h1

TY  - THES
AU  - Engels, Leonie
TI  - Modulare Enzym-Kaskaden zur Synthese von Glykanepitopen
PB  - RWTH Aachen University
VL  - Dissertation
CY  - Aachen
M1  - RWTH-2015-00909
SP  - 282 S. : graph. Darst.
PY  - 2015
N1  - Aachen, Techn. Hochsch., Diss., 2015
AB  - The aim of this work was the in vitro synthesis of the non-sulfated HNK-1 epitope (GlcA(β1-3)Gal(β1-4)GlcNAc(β1-R) and the glycan epitope 2´ fucosyllactose (2´ FL, Fuc(α1-2)Gal(β1-4)Glc) and furthermore the provision of UDP-glucuronic acid (UDP GlcA) as donor substrate for the non-sulfated HNK-1 epitope with modular enzyme-cascades.For the synthesis of UDP-GlcA two UDP-glucose-dehydrogenases, the human His6UGDH and His6KfiD of E. coli K5 revealed as suitable enzymatic tools. However, because of its higher catalytic efficiency recombinant His6UGDH was preferred. Three different synthesis strategies were tested. The one-step process with the UGDH-module was compared with two combinatorial strategies. In a one-pot system the combination of the SuSy-module with the UGDH-module and furthermore the combination of the SuSy- and UGDH-module with the UMPK-module were carried out. For the UMPK-module a human UMP-kinase was successfully produced and characterized. The functionality of all selected enzyme-module systems was proven by ESI-MS analysis of the target product UDP-GlcA.However, due to the shorter synthesis period and the chemical instability of the nucleotide sugar the one-step process was favoured. With the regeneration of the cofactor NAD+,  realized by applying His6NOX-, this strategy reveals as a cost- and time-efficient method with a yield of 92
LB  - PUB:(DE-HGF)11
UR  - https://publications.rwth-aachen.de/record/463579
ER  -