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

Abstract

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

FIG. 1

Deduced amino acid sequences for eglA from P. furiosus. The putative signal peptide is underlined. The proline- and hydroxyamino-acid-rich region is double underlined. The putative catalytic nucleophile and acid-base are identified by asterisks.

FIG. 2

Alignment of the amino acid sequences for family 12 endoglucanases in the region of the catalytic nucleophile (†) and acid-base (‡). Residues that occur in at least 10 of the 14 sequences are in boldface. The numbers refer to the amino acids in the following proteins (accession numbers, where available, are given in parentheses): pf eglA, P. furiosus endoglucanase; tm celA, T. maritima endoglucanase A (Z69341); tm celB, T. maritima endoglucanase B (Z69341); tn celA, T. neapolitana endoglucanase A (U93354); tn celB, T. neapolitana endoglucanase A (U93354); rt celA, R. marinus cellulase (U72637); ak celA, Aspergillus kawachii endoglucanase A (D12901); ao cel, Aspergillus oryzae endoglucanase (D83731); aa gun, Aspergillus aculeatus endoglucanase (P22669); tr gun, Trichoderma reesei endoglucanase (AB003694); sr, eglS, S. rochei endoglucanase S (X73953); sl celB, S. lividans cellulase B (U04629); sh celA2, Streptomyces halstedii cellulase A2 (U51222); ec celS, E. carotovora cellulase S (P16630); mt rv1090, Mycobacterium tuberculosis putative endoglucanase (AL021897). The percentages to the right indicate the amino acid sequence identities between the indicated enzyme and the P. furiosus endoglucanase as determined by using BESTFIT (gap creation penalty, 1; gap extension penalty, 0.3).

FIG. 3

HPLC analysis of the initial degradation of 10 mM cellopentaose with EglA at 98°C. Cellooligosaccharide DPs are identified by number. For hydrolysis products, DP < 5; for synthesis products DP > 5. The dotted line represents control incubated for the same period of time in the absence of enzyme. RI, refractive index.

FIG. 4

Temperature dependence for the hydrolysis of barley glucan (open circles) and PNPGlu₃ (closed circles) by EglA. Inset: Arrhenius plot for the hydrolysis of PNPGlu₃.