Hyperthermostable endoglucanase from Pyrococcus horikoshii

Abstract

An endoglucanase homolog from the hyperthermophilic archaeon Pyrococcus horikoshii was expressed in Escherichia coli, and its enzymatic characteristics were examined. The expressed protein was a hyperthermostable endoglucanase which hydrolyzes celluloses, including Avicel and carboxymethyl cellulose, as well as beta-glucose oligomers. This enzyme is the first endoglucanase belonging to glycosidase family 5 found from Pyrococcus species and is also the first hyperthermostable endoglucanase to which celluloses are the best substrates. This enzyme is expected to be useful for industrial hydrolysis of cellulose at high temperatures, particularly in biopolishing of cotton products.

FIG. 1.

Comparison of the amino acid sequences of the endoglucanases from P. horikoshii (EGPh) and from A. cellulolyticus catalytic domain (EGAc) (43% identity). The first line shows the putative signal peptide sequence of EGPh. The sequences are aligned with dashes to indicate gaps. Asterisks (*) indicate that the amino acid is identical in both EGAc and EGPh sequences. The residues conserved in the family 5 endoglucanases are indicated by number signs (#). The active-site residues are shown in boldface. The C-terminal regions of both protein (residues 401 to 430 in EGPh and 379 to 521 in EGAc) were excluded from the alignment because of their low homology in these regions.

FIG. 2.

Mode of hydrolysis of EGPh. The viscosity values measured were plotted against the reducing sugar concentrations as measured by Somogyi-Nelson reaction using glucose as the standard. A solution of CMC (3.5% in 100 mM acetate buffer, pH 5.6, 50 ml) was subjected to hydrolysis by 4 U of EGPh/ml for 30 to 120 min at 90°C. After 30, 60, and 120 min of incubation, the viscosity of the solution was measured at 40°C, using Viscometer RE110L (Toki Sangyo, Tokyo, Japan). The initial value of viscosity was 18.92 mPa.

FIG. 3.

(A) Effect of the temperature on the hydrolytic activity of EGPh on CMC. The hydrolytic activity was measured in 100 mM acetate buffer (pH 5.6). The assay was measured for 15 min. (B) Effect of heating on EGPh activity. EGPh solution was incubated at 97°C in 100 mM acetate buffer (pH 5.6). At the time shown, aliquots were taken out and the activity was measured in the same buffer at 85°C using CMC as the substrate.