TY - JOUR
T1 - Acceptor-dependent regioselectivity of glycosynthase reactions by Streptomyces E383A β-glucosidase
AU - Faijes, Magda
AU - Saura-Valls, Marc
AU - Pérez, Xavi
AU - Conti, Marta
AU - Planas, Antoni
N1 - Funding Information:
This work was supported in part by Grants BIO2001-2064-C02-02 and BFU2004-06377-C02-02 from the Ministerio de Ciencia y Tecnología, Spain. M.F. and X.P. acknowledge the doctoral fellowships from Instituto Danone and Institut Químic de Sarrià, respectively. We thank Dr. J. A. Pérez Pons, M. Vallmitjana and E. Querol, for providing the gene of the Streptomyces β-glucosidase and for helpful collaboration.
PY - 2006/9/4
Y1 - 2006/9/4
N2 - The nonnucleophilic mutant E383A β-glucosidase from Streptomyces sp. has proven to be an efficient glycosynthase enzyme, catalyzing the condensation of α-glucosyl and α-galactosyl fluoride donors to a variety of acceptors. The enzyme has maximal activity at 45 °C, and a pH-dependence reflecting general base catalysis with an apparent kinetic pKa of 7.2. The regioselectivity of the new glycosidic linkage depends unexpectedly on the acceptor substrate. With aryl monosaccharide acceptors, β-(1→3) disaccharides are obtained in good to excellent yields, thus expanding the synthetic products available with current exo-glycosynthases. With xylopyranosyl acceptor, regioselectivity is poorer and results in the formation of a mixture of β-(1→3) and β-(1→4) linkages. In contrast, disaccharide acceptors produce exclusively β-(1→4) linkages. Therefore, the presence of a glycosyl unit in subsite +II redirects regioselectivity from β-(1→3) to β-(1→4). To improve operational performance, the E383A mutant was immobilized on a Ni2+-chelating Sepharose resin. Immobilization did not increase stability to pH and organic solvents, but the operational stability and storage stability were clearly enhanced for recycling and scaling-up.
AB - The nonnucleophilic mutant E383A β-glucosidase from Streptomyces sp. has proven to be an efficient glycosynthase enzyme, catalyzing the condensation of α-glucosyl and α-galactosyl fluoride donors to a variety of acceptors. The enzyme has maximal activity at 45 °C, and a pH-dependence reflecting general base catalysis with an apparent kinetic pKa of 7.2. The regioselectivity of the new glycosidic linkage depends unexpectedly on the acceptor substrate. With aryl monosaccharide acceptors, β-(1→3) disaccharides are obtained in good to excellent yields, thus expanding the synthetic products available with current exo-glycosynthases. With xylopyranosyl acceptor, regioselectivity is poorer and results in the formation of a mixture of β-(1→3) and β-(1→4) linkages. In contrast, disaccharide acceptors produce exclusively β-(1→4) linkages. Therefore, the presence of a glycosyl unit in subsite +II redirects regioselectivity from β-(1→3) to β-(1→4). To improve operational performance, the E383A mutant was immobilized on a Ni2+-chelating Sepharose resin. Immobilization did not increase stability to pH and organic solvents, but the operational stability and storage stability were clearly enhanced for recycling and scaling-up.
KW - Enzyme specificity
KW - Glycosylation
KW - Glycosynthase
KW - Immobilization
KW - Oligosaccharide synthesis
KW - Streptomyces sp
KW - enzymatic
KW - β-Glucosidase
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UR - https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=pure_univeritat_ramon_llull&SrcAuth=WosAPI&KeyUT=WOS:000239377600010&DestLinkType=FullRecord&DestApp=WOS
U2 - 10.1016/j.carres.2006.04.049
DO - 10.1016/j.carres.2006.04.049
M3 - Article
C2 - 16716271
AN - SCOPUS:33745684043
SN - 0008-6215
VL - 341
SP - 2055
EP - 2065
JO - Carbohydrate Research
JF - Carbohydrate Research
IS - 12
ER -