Eliminating hydrolytic activity without affecting the transglycosylation of a GH1 β-glucosidase.
Unveiling the determinants for transferase and hydrolase activity in glycoside hydrolases would allow using their vast diversity for creating novel transglycosylases, thereby unlocking an extensive toolbox for carbohydrate chemists. Three different amino acid substitutions at position 220 of a GH1 β-glucosidase from Thermotoga neapolitana caused an increase of the ratio of transglycosylation to hydrolysis (rs/rh) from 0.33 to 1.45-2.71. Further increase in rs/rhwas achieved by modulation of pH of the reaction medium. The wild-type enzyme had a pH optimum for both hydrolysis and transglycosylation around 6 and reduced activity at higher pH. Interestingly, the mutants had constant transglycosylation activity over a broad pH range (5-10), while the hydrolytic activity was largely eliminated at pH 10. The results demonstrate that a combination of protein engineering and medium engineering can be used to eliminate the hydrolytic activity without affecting the transglycosylation activity of a glycoside hydrolase. The underlying factors for this success are pursued, and perturbations of the catalytic acid/base in combination with flexibility are shown to be important factors.
Department of Chemistry, Biotechnology, Lund University, P.O. Box 124, SE-221 00, Lund, Sweden.
Pontus
P
Lundemo
Department of Chemistry, Biotechnology, Lund University, P.O. Box 124, SE-221 00, Lund, Sweden.
Eva Nordberg
EN
Karlsson
Department of Chemistry, Biotechnology, Lund University, P.O. Box 124, SE-221 00, Lund, Sweden. patrick.adlercreutz@biotek.lu.se.
Patrick
P
Adlercreutz
D019943:Amino Acid Substitution; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009154:Mutation; D015202:Protein Engineering; D013379:Substrate Specificity; D046949:Thermotoga neapolitana / Q000201:enzymology; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003470:Culture Media; D001617:beta-Glucosidase
Transglycosylation; pH-dependent enzyme mechanism; β-glycosidase
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