Human milk oligosaccharides (HMO) are the third largest solid fraction in milk, yet resist digestion by the infant due to complex glycosidic linkages. These undigested glycans can then serve as a prebiotic carbon source for colonic microbes. Glycoproteins in milk have glycans that are just as complex as HMOs, in terms of glycosidic linkages and composition of monosaccharide moieties. Releasing glycans from glycoproteins could provide additional sources of unconjugated oligosaccharides for biological testing. This study investigates enzymes capable of releasing O-glycans; in particular we characterized endo-α-N-acetylgalactosaminidases from Bifidobacteria breve (BBRE_00338), B. longum subsp. longum (BLNG_02015), and B. longum subsp. infantis (BINF_02535), which have an enzymatic domain similar to GH101 and GH129 enzymes, and cleave the alpha-1,3 linkage between an O-linked glycan and the serine amino acid it is attached to. The enzymes of interest were expressed recombinantly and assayed for optimum activity conditions and substrate specificities on synthetic para-nitrophenol substrates. Optimum activity was observed at 37 °C and at a pH of 5.4. Kinetic analysis based on Michaelis-Menten assumptions estimated Km between 8.293-44.98 mM. Kinetic efficiency was calculated to be 23.393 min-1 mM -1. The observed substrate specificity of BINF_2535 is different than other GH101 and GH129 enzymes, suggesting its possible that BINF_2535 and its orthologs are a separate family from or a subgroup within GH129.
|Advisor:||Mills, David A.|
|Commitee:||Barile, Danila, German, J. B.|
|School:||University of California, Davis|
|School Location:||United States -- California|
|Source:||MAI 56/03M(E), Masters Abstracts International|
|Subjects:||Molecular biology, Microbiology, Biochemistry|
|Keywords:||Bifidobacteria, Glycosidase, Kinetics|
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