Directed evolution on the cold adapted properties of TAB5 alkaline phosphatase
peer reviewed Psychrophilic alkaline phosphatase (AP) from the Antarctic strain TAB5 was subjected to directed evolution in order to identify the key residues steering the enzyme's cold-adapted activity and stability. A round of random mutagenesis and further recombination yielded three thermos...
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Oxford University Press
2008
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Online Access: | https://orbi.uliege.be/handle/2268/16631 https://orbi.uliege.be/bitstream/2268/16631/1/PEDS_2008_AP.pdf https://doi.org/10.1093/protein/gzn009 |
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ftorbi:oai:orbi.ulg.ac.be:2268/16631 2024-10-13T14:03:16+00:00 Directed evolution on the cold adapted properties of TAB5 alkaline phosphatase Koutsioulis, D. Wang, E. Tzanodaskalaki, M. Nikiforaki, D. Deli, A. Feller, Georges Heikinheimo, P. Bouriotis, V. 2008 https://orbi.uliege.be/handle/2268/16631 https://orbi.uliege.be/bitstream/2268/16631/1/PEDS_2008_AP.pdf https://doi.org/10.1093/protein/gzn009 en eng Oxford University Press urn:issn:1741-0126 urn:issn:1741-0134 https://orbi.uliege.be/handle/2268/16631 info:hdl:2268/16631 https://orbi.uliege.be/bitstream/2268/16631/1/PEDS_2008_AP.pdf doi:10.1093/protein/gzn009 info:pmid:18411226 open access http://purl.org/coar/access_right/c_abf2 info:eu-repo/semantics/openAccess Protein Engineering, Design and Selection, 21 (5), 319-27 (2008) Alkaline Phosphatase/*chemistry/genetics Binding Sites Calorimetry Differential Scanning Dimerization Directed Molecular Evolution Kinetics Mutagenesis Site-Directed Mutation Protein Conformation Protein Denaturation Protein Engineering/*methods Protein Folding Recombination Genetic Temperature Thermodynamics Life sciences Biochemistry biophysics & molecular biology Sciences du vivant Biochimie biophysique & biologie moléculaire journal article http://purl.org/coar/resource_type/c_6501 info:eu-repo/semantics/article peer reviewed 2008 ftorbi https://doi.org/10.1093/protein/gzn009 2024-09-27T07:02:04Z peer reviewed Psychrophilic alkaline phosphatase (AP) from the Antarctic strain TAB5 was subjected to directed evolution in order to identify the key residues steering the enzyme's cold-adapted activity and stability. A round of random mutagenesis and further recombination yielded three thermostable and six thermolabile variants of the TAB5 AP. All of the isolated variants were characterised by their residual activity after heat treatment, Michaelis-Menten kinetics, activation energy and microcalorimetric parameters of unfolding. In addition, they were modelled into the structure of the TAB5 AP. Mutations which affected the cold-adapted properties of the enzyme were all located close to the active site. The destabilised variants H135E and H135E/G149D had 2- and 3-fold higher kcat, respectively, than the wild-type enzyme. Wild-type AP has a complex heat-induced unfolding pattern while the mutated enzymes loose local unfolding transitions and have large shifts of the Tm values. Comparison of the wild-type and mutated TAB5 APs demonstrates that there is a delicate balance between the enzyme activity and stability and that it is possible to improve the activity and thermostability simultaneously as demonstrated in the case of the H135E/G149D variant compared to H135E. Article in Journal/Newspaper Antarc* Antarctic University of Liège: ORBi (Open Repository and Bibliography) Antarctic The Antarctic Protein Engineering Design and Selection 21 5 319 327 |
institution |
Open Polar |
collection |
University of Liège: ORBi (Open Repository and Bibliography) |
op_collection_id |
ftorbi |
language |
English |
topic |
Alkaline Phosphatase/*chemistry/genetics Binding Sites Calorimetry Differential Scanning Dimerization Directed Molecular Evolution Kinetics Mutagenesis Site-Directed Mutation Protein Conformation Protein Denaturation Protein Engineering/*methods Protein Folding Recombination Genetic Temperature Thermodynamics Life sciences Biochemistry biophysics & molecular biology Sciences du vivant Biochimie biophysique & biologie moléculaire |
