Tuan Son Hoang , Sara-Theres Wormstall , Nam-Hai Hoang , Udo Reichl , Thomas F.T. Rexer
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引用次数: 0
Abstract
UDP-N-acetylgalactosamine (UDP-GalNAc) is an essential building block in the synthesis of glycans including O-glycans and glycosaminoglycans. For the latter, enzymatic synthesis is often a promising approach for producing multi-gram amounts. However, the high cost of UDP-GalNAc has limited this approach. This study reports on the development and optimization of a multi-enzyme cascade for synthesizing UDP-GalNAc from inexpensive substrates. Consisting of six recombinant enzymes, the cascade converts uridine (Uri) and GalNAc to UDP-GalNAc with in situ ATP regeneration using polyphosphate (PolyPn). Two rounds of Design of Experiments (DoE) optimization were performed to systematically evaluate and optimize reaction parameters including pH, temperature, MgCl2, ATP, and PolyPn concentrations. The established cascade achieved a percentage yield of 95 % and an UDP-GalNAc titer of 46.1 mM (28 g/L). This represented a 19-fold improvement over the initial conditions. Purification by anion-exchange chromatography yielded a maximum recovery of 89 % and a purity of 90 %. Overall, this scalable, low-cost enzymatic synthesis of UDP-GalNAc overcomes current limitations in availability and cost, potentially enabling new applications in the field of glycobiotechnology.
期刊介绍:
New Biotechnology is the official journal of the European Federation of Biotechnology (EFB) and is published bimonthly. It covers both the science of biotechnology and its surrounding political, business and financial milieu. The journal publishes peer-reviewed basic research papers, authoritative reviews, feature articles and opinions in all areas of biotechnology. It reflects the full diversity of current biotechnology science, particularly those advances in research and practice that open opportunities for exploitation of knowledge, commercially or otherwise, together with news, discussion and comment on broader issues of general interest and concern. The outlook is fully international.
The scope of the journal includes the research, industrial and commercial aspects of biotechnology, in areas such as: Healthcare and Pharmaceuticals; Food and Agriculture; Biofuels; Genetic Engineering and Molecular Biology; Genomics and Synthetic Biology; Nanotechnology; Environment and Biodiversity; Biocatalysis; Bioremediation; Process engineering.