Sugar Nucleotide Regeneration Systems

UDP-a-D-Gal UDP-a-D-GlcNAc GDP-a-D-Man GDP-P-l-Fuc CMP-Neu5Ac In situ regeneration systems for nucleotide sugars UDP-a-D-Glc combinatorial biocatalysis [44-49]... 

The enzymatic preparation of the activated sugar nucleotide may also involve a cofactor regeneration system. An example of this is an economic one-pot procedure, in which N-acetylneuraminic acid (NeuAc) is generated in situ from IV-acetylmannosamine (ManNac) and pyruvate with sialic acid aldolase and then converted irreversibly to CMP-NeuAc ([14], see also Sec. III). 

In brief, glycosidation systems similar to that depicted in Fig. 10.3, with regeneration of sugar nucleotides, have been developed with UDP GIcNAc, GDP Man, GDP Fuc, UDP GlcUA, and CMP NeuSAc. Enzymic sialylation will be described in detail in Section 12.4. 

In a system where UDP-Gal regeneration is the goal, the inclusion of a one-step conversion of UTP to UDP-Gal using the enzyme UDP-Gal pyrophosphorylase would be ideal. Unfortunately, this enzyme is not commercially available, nor is it easy to prepare from biological sources [8]. Luckily, an alternative biosynthetic pathway to UDP-Gal exists. In nature, UDP-Glc is a central intermediate for the biosynthesis of UDP-Gal and UDP-GlcUA, as well as other sugar nucleotides. The enzyme UDP-Glc pyrophosphorylase (UDPGP EC 2.1.1.9) required for the conversion of UTP to UDP-Glc can be purchased. The UDP-Gal 4-epimerase... 

In summary, the synthesis and in situ regeneration of nucleotide sugars by combinatorial biocatalysis suffers from the main disadvantage that each enzyme has to be produced in sufficient amounts. This affords efficient recombinant protein produchon hosts being a bottleneck for some genes [25]. However, once a multi-enzyme system has been developed, the productivity can be improved by repetitive use of the biocatalysts as demonstrated for repetitive batch syntheses with soluble enzymes [25, 38] or with immobilized enzymes [48]. The advantage... 

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