Effect of active site mutation on pH activity and transglycosylation of Sulfolobus acidocaldarius β-glycosidase

Abstract

Sulfolobus acidocaldarius β-glycosidase (BGAL_SULAC) was used as an extremophilic enzyme model to study the effect of mutations close to the catalytic residues on the enzyme activity and the pH-activity profile. We report here the results for three mutations (N211D, V212D and V212T) changing the polarity close to the putative acid/base catalyst E209. N211D was outside the H-bonding distance from E209, whereas V212D and V212T were in H-bonding distance from E209. V212D and V212T shifted the pH-activity profile towards acidic pH with both lactose and cellobiose as substrates. N211D and V212D decreased clearly the activity. Although V212T increased 6-fold the Km value with cellobiose, the mutant showed higher specific activity in high substrate concentrations. The reason was greatly reduced production of trisaccharide by V212T from cellobiose by transglycosylation. Threonine differs by the terminal oxygen from valine, indicating that additional hydrogen bonding to substrate or reaction products may affect the reaction behavior of the enzyme. Although the mutations in the active site are often harmful, the mutation V212T showed biotechnologically promising properties.