Nucleotide sugars are considered as bottleneck and expensive substrates for enzymatic glycan synthesis using Leloir-glycosyltransferases. Synthesis from cheap substrates such as monosaccharides is accomplished by multi-enzyme cascade reactions. Optimization of product yields in such enzyme modules is dependent on the interplay of multiple parameters of the individual enzymes and governed by a considerable time effort when convential analytic methods like capillary electrophoresis (CE) or HPLC are applied. We here demonstrate for the first time multiplexed CE (MP-CE) as fast analytical tool for the optimization of nucleotide sugar synthesis with multi-enzyme cascade reactions. We introduce a universal separation method for nucleotides and nucleotide sugars enabling us to analyze the composition of six different enzyme modules in a high-throughput format. Optimization of parameters (T, pH, inhibitors, kinetics, cofactors and enzyme amount) employing MP-CE analysis is demonstrated for enzyme modules for the synthesis of UDP-α-D-glucuronic acid (UDP-GlcA) and UDP-α-D-galactose (UDP-Gal). In this way we achieve high space-time-yields: 1.8 g/L⋆h for UDP-GlcA and 17 g/L⋆h for UDP-Gal. The presented MP-CE methodology has the impact to be used as general analytical tool for fast optimization of multi-enzyme cascade reactions.

In two cycles of an error‐prone PCR process, variants of formate dehydrogenase from Candida boidinii were created which revealed an up to 4.4‐fold (440%) higher residual activity after entrapment in polyacrylamide gels than the wild‐type enzyme. These were identified in an assay using single precursor molecules of polyacrylamide instead of the complete gel for selection. The stabilization resulted from an exchange of distinct lysine, glutamic acid, and cysteine residues remote from the active site, which did not affect the kinetics of the catalyzed reaction. Thermal stability increased at the exchange of lysine and glutamic acid, but decreased due the exchange of cysteine. Overall, the variants reveal very suitable properties for application in a technical synthetic process, enabling use of entrapment in polyacrylamide as an economic and versatile immobilization method.

A highly flexible enzyme module system (EMS) was developed which allows for the first time the in situ regeneration of deoxythymidine 5'-diphosphate (dTDP)-activated deoxy sugars and furthermore enables us to produce novel sorangiosides in a combinatorial biocatalytic approach using three enzyme modules. The SuSy module with the recombinant plant enzyme sucrose synthase (SuSy) and the deoxy sugar module consisting of the enzymes RmlB (4,6-dehydratase), RmlC (3,5-epimerase) and RmlD (4-ketoreductase) from the biosynthetic pathway of dTDP-b-l-rhamnose were combined with the glycosyltransferase module containing the promiscuous recombinant glycosyltransferase SorF from Sorangium cellulosum So ce12. Kinetic data and the catalytic efficiency were determined for the donor substrates of SorF: dTDP-a-d-glucose, dTDP-b-l-rhamnose, uridine diphosphate (UDP)-ad-glucose (Glc), and dTDP-6-deoxy-4-keto-a-d-glucose. The synthesis of glucosyl-sorangioside with in situ regeneration of dTDP-Glc was accomplished by combination of SuSy and SorF. The potential of the EMS is demonstrated by combining SuSy, RmlB, RmlC, RmlD with SorF in one-pot for the in situ regeneration of dTDP-activated (deoxy) sugars. The HPLC/MS analysis revealed the formation of rhamnosyl-sorangioside and glucosyl-sorangioside, demonstrating the in situ regeneration of dTDP-b-l-rhamnose and dTDP-a-d-glucose and a cycle number for dTDP higher than 9. Furthermore, NADH (reduced form of nicotinamdie adenine dinucleotide) regeneration with formate dehydrogenase in the reduction step catalyzed by the 4-ketoreductase RmlD could be integrated in the one-pot synthesis yielding similar conversion rates and cycle numbers. In summary, we have established the first in situ regeneration cycle for dTDP-activated (deoxy) sugars by a highly flexible EMS which allows simple exchange of enzymes in the deoxy sugar module and exchange of glycosyltransferases as well as aglycones in the glycosyltransferase module to synthesize new hybrid glycosylated natural products in one-pot.

Die Kosten des Downstream-Processings verursachen einen Großteil der Herstellungskosten monoklonaler Antikörper (mAbs). Im Plattform-Downstream-Processing werden vor allem chromatographische Verfahren zur Aufreinigung der mAbs aus Zellüberstand eingesetzt. Eine Alternative ist die Extraktion der mAbs mit wässrigen Zweiphasen-Systemen (aqueous two-phase systems, ATPS). Wissenschaftliche Studien haben das Potenzial der wässrigen Zweiphasen-Extraktion (ATPE) mit verschiedenen ATPS belegt. Die Aufreinigung der mAbs in einer kontinuierlichen, mehrstufigen Extraktion, die zu einer hohen finalen Reinheit des mAbs führt, wurde bisher kaum untersucht. Speziell für kontinuierliche Fermentationsprozesse bietet die ATPE einen Vorteil gegenüber der diskontinuierlichen Chromatographie.Im Rahmen dieser Arbeit wird das Anwendungspotenzial der ATPE bei der Herstellung von mAbs diskutiert. Es werden zwei Möglichkeiten betrachtet, die ATPE in bestehende Plattformprozesse zu integrieren. Diese unterscheiden sich im Einsatz der ATPE, die entweder als Ersatz oder als Unterstützung der im Capture-Schritt herkömmlich genutzten Protein-A-Chromatographie infrage kommt. Beide Möglichkeiten werden anhand experimenteller Ergebnisse diskutiert.