- Regioselectivity in β-galactosidase-catalyzed transglycosylation for the enzymatic assembly of D-galactosyl-D-mannose
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The regioselectivity of β-galactosidase derived from Bacillus circulans ATCC 31382 (β-1,3-galactosidase) in transgalactosylation reactions using D-mannose as an acceptor was investigated. This D-mannose associated regioselectivity was found to be different from reactions using either GlcNAc or GalNAc as acceptors, not only for β-1,3-galactosidase but also for β-galactosidases of different origins. The relative hydrolysis rate of Galβ-pNP and D-galactosyl-D-mannoses, of various linkages, was also measured in the presence of β-1,3-galactosidase and was found to correlate well with the ratio of disaccharides formed by transglycosylation. The unexpected regioselectivity using D-mannose can therefore be explained by an anomalous specificity in the hydrolysis reaction. By utilizing the identified characteristics of both regioselectivity and hydrolysis specificity using D-mannose, an efficient method for enzymatic synthesis of β-1,3-, β-1,4- and β-1,6-linked D-galactosyl-D-mannose was subsequently established.
- Miyasato, Mariko,Ajisaka, Katsumi
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- A novel fluorescent assay for T-synthase activity
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Loss of T-synthase (uridine diphosphate galactose:Nacetylgalactosaminyl- α1-Ser/Thr β3galactosyltransferase), a key enzyme required for the formation of mucin-type core 1 O-glycans, is observed in several human diseases, including cancer, Tn syndrome and IgA nephropathy, but current methods to assay the enzyme use radioactive substrates and complicated isolation of the product. Here we report the development of a novel fluorescent assay to measure its activity in a variety of tumor cell lines. Deficiencies in T-synthase activity correlate with mutations in the gene encoding the molecular chaperone Cosmc that is required for folding the T-synthase. This new high-throughput assay allows for facile screening of tumor specimens and other biological material for T-synthase activity and could be used diagnostically.
- Ju, Tongzhong,Xia, Baoyun,PAryal, Rajindra,Wang, Wenyi,Wang, Yingchun,Ding, Xiaokun,Mi, Rongjuan,He, Miao,Richard D. Cummings
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experimental part
p. 352 - 362
(2011/10/09)
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