Integration of a Nucleoside Triphosphate Regeneration System in the One‐pot Synthesis of UDP‐sugars and Hyaluronic Acid

Abstract

Enzymatic synthesis is a promising approach to produce hyaluronic acid (HA) products with defined molecular weight (MW) and dispersity. We here report on the evaluation of nucleoside triphosphate (NTP) regeneration system for the synthesis of the precursors uridine-5'-diphosphate α-d-N-acetylglucosamine (UDP-GlcNAc) and uridine-5'-diphosphate α-dglucuronic acid (UDP-GlcA) and integration in the one-pot synthesis of HA. By integration of polyphosphate kinase from Ruegeria pomeroyi (RpPPK2-3) into the enzyme cascade to synthesize UDP-GlcA we reached 89 % yield after 1 h with a regeneration cycle number (RCN) of 17. With the UDP-GlcNAc cascade, the ATP concentration was lowered 250-fold and reached 94 % yield after 1 h with an RCN of 234. Integration of the RpPPK2-3 regeneration system in one-pot HA synthesis afforded optimization of MgCl 2 and ATP starting concentrations. With 25 mM MgCl 2 and 0.1 mM ATP, a HA concentration of 0.81 g L À 1 with an average MW of 1.17 MDa and dispersity of about 1.08 and an RCN of 75 was obtained. We conclude that integration of NTP regeneration with RpPPK2-3 expanded the enzymatic possibilities to produce HA with lower range MW.

Polyphosphate Kinase 2 (PPK2) Enzymes: Structure, Function, and Roles in Bacterial Physiology and Virulence

“…This marks the first time PPK2s have been shown to use all the natural nucleotides—including thymidine—as substrates, highlighting the potential of these so-called universal PPK2s as tools for biotechnological NTP regeneration [ 66 ]. Specifically, universal PPK2s could be a boon for the regeneration of UTP and CTP required for the enzymatic synthesis of complex glycans [ 59 , 67 , 68 ], in addition to more traditional ATP regeneration systems (reviewed in [ 63 ]).…”

Repetitive Synthesis of High‐Molecular‐Weight Hyaluronic Acid with Immobilized Enzyme Cascades

“…Furthermore, the strong substrate inhibition by UDP‐GlcA limits the substrate concentration for high PmHAS 1–703 activity. Therefore, the UDP‐sugar ratio dictates the M W of HA for soluble PmHAS 1–703 by the interplay between de novo synthesis and polymerization at the early beginning of the reaction [37,39] . In general, polymerization is faster and preferred over the de novo synthesis by soluble PmHAS 1–703 [10,11] .…”

“…In general, each cycle reached high yields of 96-98 % and a high ATP recycling efficiency (> 91 %) (Table 3) Surprisingly, the immobilized EM UDP-GlcNAc module was still efficient with 20 mm PolyP concentrations which is a significant improvement in comparison to the performance of EM UDP-GlcNAc in the soluble one-pot synthesis, where 20 mm PolyP led to reduced UDP-GlcNAc synthesis. [39] In summary, the syntheses of UDP-GlcA and UDP-GlcNAc with immobilized enzyme cascades including ATP regeneration was highly efficient. With high process stability of all immobilized enzymes for at least two days and high product yields after 2-3 h, the immobilized enzyme cascades shall be utilized in multiple cycles within two days.…”

“…[38] RpPPK2-3 is probably occupied with the ATP regeneration in the early beginning of the reaction, and later on, it could be that not enough PolyP is present for efficient UDP regeneration as we have seen earlier. [39] A comparison with our previous studies [37][38][39] is necessary to rationalize the outcome of HA synthesis with immobilized enzyme modules. With soluble enzyme cascades a high UDP-GlcNAc concentration gives high M W of HA which is due to a high K M for UDP-GlcNAc of PmHAS .…”

Immobilization of CMP‐Sialic Acid Synthetase and α2,3‐Sialyltransferase for Cascade Synthesis of 3′‐Sialyl β‐D‐Galactoside with Enzyme Reuse

“…The current study therefore presents progress towards CSS and SiaT cascade reactions on solid support for the practical synthesis of sialo‐oligosaccharides. The results can have relevance for cascade syntheses of oligosaccharides and sugar nucleotides showing potential for production [40–44] …”