Glycosynthases from Thermotoga neapolitana beta-glucosidase 1A: A comparison of alpha-glucosyl fluoride and in situ-generated alpha-glycosyl formate donors
Pozzo, Tania LU ; Plaza, Merichel LU ; Romero-Garcia, Javier ; Faijes, Magda ; Nordberg Karlsson, Eva LU
and Planas, Antoni
(2014)
In Journal of Molecular Catalysis B: Enzymatic
107.
p.132-139
- Abstract
- TnBgl1A from the thermophile Thermotoga neapolitana is a dimeric beta-glucosidase that belongs to glycoside hydrolase family 1 (GH1), with hydrolytic activity through the retaining mechanism, and a broad substrate specificity acting on beta-1,4-, beta-1,3- and beta-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 beta-D-glucopyranoside (4NPGIc) as an acceptor molecule: the first using an alpha-glucosyl fluoride donor at low temperature (35... (More)
- TnBgl1A from the thermophile Thermotoga neapolitana is a dimeric beta-glucosidase that belongs to glycoside hydrolase family 1 (GH1), with hydrolytic activity through the retaining mechanism, and a broad substrate specificity acting on beta-1,4-, beta-1,3- and beta-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 beta-D-glucopyranoside (4NPGIc) as an acceptor molecule: the first using an alpha-glucosyl fluoride donor at low temperature (35 degrees C) in a classical glycosynthase reaction, and the second by in situ generation of the glycosyl donor with (4NPGIc), where formate served as the exogenous nucleophile under higher temperature (70 degrees C). Using the first approach, TnBgl1A_E349G and TnBgl1A_E349A synthesized disaccharides with beta-1,3-linkages in good yields (up to 61%) after long incubations (15 h). However, the GH1 glycosynthase Bg13_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 beta-1,3-linkages (up to 45%) but also beta-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 4NPGIc as the acceptor. (C) 2014 Elsevier B.V. All rights reserved. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/4648917
- author
- Pozzo, Tania
LU
; Plaza, Merichel
LU
; Romero-Garcia, Javier
; Faijes, Magda
; Nordberg Karlsson, Eva
LU
and Planas, Antoni
- organization
- publishing date
- 2014
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- beta-Glucosidase, alpha-Glucosyl fluoride, Formate, 4-Nitrophenyl, beta-D-glucopyranoside, Quercetin
- in
- Journal of Molecular Catalysis B: Enzymatic
- volume
- 107
- pages
- 132 - 139
- publisher
- Elsevier
- external identifiers
-
- wos:000340698300019
- scopus:84903720920
- ISSN
- 1873-3158
- DOI
- 10.1016/j.molcatb.2014.05.021
- language
- English
- LU publication?
- yes
- id
- 019d420c-82c6-4133-ba98-712b190e5640 (old id 4648917)
- date added to LUP
- 2016-04-01 10:43:25
- date last changed
- 2022-04-28 00:49:58
@article{019d420c-82c6-4133-ba98-712b190e5640,
abstract = {{TnBgl1A from the thermophile Thermotoga neapolitana is a dimeric beta-glucosidase that belongs to glycoside hydrolase family 1 (GH1), with hydrolytic activity through the retaining mechanism, and a broad substrate specificity acting on beta-1,4-, beta-1,3- and beta-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 beta-D-glucopyranoside (4NPGIc) as an acceptor molecule: the first using an alpha-glucosyl fluoride donor at low temperature (35 degrees C) in a classical glycosynthase reaction, and the second by in situ generation of the glycosyl donor with (4NPGIc), where formate served as the exogenous nucleophile under higher temperature (70 degrees C). Using the first approach, TnBgl1A_E349G and TnBgl1A_E349A synthesized disaccharides with beta-1,3-linkages in good yields (up to 61%) after long incubations (15 h). However, the GH1 glycosynthase Bg13_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 beta-1,3-linkages (up to 45%) but also beta-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 4NPGIc as the acceptor. (C) 2014 Elsevier B.V. All rights reserved.}},
author = {{Pozzo, Tania and Plaza, Merichel and Romero-Garcia, Javier and Faijes, Magda and Nordberg Karlsson, Eva and Planas, Antoni}},
issn = {{1873-3158}},
keywords = {{beta-Glucosidase; alpha-Glucosyl fluoride; Formate; 4-Nitrophenyl; beta-D-glucopyranoside; Quercetin}},
language = {{eng}},
pages = {{132--139}},
publisher = {{Elsevier}},
series = {{Journal of Molecular Catalysis B: Enzymatic}},
title = {{Glycosynthases from Thermotoga neapolitana beta-glucosidase 1A: A comparison of alpha-glucosyl fluoride and in situ-generated alpha-glycosyl formate donors}},
url = {{http://dx.doi.org/10.1016/j.molcatb.2014.05.021}},
doi = {{10.1016/j.molcatb.2014.05.021}},
volume = {{107}},
year = {{2014}},
}