Combination and immobilization of enzyme module systems for the synthesis of hyaluronic acid

• Kombination und Immobilisierung von Enzymmodulsystemen zur Synthese von Hyaluronsäure

Gottschalk, Johannes; Elling, Lothar (Thesis advisor); Schwaneberg, Ulrich (Thesis advisor)

Aachen : RWTH Aachen University (2021, 2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021

Abstract

This work aimed to establish a one-pot synthesis that produces not only the needed UDP sugars but also high MW HA with low dispersity. For this, a new EM UDP-GlcA had to be established. The EM UDP-GlcA contains the enzymes AtGlcAK, AtUSP, and PmPpA. All enzymes in his work were successfully expressed in E. coli and can be purified with IMAC. The new enzymes were characterized for their kinetic parameters as well as optimal conditions. Especially the substrate inhibition of AtGlcAK and the activity increase by K+ were new findings for this plant glucuronokinase. In this work, the first characterization of PmPpA was implemented and here a substrate inhibition was detected. The cofactor, pH, and temperature optima were investigated with the result that these three enzymes are compatible. The EM UDP-GlcA performed very well with a UDP-GlcA yield of 84 % after 1 h. Since AtGlcAK is the bottleneck of this EM, the addition of K+ also improved the EM UDP-GlcA performance. PmHAS1-703 was also further analyzed and here the K+ activation was confirmed. The EM UDP GlcA was first combined with the EM HA using different starting concentrations of GlcA. This experiment revealed that a certain GlcA concentration is needed for a high MW of HA. It was the first prove in this work for the following hypothesis that accompanies most of the results here. In previous studies but also here the activity of PmHAS1-703 can be controlled with the UDP-sugar concentration. A high UDP-GlcNAc concentration and a UDP-GlcA concentration between 6-8 mM led to high activity of PmHAS1-703. The synthesis of HA by PmHAS1-703 must be divided into two phases: de novo synthesis and polymerization. It was shown that polymerization is a lot faster than de novo synthesis. A higher overall activity of PmHAS1-703 means that more polymerizations happen, and the final HA size is bigger. This hypothesis was further confirmed when all EMs were combined to the one-pot synthesis. The MW of HA increases gradually with time. Stopping the reaction earlier results in a tailored HA product but the concentration was relatively low. Surprisingly two other key factors pH and MgCl2 concentration had a great impact on the UDP-sugar synthesis. For example, a pH of 8.5 or 9 or a high MgCl2 concentration leads to an enhanced UDP-GlcNAc and a reduced UDP GlcA synthesis. This again increases the overall activity of PmHAS1-703 and the final MW of HA. With 25 mM MgCl2, an average HMW HA (1.54 MDa) with low dispersity (1.05) and high concentration (1.4 g L-1) were produced. These results are according to the hypothesis. In conclusion, two easily operated key factors were found by which the HA product can be tailored, and the HA concentration is gram-scale. The next step was to integrate an NTP regeneration system. The PolyP/RpPPK2-3 system was chosen since the phosphate donor PolyP is cheap and stable under aqueous conditions, unlike other phosphate donors. For the first time, kinetic parameters for ADP were determined with the result that RpPPK2-3 showed a higher affinity towards ADP than UDP. Implementing RpPPK2-3 into the EM UDP-GlcA and EM UDP-GlcNAc revealed that the efficiency of ATP regeneration is directly dependent on the applied kinases. BlNaHK has a higher affinity towards ATP than AtGlcAK. Therefore, the regeneration numbers extremely differ (EM UDP-GlcA: 17; EM UDP GlcNAc: 234). However, for both EMs the ATP amount could be decreased, and the yields were unchanged. For the one-pot synthesis, the PolyP concentration had to be increased, which causes that the UDP-GlcNAc synthesis was greatly reduced because of the metal ion scavenging effect of PolyP. That fits the observation that the UDP-GlcNAc synthesis is reduced at low MgCl2 concentration. The lower UDP-GlcNAc concentration decreased the PmHAS1-703 activity and according to the hypothesis also lower MW of HA were synthesized. Nevertheless, this experiment also showed, that the UDP-sugar concentration can also be controlled with the applied ATP concentration. A low ATP concentration enhances UDP-GlcNAc and decreases UDP-GlcA synthesis. With 0.1 mM ATP and 25 mM MgCl2, HA with an average MW of 1.17 MDa and a dispersity of 1.08 was produced in a concentration of 0.8 g L 1. The regeneration number was 75. With this experiment, the possibilities to synthesize lower HMW HA was extended. Further on, all enzymes were non-covalent immobilized via His-tag and NTA-Ni2+ linker on magnetic beads. The immobilization of enzymes had a tremendous effect on the kinetic parameters of the enzymes. Especially PmHAS1-703 showed a higher affinity towards UDP GlcNAc and a general higher activity with UDP-GlcA. The repetitive synthesis of UDP GlcA and UDP-GlcNAc with ATP regeneration was successful. However, a significant decrease in the performance has been noticed probably due to the mechanical forces the enzymes are exposed to. Nevertheless, according to the hypothesis, a higher PmHAS1-703 activity should lead to a higher MW of HA, and indeed in the one-pot synthesis, the average MW of HA was in the first cycle higher (3.6 MDa) and the dispersity is still very low (1.03). The concentration (0.5 g L-1) was lower compared to the soluble approaches. But also here, the mechanical forces diminish the results for further cycles. In summary, this work has successfully shown that the enzymatic production of HMW HA with low dispersity is possible and controllable. Compared to industrial processes HA products with higher quality were synthesized. Applying different conditions, either with pH, MgCl2, ATP, or immobilization can control the UDP-sugar synthesis and/or the PmHAS1-703 activity which determines the final HA product. This work lays the ground for future studies involving the in vitro synthesis of HA to not only compete in quality but also quantity with common HA productions.

Institutions

• Department of Biology [160000]
• Laboratory for Biomaterials [162820]