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

Lundemo, Pontus LU ; Karlsson, Eva Nordberg LU and Adlercreutz, Patrick LU (2016) In Applied Microbiology and Biotechnology p.1-11
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... (More)

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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author
organization
publishing date
type
Contribution to journal
publication status
epub
subject
keywords
pH-dependent enzyme mechanism, Transglycosylation, β-glycosidase
in
Applied Microbiology and Biotechnology
pages
11 pages
publisher
Springer
external identifiers
  • scopus:84988723863
  • wos:000392502600018
ISSN
0175-7598
DOI
10.1007/s00253-016-7833-9
language
English
LU publication?
yes
id
43a90a6d-8880-40ee-af3a-0e066852137e
date added to LUP
2016-10-31 13:31:11
date last changed
2017-09-18 11:28:48
@article{43a90a6d-8880-40ee-af3a-0e066852137e,
  abstract     = {<p>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 (r<sub>s</sub>/r<sub>h</sub>) from 0.33 to 1.45–2.71. Further increase in r<sub>s</sub>/r<sub>h</sub> 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.</p>},
  author       = {Lundemo, Pontus and Karlsson, Eva Nordberg and Adlercreutz, Patrick},
  issn         = {0175-7598},
  keyword      = {pH-dependent enzyme mechanism,Transglycosylation,β-glycosidase},
  language     = {eng},
  month        = {09},
  pages        = {1--11},
  publisher    = {Springer},
  series       = {Applied Microbiology and Biotechnology},
  title        = {Eliminating hydrolytic activity without affecting the transglycosylation of a GH1 β-glucosidase},
  url          = {http://dx.doi.org/10.1007/s00253-016-7833-9},
  year         = {2016},
}