Multiplexed Capillary Electrophoresis as Analytical Tool for Fast Optimization of Multi-Enzyme Cascade Reactions - Synthesis of Nucleotide Sugars: Dedicated to Prof. Dr. Vladimir Křen on the occasion of his 60thbirthday.
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.
Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Aachen, Germany.
Claudia
C
Wahl
Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Aachen, Germany.
Dennis
D
Hirtz
Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Aachen, Germany. l.elling@biotec.rwth-aachen.de.
Lothar
L
Elling
D019075:Electrophoresis, Capillary / Q000379:methods*; D004798:Enzymes / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods; D007700:Kinetics; D009702:Nucleoside Diphosphate Sugars / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification*; D009711:Nucleotides / Q000032:analysis*
D004798:Enzymes; D009702:Nucleoside Diphosphate Sugars; D009711:Nucleotides
Analytical biotechnology; Biocatalysis; Multi-enzyme cascade; Nucleotide sugar; Synthetic biology