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

Research output: Contribution to journalArticle

Abstract

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.

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Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Biochemistry and Molecular Biology
  • Biocatalysis and Enzyme Technology

Keywords

  • pH-dependent enzyme mechanism, Transglycosylation, β-glycosidase
Original languageEnglish
Pages (from-to)1121-1131
JournalApplied Microbiology and Biotechnology
Volume101
Issue number3
Early online date2016 Sep 27
Publication statusPublished - 2017 Feb
Publication categoryResearch
Peer-reviewedYes