- Glycosynthases from Thermotoga neapolitana β-glucosidase 1A: A comparison of α-glucosyl fluoride and in situ-generated α-glycosyl formate donors
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TnBgl1A from the thermophile Thermotoga neapolitana is a dimeric β-glucosidase that belongs to glycoside hydrolase family 1 (GH1), with hydrolytic activity through the retaining mechanism, and a broad substrate specificity acting on β-1,4-, β-1,3- and β-1,6-linkages over a range of glyco-oligosaccharides. Three variants of the enzyme (TnBgl1A-E349G, TnBgl1A-E349A and TnBgl1A-E349S), mutated at the catalytic nucleophile, were constructed to evaluate their glycosynthase activity towards oligosaccharide synthesis. Two approaches were used for the synthesis reactions, both of which utilized 4-nitrophenyl β-d-glucopyranoside (4NPGlc) as an acceptor molecule: the first using an α-glucosyl fluoride donor at low temperature (35 °C) in a classical glycosynthase reaction, and the second by in situ generation of the glycosyl donor with (4NPGlc), where formate served as the exogenous nucleophile under higher temperature (70 °C). Using the first approach, TnBgl1A-E349G and TnBgl1A-E349A synthesized disaccharides with β-1,3-linkages in good yields (up to 61%) after long incubations (15 h). However, the GH1 glycosynthase Bgl3-E383A from a mesophilic Streptomyces sp., used as reference enzyme, generated a higher yield at the same temperature with both a shorter reaction time and a lower enzyme concentration. The second approach yielded disaccharides for all three mutants with predominantly β-1,3-linkages (up to 45%) but also β-1,4-linkages (up to 12.5%), after 7 h reaction time. The TnBgl1A glycosynthases were also used for glycosylation of flavonoids, using the two described approaches. Quercetin-3-glycoside was tested as an acceptor molecule and the resultant product was quercetin-3,4′-diglycosides in significantly lower yields, indicating that TnBgl1A preferentially selects 4NPGlc as the acceptor.
- Pozzo, Tania,Plaza, Merichel,Romero-Garcia, Javier,Faijes, Magda,Karlsson, Eva Nordberg,Planas, Antoni
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p. 132 - 139
(2014/07/21)
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- Synthesis of quercetin glycosides and their melanogenesis stimulatory activity in B16 melanoma cells
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4′-O-β-d-Glucopyranosyl-quercetin-3-O-β-d-glucopyranosyl- (1→4)-β-d-glucopyra-noside (3) was isolated from Helminthostachys zeylanica root extract as a melanogenesis acceleration compound and was synthesized using rutin as the starting material Related compounds were also synthesized to understand the structure-activity relationships in melanin biosynthesis Melanogenesis activities of the glycosides were determined by measuring intracellular melanin content in B16 melanoma cells Among the synthesized quercetin glycosides, quercetin-3-O-β-d-glucopyranoside (1), quercetin-3-O-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside (2), and 3 showed more potent intracellular melanogenesis acceleration activities than theophyline used as positive control in a dose-dependent manner with no cytotoxic effect
- Yamauchi, Kosei,Mitsunaga, Tohru,Batubara, Irmanida
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p. 937 - 944
(2014/02/14)
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- Glucosylation of Quercetin by a Cell Suspension Culture of Vitis sp.
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A cell suspension culture of a Vitis hybrid converted quercetin to six glucosides.Their structures were identified as quercetin 3-O-β-D-glucopyranoside, quercetin 3,4'-di-O-β-D-glucopyranoside, quercetin 3,7-di-O-β-D-glucopyranoside, isorhamnetin 3-O-β-D-glucopyranoside, isorhamnetin 3,4'-di-O-β-D-glucopyranoside, and isorhamnetin 3,7-di-O-β-D-glucopyranoside by UV, FD-MS, 1H-NMR, 13C-NMR spectroscopy and TLC analysis.The course of conversion was also investigated and it was shown that quercetin 3-O-glucoside reached the maximum yield of 31 percent in 24 hr and then gradually disappeared accompanied by the production of quercetin 3,4'- and 3,7-di-O-glucosides.Although the same rise and fall relationship was observed between isorhamnetin 3-O-glucoside and isorhamnetin 3,4'- or 3,7-di-O-glucoside, their conversion ratios were much lower than those of quercetin glucosides.
- Kodama, Tohru,Ishida, Hidekatsu,Kokubo, Tetsuro,Yamakawa, Takashi,Noguchi, Hiroshi
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p. 3283 - 3288
(2007/10/02)
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