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Engineering of pyranose 2-oxidase: Improvement for biofuel cell and food applications through semi-rational protein design

Spadiut, Oliver; Pisanelli, Ines; Maischberger, Thomas; Peterbauer, Clemens; Gorton, Lo LU ; Chaiyen, Pimchai and Haltrich, Dietmar (2009) In Journal of Biotechnology 139(3). p.250-257
Abstract
Pyranose 2-oxidase (P20x) has several proposed biotechnological applications such as a bio-component in biofuel cells or for carbohydrate transformations. To improve some of the catalytic properties of P20x from Trametes multicolor, we selected a semi-rational approach of enzyme engineering, saturation mutagenesis of active-site residues and subsequent screening of mutant libraries for improved activity. One of the active-site mutants with improved catalytic characteristics identified was V546C. which showed catalytic constants increased by up to 5.7-fold for both the sugar substrates (D-glucose and D-galactose) and alternative electron acceptors (1,4-benzoquinone, BQ and ferricenium ion, Fc(+)], albeit at the expense of increased... (More)
Pyranose 2-oxidase (P20x) has several proposed biotechnological applications such as a bio-component in biofuel cells or for carbohydrate transformations. To improve some of the catalytic properties of P20x from Trametes multicolor, we selected a semi-rational approach of enzyme engineering, saturation mutagenesis of active-site residues and subsequent screening of mutant libraries for improved activity. One of the active-site mutants with improved catalytic characteristics identified was V546C. which showed catalytic constants increased by up to 5.7-fold for both the sugar substrates (D-glucose and D-galactose) and alternative electron acceptors (1,4-benzoquinone, BQ and ferricenium ion, Fc(+)], albeit at the expense of increased Michaelis constants. By combining V546C with other amino acid replacements, we obtained P20x variants that are of interest for biofuel cell applications due to their increased k(cat) for both BQ and Fc(+), eg., V546C/E542K showed 4.4- and 17-fold increased k(cat) for BQ compared to the wild-type enzyme when D-glucose and D-galactose, respectively, were the saturating substrates, while V546C/T169G showed approx. 40- and 50-fold higher k(cat) for BQ and Fc(+), respectively, with D-galactose in excess. This latter variant also shows significantly modulated sugar substrate selectivity, due to an increase in k(cat)/K-M for D-galactose and a decrease in k(cat)/K-M for D-glucose when oxygen is the electron acceptor, as well as improved catalytic efficiencies for D-galactose, regardless of the electron acceptor used. While the wildtype enzyme strongly prefers D-glucose over D-galactose as its substrate, V546C/T169G converts both sugars equally well as was shown by the kinetic constants determined as well as by biotransformation experiments. (C) 2008 Elsevier B.V. All rights reserved. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Anodic bio-component, cells, Biofuel, Enzyme engineering, Pyranose oxidase, Saturation mutagenesis
in
Journal of Biotechnology
volume
139
issue
3
pages
250 - 257
publisher
Elsevier
external identifiers
  • wos:000263742300008
  • scopus:58849128590
ISSN
1873-4863
DOI
10.1016/j.jbiotec.2008.11.004
language
English
LU publication?
yes
id
83ffdd40-2991-4328-846a-d600b98e775a (old id 1371126)
date added to LUP
2009-05-08 13:51:04
date last changed
2017-07-30 03:45:19
@article{83ffdd40-2991-4328-846a-d600b98e775a,
  abstract     = {Pyranose 2-oxidase (P20x) has several proposed biotechnological applications such as a bio-component in biofuel cells or for carbohydrate transformations. To improve some of the catalytic properties of P20x from Trametes multicolor, we selected a semi-rational approach of enzyme engineering, saturation mutagenesis of active-site residues and subsequent screening of mutant libraries for improved activity. One of the active-site mutants with improved catalytic characteristics identified was V546C. which showed catalytic constants increased by up to 5.7-fold for both the sugar substrates (D-glucose and D-galactose) and alternative electron acceptors (1,4-benzoquinone, BQ and ferricenium ion, Fc(+)], albeit at the expense of increased Michaelis constants. By combining V546C with other amino acid replacements, we obtained P20x variants that are of interest for biofuel cell applications due to their increased k(cat) for both BQ and Fc(+), eg., V546C/E542K showed 4.4- and 17-fold increased k(cat) for BQ compared to the wild-type enzyme when D-glucose and D-galactose, respectively, were the saturating substrates, while V546C/T169G showed approx. 40- and 50-fold higher k(cat) for BQ and Fc(+), respectively, with D-galactose in excess. This latter variant also shows significantly modulated sugar substrate selectivity, due to an increase in k(cat)/K-M for D-galactose and a decrease in k(cat)/K-M for D-glucose when oxygen is the electron acceptor, as well as improved catalytic efficiencies for D-galactose, regardless of the electron acceptor used. While the wildtype enzyme strongly prefers D-glucose over D-galactose as its substrate, V546C/T169G converts both sugars equally well as was shown by the kinetic constants determined as well as by biotransformation experiments. (C) 2008 Elsevier B.V. All rights reserved.},
  author       = {Spadiut, Oliver and Pisanelli, Ines and Maischberger, Thomas and Peterbauer, Clemens and Gorton, Lo and Chaiyen, Pimchai and Haltrich, Dietmar},
  issn         = {1873-4863},
  keyword      = {Anodic bio-component,cells,Biofuel,Enzyme engineering,Pyranose oxidase,Saturation mutagenesis},
  language     = {eng},
  number       = {3},
  pages        = {250--257},
  publisher    = {Elsevier},
  series       = {Journal of Biotechnology},
  title        = {Engineering of pyranose 2-oxidase: Improvement for biofuel cell and food applications through semi-rational protein design},
  url          = {http://dx.doi.org/10.1016/j.jbiotec.2008.11.004},
  volume       = {139},
  year         = {2009},
}