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Volume 1 (2004), Issue 4, Pages 285-292
Research Article

Hydrolase and glycosynthase activity of endo-1,3-β-glucanase from the thermophile Pyrococcus furiosus

1Laboratory for Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT, Wageningen, The Netherlands
2Laboratory of Biochemistry, Institut Químic de Sarriá, Universitat Ramon Llull, Via Augusta 390, 08017 Barcelona, Spain

Received 21 June 2004; Accepted 4 August 2004

Copyright © 2004 Hindawi Publishing Corporation. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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.