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Increasing the thermostability of sucrose phosphorylase by a combination of sequence- and structure-based mutagenesis

An Cerdobbel (UGent) , Karel De Winter (UGent) , Dirk Aerts (UGent) , Remko Kuipers, Henk-Jan Joosten, Wim Soetaert (UGent) and Tom Desmet (UGent)
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Biotechnology for a sustainable economy (Bio-Economy)
Abstract
Sucrose phosphorylase is a promising biocatalyst for the glycosylation of a wide variety of acceptor molecules, but its low thermostability is a serious drawback for industrial applications. In this work, the stability of the enzyme from Bifidobacterium adolescentis has been significantly improved by a combination of smart and rational mutagenesis. The former consists of substituting the most flexible residues with amino acids that occur more frequently at the corresponding positions in related sequences, while the latter is based on a careful inspection of the enzyme's crystal structure to promote electrostatic interactions. In this way, a variant enzyme could be created that contains six mutations and whose half-life at the industrially relevant temperature of 60 degrees C has more than doubled compared with the wild-type enzyme. An increased stability in the presence of organic co-solvents could also be observed, although these effects were most noticeable at low temperatures.
Keywords
DIRECTED EVOLUTION, ENHANCED PROTEIN THERMOSTABILITY, thermostability, sucrose phosphorylase, enzyme engineering, solvent stability, RATIONAL DESIGN, STABILITY, ENZYME, MUTATIONS

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Chicago
Cerdobbel, An, Karel De Winter, Dirk Aerts, Remko Kuipers, Henk-Jan Joosten, Wim Soetaert, and Tom Desmet. 2011. “Increasing the Thermostability of Sucrose Phosphorylase by a Combination of Sequence- and Structure-based Mutagenesis.” Protein Engineering Design & Selection 24 (11): 829–834.
APA
Cerdobbel, A., De Winter, K., Aerts, D., Kuipers, R., Joosten, H.-J., Soetaert, W., & Desmet, T. (2011). Increasing the thermostability of sucrose phosphorylase by a combination of sequence- and structure-based mutagenesis. PROTEIN ENGINEERING DESIGN & SELECTION, 24(11), 829–834.
Vancouver
1.
Cerdobbel A, De Winter K, Aerts D, Kuipers R, Joosten H-J, Soetaert W, et al. Increasing the thermostability of sucrose phosphorylase by a combination of sequence- and structure-based mutagenesis. PROTEIN ENGINEERING DESIGN & SELECTION. 2011;24(11):829–34.
MLA
Cerdobbel, An, Karel De Winter, Dirk Aerts, et al. “Increasing the Thermostability of Sucrose Phosphorylase by a Combination of Sequence- and Structure-based Mutagenesis.” PROTEIN ENGINEERING DESIGN & SELECTION 24.11 (2011): 829–834. Print.
@article{2121933,
  abstract     = {Sucrose phosphorylase is a promising biocatalyst for the glycosylation of a wide variety of acceptor molecules, but its low thermostability is a serious drawback for industrial applications. In this work, the stability of the enzyme from Bifidobacterium adolescentis has been significantly improved by a combination of smart and rational mutagenesis. The former consists of substituting the most flexible residues with amino acids that occur more frequently at the corresponding positions in related sequences, while the latter is based on a careful inspection of the enzyme's crystal structure to promote electrostatic interactions. In this way, a variant enzyme could be created that contains six mutations and whose half-life at the industrially relevant temperature of 60 degrees C has more than doubled compared with the wild-type enzyme. An increased stability in the presence of organic co-solvents could also be observed, although these effects were most noticeable at low temperatures.},
  author       = {Cerdobbel, An and De Winter, Karel and Aerts, Dirk and Kuipers, Remko and Joosten, Henk-Jan and Soetaert, Wim and Desmet, Tom},
  issn         = {1741-0126},
  journal      = {PROTEIN ENGINEERING DESIGN \& SELECTION},
  keyword      = {DIRECTED EVOLUTION,ENHANCED PROTEIN THERMOSTABILITY,thermostability,sucrose phosphorylase,enzyme engineering,solvent stability,RATIONAL DESIGN,STABILITY,ENZYME,MUTATIONS},
  language     = {eng},
  number       = {11},
  pages        = {829--834},
  title        = {Increasing the thermostability of sucrose phosphorylase by a combination of sequence- and structure-based mutagenesis},
  url          = {http://dx.doi.org/10.1093/protein/gzr042},
  volume       = {24},
  year         = {2011},
}

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