Acceptor-dependent regioselectivity of glycosynthase reactions by Streptomyces E383A beta-glucosidase.
The nonnucleophilic mutant E383A beta-glucosidase from Streptomyces sp. has proven to be an efficient glycosynthase enzyme, catalyzing the condensation of alpha-glucosyl and alpha-galactosyl fluoride donors to a variety of acceptors. The enzyme has maximal activity at 45 degrees 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, beta-(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 beta-(1-->3) and beta-(1-->4) linkages. In contrast, disaccharide acceptors produce exclusively beta-(1-->4) linkages. Therefore, the presence of a glycosyl unit in subsite +II redirects regioselectivity from beta-(1-->3) to beta-(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.
Faijes
Magda
M
Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull, Via Augusta 390, E-08017 Barcelona, Spain.
Saura-Valls
Marc
M
Pérez
Xavi
X
Conti
Marta
M
Planas
Antoni
A
D019943:Amino Acid Substitution / Q000235:genetics*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002384:Catalysis; D004187:Disaccharides / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D013237:Stereoisomerism; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004187:Disaccharides; D001617:beta-Glucosidase