In Vitro One-Pot Enzymatic Synthesis of Hyaluronic Acid from Sucrose and N-Acetylglucosamine: Optimization of the Enzyme Module System and Nucleotide Sugar Regeneration

摘要

Enzymatic in vitro synthesis of hyaluronic acid (HA) is the technology of choice for the production of defined HA preparations under controlled conditions. One drawback is the high costs for the substrates uridine diphosphate (UDP)-GlcA and UDP-GlcNAc. We developed an economic multi-enzyme strategy for the in vitro one-pot synthesis of high-molecular-weight (M-w)-HA from cheap and sustainable substrates sucrose and N-acetylglucosamine (GlcNAc) with in situ regeneration of nucleotide sugars. Multiplexed capillary electrophoresis was used for analysis and optimization of the reaction cascades. We here present novel parameters influencing the polymerization rate and M-w of HA synthesized by Pasteurella multocida hyaluronan synthase (pmHAS(1-703)-His(6)), including cofactors, kinetics, and substrate ratio. UDP-GlcA regeneration by sucrose synthase proved to be favorable for kinetic and economic reasons. With optimized reaction conditions, HA with a M-w2MDa is synthesized from catalytic UDP concentrations and 10mm GlcNAc in less than 10h.