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Volume 272, Number 50, Issue of December 12, 1997 pp. 31258-31264

Molecular and Biochemical Characterization of an Endo--1,3-glucanase of the Hyperthermophilic Archaeon Pyrococcus furiosus

(Received for publication, March 28, 1997, and in revised form, August 7, 1997)

From the Bacterial Genetics Group, Department of Microbiology, Wageningen Agricultural University, Hesselink van Suchtelenweg 4, NL-6703 CT Wageningen, The Netherlands

We report here the first molecular characterization of an endo--1,3-glucanase from an archaeon. Pyrococcus furiosus is a hyperthermophilic archaeon that is capable of saccharolytic growth. The isolated lamA gene encodes an extracellular enzyme that shares homology with both endo--1,3- and endo--1,3-1,4-glucanases of the glycosyl hydrolase family 16. After deletion of the N-terminal leader sequence, a lamA fragment encoding an active endo--1,3-glucanase was overexpressed in Escherichia coli using the T7-expression system. The purified P. furiosus endoglucanase has highest hydrolytic activity on the -1,3-glucose polymer laminarin and has some hydrolytic activity on the -1,3-1,4 glucose polymers lichenan and barley -glucan. The enzyme is the most thermostable endo--1,3-glucanase described up to now; it has optimal activity at 100-105 °C. In the predicted active site of glycosyl hydrolases of family 16 that show predominantly endo--1,3-glucanase activity, an additional methionine residue is present. Deletion of this methionine did not change the substrate specificity of the endoglucanase, but it did cause a severe reduction in its catalytic activity, suggesting a structural role of this residue in constituting the active site. High performance liquid chromatography analysis showed in vitro hydrolysis of laminarin by the endo--1,3-glucanase proceeds more efficiently in combination with an exo--glycosidase from P. furiosus (CelB). This most probably reflects the physiological role of these enzymes: cooperation during growth of P. furiosus on -glucans.

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