An endoglucanase, eglA, from the hyperthermophilic archaeon Pyrococcus furiosus hydrolyzes beta-1,4 bonds in mixed-linkage (1 -> 3),(1 -> 4)-beta-D-glucans and cellulose

The eglA gene, encoding a thermostable endoglucanase from the hyperthermoph ilic archaeon Pyrococcus furiosus, was cloned and expressed in Escherichia coil. The nucleotide sequence of the gene predicts a 319-amino-acid protein with a calculated molecular mass of 35.9 kDa. The endoglucanase has a 19-a mino-acid signal peptide hut not cellulose-binding domain. The P. furiosus endoglucanase has significant amino acid sequence similarities, including t he conserved catalytic nucleophile and proton donor, with endoglucanases fr om glucosyl hydrolase family 12. The purified recombinant enzyme hydrolyzed beta-1,4 but not beta-1,3 glucosidic linkages and had the highest specific activity on cellopentaose (degree of polymerization [DP] = 5) and cellohex aose (DP = 6) oligosaccharides. To a lesser extent, EglA also hydrolyzed sh orter cellodextrins (DP < 5) as well as the amorphous portions of polysacch arides which contain only beta.1,4 bonds such as carboxymethyl cellulose, m icrocrystalline cellulose, Whatman paper, and cotton linter. The highest sp ecific activity toward polysaccharides occurred with mixed-linkage beta-glu cans such as barley beta-glucan and lichenan. Kinetics studies with cellool iogsaccharides and p-nitrophenyl-cellooligosaccharides indicated that the e nzyme had three glucose binding subsites (-I, -II, and -III) for the nonred ucing end and two glucose binding subsites (+I and +II) for the reducing en d from the scissile glycosidic linkage. The enzyme had temperature and pH o ptima of 100 degrees C and 6.0, respectively; a half-life of 40 h at 95 deg rees C; and a denaturing temperature of 112 degrees C as determined by diff erential scanning calorimetry. The discovery of a thermostable enzyme with this substrate specificity has implications for both the evolution of enzym es involved in polysaccharide hydrolysis and the occurrence of growth subst rates in hydrothermal vent environments.