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Archaea, 1:285–292
© 2004 Heron Publishing—Victoria, Canada
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Hydrolase and glycosynthase activity of endo-1,3-β-glucanase from the thermophile Pyrococcus furiosus

J. van Lieshout (1, 2), M. Faijes (3), J. Neito (1), J. van der Oost (1) and A. Planas (2, 3)

1. Laboratory for Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT, Wageningen, The Netherlands / 2. Corresponding authors ([email protected], [email protected]) / 3. Laboratory of Biochemistry, Institut Químic de Sarriá, Universitat Ramon Llull, Via Augusta 390, 08017 Barcelona, Spain / Received June 21, 2004; accepted August 4, 2004; published online September 13, 2004


Pyrococcus furiosus laminarinase (LamA, PF0076) is an endo-glycosidase that hydrolyzes β-1,3-gluco-oligosaccharides, but not β-1,4-gluco-oligosaccharides. We studied the specificity of LamA towards small saccharides by using 4-methylumbelliferyl β-glucosides with different linkages. Besides endo-activity, wild-type LamA has some exo-activity, and catalyzes the hydrolysis of mixed-linked oligosaccharides (Glcβ4Glcβ3Glcβ-MU (Glc = glucosyl, MU = 4-methylumbelliferyl)) with both β-1,4 and β-1,3 specificities. The LamA mutant E170A had severely reduced hydrolytic activity, which is consistent with Glu170 being the catalytic nucleophile. The E170A mutant was active as a glycosynthase, catalyzing the condensation of α-laminaribiosyl fluoride to different acceptors. The best condensation yields were found at pH 6.5 and 50 °C, but did not exceed 30%. Depending on the acceptor, the synthase generated either a β-1,3 or a β-1,4 linkage.

Keywords: enzymatic synthesis, glycosyl fluoride, glycosylation, glycosynthase mutant, hydrolysis, laminarinase, nucleophile.

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