Glycosyltransferases (GTs) catalyze the reaction between an activated sugar donor and an acceptor to form a new glycosidic linkage. GTs are responsible for the assembly of oligosaccharides in vivo and are also important for the in vitro synthesis of these biomolecules. However, the functional identification and characterization of new GTs are both difficult and tedious. This paper describes an approach that combines arrays of reactions on an immobilized array of acceptors with analysis by mass spectrometry to screen putative GTs. A total of 14,280 combinations of GT, acceptor and donor in four buffer conditions were screened and led to the identification and characterization of four new GTs. This work is significant because it provides a label-free method for the rapid functional annotation of putative enzymes.
Enzymatic synthesis using glycosyltransferases is a powerful approach to building polysaccharides with high efficiency and selectivity. Sugar nucleotides are fundamental donor molecules in enzymatic glycosylation reactions by Leloir-type glycosyltransferases. The applications of these donors are restricted by their limited availability. In this protocol, N-acetylglucosamine (GlcNAc)/N-acetylgalactosamine (GalNAc) are phosphorylated by N-acetylhexosamine 1-kinase (NahK) and subsequently pyrophosphorylated by N-acetylglucosamine uridyltransferase (GlmU) to give UDP–GlcNAc/GalNAc. Other UDP–GlcNAc/GalNAc analogues can also be prepared depending on the tolerance of these enzymes to the modified sugar substrates. Starting from l-fucose, GDP–fucose is constructed by one bifunctional enzyme l-fucose pyrophosphorylase (FKP) via two reactions.
Clostridium butyricum is known as a butyrate producer and a regulator of gut health, but whether it exerts a beneficial effect as a dietary supplement via modulating the intestinal microbiota remains elusive. This study investigated the impact of C. butyricum on the fecal microbiota composition and their metabolites 14 and 28 days after weaning with 10 g/kg dietary supplementation of C. butyricum. Dynamic changes of microbial compositions showed dramatically increasing Selenomonadales and decreasing Clostridiales on days 14 and 28. Within Selenomonadales, Megasphaera became the main responder by increasing from 3.79 to 11.31%. Following the prevalence of some acetate producers ( Magasphaera) and utilizers ( Eubacterium_hallii) at the genus level and even with a significant decrease in fecal acetate on day 28, the present data suggested that C. butyricum influenced microbial metabolism by optimizing the structure of microbiota and enhancing acetate production and utilization for butyrate production.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.