Improved oligosaccharide synthesis by protein engineering of β-glucosidase CelB from hyperthermophilic Pyrococcus furiosus
Hansson, Therese LU ; Kaper, Thijs ; Van Oost, John Der ; De Vos, Willem M. and Adlercreutz, Patrick LU
(2001)
In Biotechnology and Bioengineering
73(3).
p.203-210
- Abstract
Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type β-glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type β-mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out in aqueous solution at 95°C at different lactose concentrations and pH values. The results showed enhanced synthetic properties of the CelB mutant enzymes. An exchange of one phenylalanine to tyrosine (F426Y) increased the oligosaccharide yield (45%) compared with the wild-type CelB (40%). Incorporation of a positively charged group in the... (More)
Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type β-glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type β-mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out in aqueous solution at 95°C at different lactose concentrations and pH values. The results showed enhanced synthetic properties of the CelB mutant enzymes. An exchange of one phenylalanine to tyrosine (F426Y) increased the oligosaccharide yield (45%) compared with the wild-type CelB (40%). Incorporation of a positively charged group in the active site (M424K) increased the pH optimum of transglycosylation reaction of CelB. The double mutant, M424K/ F426Y, showed much better transglycosylation properties at low (10-20%) lactose concentrations compared to the wild-type. At a lactose concentration of 10%, the oligosaccharide yield for the mutant was 40% compared to 18% for the wild-type. At optimal reaction conditions, a higher ratio of tetrasaccharides to trisaccharides was obtained with the double mutant (0.42, 10% lactose) compared to the wild-type (0.19, 70% lactose). At a lactose concentration as Iow as 10%, only trisaccharides were synthesized by CelB wild-type. The β-mannosidase BmnA from P. furiosus showed both β-glucosidase and β-galactosidase activity and in the transglycosylation of lactose the maximal oligosaccharide yield of BmnA was 44%. The oligosaccharide yields obtained in this study are high compared to those reported with other transglycosylating β-glycosidases in oligosaccharide synthesis from lactose.
(Less)
- author
- Hansson, Therese
LU
; Kaper, Thijs
; Van Oost, John Der
; De Vos, Willem M.
and Adlercreutz, Patrick
LU
- organization
- publishing date
- 2001-05-05
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- β-glycosidase, Hyperthermophilic, Lactose, Oligosaccharides, Protein engineering, Transglycosylation
- in
- Biotechnology and Bioengineering
- volume
- 73
- issue
- 3
- pages
- 8 pages
- publisher
- John Wiley & Sons Inc.
- external identifiers
-
- pmid:11257602
- scopus:0035810688
- ISSN
- 0006-3592
- DOI
- 10.1002/bit.1052
- language
- English
- LU publication?
- yes
- id
- 18c38cc7-6097-407c-9e90-c424fceef50a
- date added to LUP
- 2019-06-20 15:46:48
- date last changed
- 2024-01-01 11:10:08
@article{18c38cc7-6097-407c-9e90-c424fceef50a,
abstract = {{<p>Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type β-glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type β-mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out in aqueous solution at 95°C at different lactose concentrations and pH values. The results showed enhanced synthetic properties of the CelB mutant enzymes. An exchange of one phenylalanine to tyrosine (F426Y) increased the oligosaccharide yield (45%) compared with the wild-type CelB (40%). Incorporation of a positively charged group in the active site (M424K) increased the pH optimum of transglycosylation reaction of CelB. The double mutant, M424K/ F426Y, showed much better transglycosylation properties at low (10-20%) lactose concentrations compared to the wild-type. At a lactose concentration of 10%, the oligosaccharide yield for the mutant was 40% compared to 18% for the wild-type. At optimal reaction conditions, a higher ratio of tetrasaccharides to trisaccharides was obtained with the double mutant (0.42, 10% lactose) compared to the wild-type (0.19, 70% lactose). At a lactose concentration as Iow as 10%, only trisaccharides were synthesized by CelB wild-type. The β-mannosidase BmnA from P. furiosus showed both β-glucosidase and β-galactosidase activity and in the transglycosylation of lactose the maximal oligosaccharide yield of BmnA was 44%. The oligosaccharide yields obtained in this study are high compared to those reported with other transglycosylating β-glycosidases in oligosaccharide synthesis from lactose.</p>}},
author = {{Hansson, Therese and Kaper, Thijs and Van Oost, John Der and De Vos, Willem M. and Adlercreutz, Patrick}},
issn = {{0006-3592}},
keywords = {{β-glycosidase; Hyperthermophilic; Lactose; Oligosaccharides; Protein engineering; Transglycosylation}},
language = {{eng}},
month = {{05}},
number = {{3}},
pages = {{203--210}},
publisher = {{John Wiley & Sons Inc.}},
series = {{Biotechnology and Bioengineering}},
title = {{Improved oligosaccharide synthesis by protein engineering of β-glucosidase CelB from hyperthermophilic Pyrococcus furiosus}},
url = {{http://dx.doi.org/10.1002/bit.1052}},
doi = {{10.1002/bit.1052}},
volume = {{73}},
year = {{2001}},
}