Entwicklung und Optimierung eines Enzymmodulsystems zur Synthese von Hyaluronsäure
- Development and optimization of an enzyme module system for the synthesis of hyaluronic acid
Eisele, Anna; Elling, Lothar (Thesis advisor); Blank, Lars M. (Thesis advisor)
Dissertation / PhD Thesis
Dissertation, RWTH Aachen University, 2020
In this thesis a novel enzyme module system (EMS) for the in vitro synthesis of hyaluronic acid (HA) from sucrose and GlcNAc was established. It consists of two enzyme modules (EMs) for the synthesis of the HA precursors UDP-GlcA and UDP-GlcNAc, of a third EM containing the polymerizing catalyst HA synthase (HAS) and of in situ regeneration systems for both UDP-sugars. The reaction analysis was based on multiplexed capillary electrophoresis (MP-CE) for detection of nucleotides and nucleotide sugars. It was complemented with HA polymer analysis by agarose gel electrophoresis, turbidity measurement and size exclusion chromatography. MP-CE allowed a high throughput screening and optimization of reaction parameters for single enzymes and EMs. Special focus was placed on the characterization of Pasteurella multocida HAS (pmHAS1-703-His6). For pmHAS1-703-His6 a substrate inhibition by UDP-GlcA (>8 mM) and a high Km value for UDP-GlcNAc (23,4 mM) were revealed. It was shown that the UDP-sugar ratio is a tool for controlling pmHAS1-703-His6 reaction. K+ was identified as a novel metal coactivator of pmHAS1-703-His6 increasing its polymerization rate and HA molecular weight (Mw). Optimized HA synthesis from UDP-sugars resulted in a 3 MDa polymer after less than 8 h. In the EMS, sucrose synthase mediated UDP-GlcA regeneration turned out to be highly beneficial not only from an economic, but also from a kinetic viewpoint. By using a low or catalytic UDP starting concentration, a favorable UDP-sugar ratio is adjusted. As a consequence, polymerization rate and Mw of HA are enhanced. With 0.02 mM UDP, 200 mM sucrose and 10 mM GlcNAc 4 mg/mL HA with a Mw of 2.3 MDa were synthesized within 9 hours. In contrast, UDP-GlcNAc regeneration proved to be kinetically unfavorable and resulted in low Mw of HA and long reaction times. Considering the costs for the PEP/PK regeneration system, this approach was dismissed. However, general feasibility of HA synthesis with regeneration of both UDP-sugars was shown. Finally, soluble pmHAS1-703-His6 could be flexibly exchanged by a membrane-associated HAS from Streptococcus zooepidemicus in the EMS using HAS producing cells as biocatalysts instead of isolated enzyme. In conclusion, this work defined precise conditions for the synthesis of high-Mw HA with pmHAS1-703-His6 and laid the foundation for an enzymatic HA production process. As future steps, efficient scale-up and downstream processing need to be established. The presented EMS can be used as a lab-scale alternative for HA production or serve as model for the metabolic engineering of a HA producing host.