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Thermostabilization of an esterase by alignment-guided focussed directed evolution

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Abstract
Site-saturation libraries of the Pseudomonas fluorescens esterase were created targeting three surface positions to increase its thermostability on the basis of the B-factor iterative test principle. All three positions were saturated simultaneously using our recently developed protocol for the design of 'small, but smart' mutant libraries bearing only consensus-like mutations. Hence, the library size could be significantly reduced while ensuring a high hit rate. Variants could be identified that showed significantly improved stability (8 degrees C higher compared with the wild type) without compromising specific activity. Subsequent iterative saturation mutagenesis gave an esterase mutant with a 9 degrees C increased melting point, but unchanged catalytic properties.
Keywords
site-saturation mutagenesis, thermostability, Pseudomonas fluorescens esterase, directed evolution, B-FIT, DESIGN, RESIDUES, HYDROLASE, DEHYDROGENASE, STABILIZATION, ENANTIOSELECTIVITY, ENZYME STABILITY, PSEUDOMONAS-FLUORESCENS, PROTEIN STABILITY

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Citation

Please use this url to cite or link to this publication:

Chicago
Jochens, Helge, Dirk Aerts, and Uwe Bornscheuer. 2010. “Thermostabilization of an Esterase by Alignment-guided Focussed Directed Evolution.” Protein Engineering Design & Selection 23 (12): 903–909.
APA
Jochens, H., Aerts, D., & Bornscheuer, U. (2010). Thermostabilization of an esterase by alignment-guided focussed directed evolution. PROTEIN ENGINEERING DESIGN & SELECTION, 23(12), 903–909.
Vancouver
1.
Jochens H, Aerts D, Bornscheuer U. Thermostabilization of an esterase by alignment-guided focussed directed evolution. PROTEIN ENGINEERING DESIGN & SELECTION. 2010;23(12):903–9.
MLA
Jochens, Helge, Dirk Aerts, and Uwe Bornscheuer. “Thermostabilization of an Esterase by Alignment-guided Focussed Directed Evolution.” PROTEIN ENGINEERING DESIGN & SELECTION 23.12 (2010): 903–909. Print.
@article{1891142,
  abstract     = {Site-saturation libraries of the Pseudomonas fluorescens esterase were created targeting three surface positions to increase its thermostability on the basis of the B-factor iterative test principle. All three positions were saturated simultaneously using our recently developed protocol for the design of 'small, but smart' mutant libraries bearing only consensus-like mutations. Hence, the library size could be significantly reduced while ensuring a high hit rate. Variants could be identified that showed significantly improved stability (8 degrees C higher compared with the wild type) without compromising specific activity. Subsequent iterative saturation mutagenesis gave an esterase mutant with a 9 degrees C increased melting point, but unchanged catalytic properties.},
  author       = {Jochens, Helge and Aerts, Dirk and Bornscheuer, Uwe},
  issn         = {1741-0126},
  journal      = {PROTEIN ENGINEERING DESIGN \& SELECTION},
  keyword      = {site-saturation mutagenesis,thermostability,Pseudomonas fluorescens esterase,directed evolution,B-FIT,DESIGN,RESIDUES,HYDROLASE,DEHYDROGENASE,STABILIZATION,ENANTIOSELECTIVITY,ENZYME STABILITY,PSEUDOMONAS-FLUORESCENS,PROTEIN STABILITY},
  language     = {eng},
  number       = {12},
  pages        = {903--909},
  title        = {Thermostabilization of an esterase by alignment-guided focussed directed evolution},
  url          = {http://dx.doi.org/10.1093/protein/gzq071},
  volume       = {23},
  year         = {2010},
}

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