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

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• The beta-glucosidase (CelB) from Pyrococcus furiosus is a highly thermostable and active family 1 glycosylhydrolase.
• Understanding molecular adaptations to high temperatures is crucial for enzyme engineering.

Purpose of the Study:

• To investigate molecular determinants of high-temperature adaptation in CelB.
• To optimize the low-temperature activity of the hyperthermostable CelB enzyme.

Main Methods:

• Generated a random mutant library of CelB in Escherichia coli.
• Screened mutants for increased activity on p-nitrophenyl-beta-D-glucopyranoside at room temperature.
• Detailed characterization of selected CelB variants, including kinetic analysis and substrate specificity determination.

Main Results:

• Identified multiple CelB variants with up to 3-fold increased aryl glucoside hydrolysis rates at room temperature.
• Amino acid substitutions were found in various regions, including the active site and enzyme surface.
• Mutants exhibited altered substrate specificity and increased flexibility, sometimes at the expense of high-temperature activity.

Conclusions:

• Enzyme engineering can enhance low-temperature activity of hyperthermostable enzymes like CelB.
• Modifications in active-site and surface residues can significantly impact enzyme kinetics and substrate specificity.
• Specific mutations, like N415S, dramatically alter substrate preference, demonstrating targeted optimization potential.