and purification of uridine diphosphate galactose

Chembiochem. 2021 Oct 12. doi: 10.1002/cbic.202100361. Online ahead of print.ABSTRACTHigh costs and low availability of UDP-galactose hampers the enzymatic synthesis of valuable oligosaccharides such as human milk oligosaccharides. Here, we report the development of a platform for the scalable, biocatalytic synthesis and purification of UDP-galactose. UDP-galactose was produced with a titer of 48 mM (27.2 g/L) in a small-scale batch process (200 µL) within 24 h using 0.02 g enzyme /g product . Through in-situ ATP regeneration, the amount of ATP (0.6 mM) supplemented was around 240-fold lower than the stoichiometric equivalent required to achieve the final product yield. Chromatographic purification using porous graphic carbon adsorbent yielded UDP-galactose with a purity of 92%. The synthesis was transferred to 1 L preparative scale production in a stirred tank bioreactor. To further reduce the synthesis costs here, the supernatant of cell lysates was used bypassing expensive purification of enzymes. Here, 23.4 g/L UDP-galactose were produced within 23 h with a synthesis yield of 71% and a biocatalyst load of 0.05 g total_protein /g product . The costs for substrates per gram of UDP-galactose synthesized were around 0.26 €/g.PMID:34637168 | DOI:10.1002/cbic.202100361

Source: Chembiochem - October 12, 2021 Category: Biochemistry Authors: Reza Mahour Ju Weon Lee Pia Grimpe Simon Boecker Valerian Grote Steffen Klamt Andreas Seidel-Morgenstern Thomas F T Rexer Udo Reichl Source Type: research