Eliminating hydrolytic activity without affecting the transglycosylation of a GH1 β-glucosidase
Lundemo, Pontus LU ; Karlsson, Eva Nordberg LU
and Adlercreutz, Patrick
LU
(2017)
In Applied Microbiology and Biotechnology
101(3).
p.1121-1131
- 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.
(Less)
- author
- Lundemo, Pontus
LU
; Karlsson, Eva Nordberg
LU
and Adlercreutz, Patrick
LU
- organization
- publishing date
- 2017-02
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- pH-dependent enzyme mechanism, Transglycosylation, β-glycosidase
- in
- Applied Microbiology and Biotechnology
- volume
- 101
- issue
- 3
- pages
- 1121 - 1131
- publisher
- Springer
- external identifiers
-
- scopus:84988723863
- pmid:27678115
- 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
- 2024-05-31 15:55:04
@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}},
keywords = {{pH-dependent enzyme mechanism; Transglycosylation; β-glycosidase}},
language = {{eng}},
number = {{3}},
pages = {{1121--1131}},
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}},
doi = {{10.1007/s00253-016-7833-9}},
volume = {{101}},
year = {{2017}},
}