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Enhanced transglucosylation/hydrolysis ratio of mutants of Pyrococcus furiosus β-glucosidase : Effects of donor concentration, water content, and temperature on activity and selectivity in hexanol

Hansson, Therese LU and Adlercreutz, Patrick LU (2001) In Biotechnology and Bioengineering 75(6). p.656-665
Abstract

The transglucosylation reaction catalyzed by wild-type β-glucosidase CelB from hyperthermophilic Pyrococcus furiosus and active site mutants (M424K, F426Y, M424K/F426Y) was studied. The conversion of pentyl-β-glucoside to hexyl-β-glucoside in hexanol was used as a model transglucosylation reaction. Hydrolysis to glucose was a side reaction. The activity (rates of hydrolysis and transglucosylation) and the selectivity (S value) were measured as a function of pentyl-β-glucoside concentration (5-240 mM), water content (1-100% v/v), and temperature (50-95°C). All mutants had lower activity than the wild-type enzyme, but they had higher selectivity, which means that they provided a higher ratio of transglucosylation product to hydrolysis... (More)

The transglucosylation reaction catalyzed by wild-type β-glucosidase CelB from hyperthermophilic Pyrococcus furiosus and active site mutants (M424K, F426Y, M424K/F426Y) was studied. The conversion of pentyl-β-glucoside to hexyl-β-glucoside in hexanol was used as a model transglucosylation reaction. Hydrolysis to glucose was a side reaction. The activity (rates of hydrolysis and transglucosylation) and the selectivity (S value) were measured as a function of pentyl-β-glucoside concentration (5-240 mM), water content (1-100% v/v), and temperature (50-95°C). All mutants had lower activity than the wild-type enzyme, but they had higher selectivity, which means that they provided a higher ratio of transglucosylation product to hydrolysis product. The largest increase in S-value (2.6 fold) was obtained by the F426Y mutant, which resulted in increased hexyl-β-glucoside yield from 56% to 69%. In addition, the F426Y enzyme had higher selectivity over the wide range of temperatures tested. The activity of CelB wild-type and CelB F426Y increased as a function of water activity (aw), and complete activation by the water was obtained in a two-phase system with 20% water phase. In contrast to CelB wild-type, the F426Y mutant had transferase activity as low as aw = 0.29. Surprisingly, the S value increased with increasing water activity up to aw = 0.92. At still higher water content the S value decreased.

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Contribution to journal
publication status
published
subject
keywords
βglucosidase, Activity, Hexanol, Selectivity, Transglucosylation
in
Biotechnology and Bioengineering
volume
75
issue
6
pages
10 pages
publisher
John Wiley and Sons
external identifiers
  • pmid:11745143
  • scopus:0035924115
ISSN
0006-3592
DOI
10.1002/bit.10043
language
English
LU publication?
yes
id
5ab4523a-b7dd-45a5-b73a-7e1c79fea249
date added to LUP
2019-06-20 15:48:18
date last changed
2021-06-08 04:57:26
@article{5ab4523a-b7dd-45a5-b73a-7e1c79fea249,
  abstract     = {<p>The transglucosylation reaction catalyzed by wild-type β-glucosidase CelB from hyperthermophilic Pyrococcus furiosus and active site mutants (M424K, F426Y, M424K/F426Y) was studied. The conversion of pentyl-β-glucoside to hexyl-β-glucoside in hexanol was used as a model transglucosylation reaction. Hydrolysis to glucose was a side reaction. The activity (rates of hydrolysis and transglucosylation) and the selectivity (S value) were measured as a function of pentyl-β-glucoside concentration (5-240 mM), water content (1-100% v/v), and temperature (50-95°C). All mutants had lower activity than the wild-type enzyme, but they had higher selectivity, which means that they provided a higher ratio of transglucosylation product to hydrolysis product. The largest increase in S-value (2.6 fold) was obtained by the F426Y mutant, which resulted in increased hexyl-β-glucoside yield from 56% to 69%. In addition, the F426Y enzyme had higher selectivity over the wide range of temperatures tested. The activity of CelB wild-type and CelB F426Y increased as a function of water activity (a<sub>w</sub>), and complete activation by the water was obtained in a two-phase system with 20% water phase. In contrast to CelB wild-type, the F426Y mutant had transferase activity as low as a<sub>w</sub> = 0.29. Surprisingly, the S value increased with increasing water activity up to a<sub>w</sub> = 0.92. At still higher water content the S value decreased.</p>},
  author       = {Hansson, Therese and Adlercreutz, Patrick},
  issn         = {0006-3592},
  language     = {eng},
  month        = {12},
  number       = {6},
  pages        = {656--665},
  publisher    = {John Wiley and Sons},
  series       = {Biotechnology and Bioengineering},
  title        = {Enhanced transglucosylation/hydrolysis ratio of mutants of Pyrococcus furiosus β-glucosidase : Effects of donor concentration, water content, and temperature on activity and selectivity in hexanol},
  url          = {http://dx.doi.org/10.1002/bit.10043},
  doi          = {10.1002/bit.10043},
  volume       = {75},
  year         = {2001},
}