Improving low-temperature catalysis in the hyperthermostable Pyrococcus furiosus beta-glucosidase CelB by directed evolution

The beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosu s (CelB) is the most thermostable and thermoactive family 1 glycosylhydrola se described to date. To obtain more insight in the molecular determinants of adaptations to high temperatures and study the possibility of optimizing low-temperature activity of a hyperthermostable enzyme, we generated a lib rary of random CelB mutants in Escherichia coli. This library was screened for increased activity on p-nitrophenyl-beta-D-glucopyranoside at room temp erature. Multiple CelB variants were identified with up to 3-fold increased rates of hydrolysis of this aryl glucoside, and 10 of them were characteri zed in detail. Amino acid substitutions were identified in the active-site region, at subunit interfaces, at the enzyme surface, and buried in the int erior of the monomers. Characterization of the mutants revealed that the in crease in low-temperature activity was achieved in different ways, includin g altered substrate specificity and increased flexibility by an apparent ov erall destabilization of the enzyme. Kinetic characterization of the active -site mutants showed that in all cases the catalytic efficiency at 20 degre es C on p-nitrophenyl-beta-D-glucose, as well as on the disaccharide cellob iose, was increased up to 2-fold. In most cases, this was achieved at the e xpense of beta-galactosidase activity at 20 degrees C and total catalytic e fficiency at 90 degrees C. Substrate specificity was found to be affected b y many of the observed amino acid substitutions, of which only some are loc ated in the vicinity of the active site. The largest effect on substrate sp ecificity was observed with the CelB variant N415S that showed a 7.5-fold i ncrease in the ratio of p-nitrophenyl-beta-D-glucopyranoside/p-nitrophenyl- beta-D-galactopyranoside hydrolysis. This asparagine at position 415 is pre dicted to interact with active-site residues that stabilize the hydroxyl gr oup at the C4 position of the substrate, the conformation of which is equat orial in glucose-containing substrates and axial in galactose-containing su bstrates.