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

作者：J Van Lieshout; M Faijes; J Nieto; J Van Der Oost; A Planas

来源：Archaea, 2004.

DOI：10.1155/2004/731548

Pyrococcus furiosus laminarinase (LamA, PF0076) is an endo-glycosidase that hydrolyzes %26#946;-1,3-gluco-oligosaccharides, but not %26#946;-1,4-gluco-oligosaccharides. We studied the specificity of LamA towards small saccharides by using 4-methylumbelliferyl %26#946;-glucosides with different linkages. Besides endo-activity, wild-type LamA has some exo-activity, and catalyzes the hydrolysis of mixed-linked oligosaccharides (Glc%26#946;4Glc%26#946;3Glc%26#946;-MU (Glc = glucosyl, MU = 4-methylumbelliferyl)) with both %26#946;-1,4 and %26#946;-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 %26#945;-laminaribiosyl fluoride to different acceptors. The best condensation yields were found at pH 6.5 and 50 %26#176;C, but did not exceed 30%. Depending on the acceptor, the synthase generated either a %26#946;-1,3 or a %26#946;-1,4 linkage.