An Endoglucanase, EglA, from the Hyperthermophilic ArchaeonPyrococcus furiosus Hydrolyzes β-1,4 Bonds in Mixed-Linkage (1→3),(1→4)-β-d-Glucans and Cellulose

摘要: The eglA gene, encoding a thermostable endoglucanase from the hyperthermophilic archaeon Pyrococcus furiosus, was cloned and expressed in Escherichia coli. nucleotide sequence of gene predicts 319-amino-acid protein with calculated molecular mass 35.9 kDa. has 19-amino-acid signal peptide but not cellulose-binding domain. P. furiosus significant amino acid similarities, including conserved catalytic nucleophile proton donor, endoglucanases glucosyl hydrolase family 12. purified recombinant enzyme hydrolyzed β-1,4 β-1,3 glucosidic linkages had highest specific activity on cellopentaose (degree polymerization [DP] = 5) cellohexaose (DP 6) oligosaccharides. To lesser extent, EglA also shorter cellodextrins < as well amorphous portions polysaccharides which contain only bonds such carboxymethyl cellulose, microcrystalline Whatman paper, cotton linter. toward occurred mixed-linkage β-glucans barley β-glucan lichenan. Kinetics studies cellooliogsaccharides p-nitrophenyl-cellooligosaccharides indicated that three glucose binding subsites (−I, −II, −III) for nonreducing end two (+I +II) reducing scissile glycosidic linkage. temperature pH optima 100°C 6.0, respectively; half-life 40 h at 95°C; denaturing 112°C determined by differential scanning calorimetry. discovery this substrate specificity implications both evolution enzymes involved polysaccharide hydrolysis occurrence growth substrates hydrothermal vent environments.