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

• Published in: Journal of molecular catalysis. B, Enzymatic Vol. 118; pp. 62 - 69
• Main Authors: Anbarasan, Sasikala, Timoharju, Tommi, Barthomeuf, Janice, Pastinen, Ossi, Rouvinen, Juha, Leisola, Matti, Turunen, Ossi
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01-08-2015
• Subjects: Active site mutations; Cellobiose hydrolysis; pH-activity; Sulfolobus acidocaldarius; Transglycosylation; β-Glycosidase; Transglycosylation; Sulfolobus acidocaldarius; pH-activity; Active site mutations; Cellobiose hydrolysis; β-Glycosidase
• ISSN: 1381-1177; 1873-3158
• DOI: 10.1016/j.molcatb.2015.05.002

•Mutations V212T, V212D and N211D were engineered close to the acid/base catalyst.•V212T and V212D shifted the pH optimum towards acidic pH.•V212T showed elevated hydrolysis of cellobiose at high substrate concentrations.•V212T decreased remarkably the transglycosylation activity.
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.