Scheme 11 Production of superbeads and biosynthetic pathway of globotriose with regeneration of UDP-Gal., GalPUT, GalU, and PykF. The feasibility of the superbead approach was demonstrated by gram scale synthesis of Gala1,3Galp1,4GlcOBn with a truncated bovine a-1,3-galactosyltransferase (a1,3GalT) expressed in E. coli. A yield of 72% was achieved based on acceptor LacOBn with 90% enzyme activity retained after reusing three times (yields were 71%, 69%, and 66%, respectively) within three weeks. The versatility was demonstrated in the synthesis of a variety of oligosaccharides such as Galp1,4GlcNAc (92% yield) and globotriose (86% yield). Recently, an improved three-enzyme system (GalE, GalU, and a1,3GalT) to synthesize isoglobotriose using commercially inexpensive UTP has been reported (115).

Larger oligosaccharide synthesis will involve more corresponding glycosyl-transferases coupled with necessary sugar nucleotide regenerating beads. Thus, these superbeads will likely become a new generation of bioreagents for the production of glycoconjugates and their derivatives.

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