Eliminating hydrolytic activity without affecting the transglycosylation of a GH1 β-glucosidase

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Eliminating hydrolytic activity without affecting the transglycosylation of a GH1 β-glucosidase. / Lundemo, Pontus; Karlsson, Eva Nordberg; Adlercreutz, Patrick.

I: Applied Microbiology and Biotechnology, Vol. 101, Nr. 3, 02.2017, s. 1121-1131.

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T1 - Eliminating hydrolytic activity without affecting the transglycosylation of a GH1 β-glucosidase

AU - Lundemo, Pontus

AU - Karlsson, Eva Nordberg

AU - Adlercreutz, Patrick

PY - 2017/2

Y1 - 2017/2

N2 - 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/rh was 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.

AB - 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/rh was 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.

KW - pH-dependent enzyme mechanism

KW - Transglycosylation

KW - β-glycosidase

UR - http://www.scopus.com/inward/record.url?scp=84988723863&partnerID=8YFLogxK

U2 - 10.1007/s00253-016-7833-9

DO - 10.1007/s00253-016-7833-9

M3 - Article

C2 - 27678115

AN - SCOPUS:84988723863

VL - 101

SP - 1121

EP - 1131

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

SN - 1432-0614

IS - 3

ER -