Characterization of a new UDP-sugar pyrophosphorylase from Hordeum vulgare (barley)

•New UDP-sugar pyrophosphorylase was discovered in Hordeum vulgare (barley).•HvUSP shows a high stability under synthesis conditions.•HvUSP shows broad biochemical tolerances towards pH, temperature and metal ions.•HvUSP has a promiscuous substrate spectrum.•Optimization of three-enzymes-cascade-reaction leads to high space-time yield of UDP-Gal.

The broad substrate spectrum of UDP-sugar pyrophosphorylases from plant salvage pathways is of high interest for the synthesis of expensive nucleotide sugars by straightforward enzyme cascade reactions in combination with monosaccharide kinases. We here present a new UDP-sugar pyrophosphorylase from Hordeum vulgare with favorable biochemical properties like broad pH and temperature tolerances as well as a broad substrate spectrum and high synthesis stability. Enzyme properties were determined and reaction conditions were optimized by high-through-put multiplexed capillary electrophoresis analysis. In combination with a galactokinase UDP-α-d-galactose (UDP-Gal) was efficiently synthesized with a space-time-yield of 17 g/L*h for full conversion of 10 mM substrate within 20 min by 1.2 U of each enzyme.

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