spellingShingle |
Alkaline Phosphatase/*chemistry/genetics Binding Sites Calorimetry Differential Scanning Dimerization Directed Molecular Evolution Kinetics Mutagenesis Site-Directed Mutation Protein Conformation Protein Denaturation Protein Engineering/*methods Protein Folding Recombination Genetic Temperature Thermodynamics Life sciences Biochemistry biophysics & molecular biology Sciences du vivant Biochimie biophysique & biologie moléculaire Koutsioulis, D. Wang, E. Tzanodaskalaki, M. Nikiforaki, D. Deli, A. Feller, Georges Heikinheimo, P. Bouriotis, V. Directed evolution on the cold adapted properties of TAB5 alkaline phosphatase |
topic_facet |
Alkaline Phosphatase/*chemistry/genetics Binding Sites Calorimetry Differential Scanning Dimerization Directed Molecular Evolution Kinetics Mutagenesis Site-Directed Mutation Protein Conformation Protein Denaturation Protein Engineering/*methods Protein Folding Recombination Genetic Temperature Thermodynamics Life sciences Biochemistry biophysics & molecular biology Sciences du vivant Biochimie biophysique & biologie moléculaire |
description |
peer reviewed Psychrophilic alkaline phosphatase (AP) from the Antarctic strain TAB5 was subjected to directed evolution in order to identify the key residues steering the enzyme's cold-adapted activity and stability. A round of random mutagenesis and further recombination yielded three thermostable and six thermolabile variants of the TAB5 AP. All of the isolated variants were characterised by their residual activity after heat treatment, Michaelis-Menten kinetics, activation energy and microcalorimetric parameters of unfolding. In addition, they were modelled into the structure of the TAB5 AP. Mutations which affected the cold-adapted properties of the enzyme were all located close to the active site. The destabilised variants H135E and H135E/G149D had 2- and 3-fold higher kcat, respectively, than the wild-type enzyme. Wild-type AP has a complex heat-induced unfolding pattern while the mutated enzymes loose local unfolding transitions and have large shifts of the Tm values. Comparison of the wild-type and mutated TAB5 APs demonstrates that there is a delicate balance between the enzyme activity and stability and that it is possible to improve the activity and thermostability simultaneously as demonstrated in the case of the H135E/G149D variant compared to H135E. |
format |
Article in Journal/Newspaper |
author |
Koutsioulis, D. Wang, E. Tzanodaskalaki, M. Nikiforaki, D. Deli, A. Feller, Georges Heikinheimo, P. Bouriotis, V. |
author_facet |
Koutsioulis, D. Wang, E. Tzanodaskalaki, M. Nikiforaki, D. Deli, A. Feller, Georges Heikinheimo, P. Bouriotis, V. |
author_sort |
Koutsioulis, D. |
title |
Directed evolution on the cold adapted properties of TAB5 alkaline phosphatase |
title_short |
Directed evolution on the cold adapted properties of TAB5 alkaline phosphatase |
title_full |
Directed evolution on the cold adapted properties of TAB5 alkaline phosphatase |
title_fullStr |
Directed evolution on the cold adapted properties of TAB5 alkaline phosphatase |
title_full_unstemmed |
Directed evolution on the cold adapted properties of TAB5 alkaline phosphatase |
title_sort |
directed evolution on the cold adapted properties of tab5 alkaline phosphatase |
publisher |
Oxford University Press |
publishDate |
2008 |
url |
https://orbi.uliege.be/handle/2268/16631 https://orbi.uliege.be/bitstream/2268/16631/1/PEDS_2008_AP.pdf https://doi.org/10.1093/protein/gzn009 |
geographic |
Antarctic The Antarctic |
geographic_facet |
Antarctic The Antarctic |
genre |
Antarc* Antarctic |
genre_facet |
Antarc* Antarctic |
op_source |
Protein Engineering, Design and Selection, 21 (5), 319-27 (2008) |
op_relation |
urn:issn:1741-0126 urn:issn:1741-0134 https://orbi.uliege.be/handle/2268/16631 info:hdl:2268/16631 https://orbi.uliege.be/bitstream/2268/16631/1/PEDS_2008_AP.pdf doi:10.1093/protein/gzn009 info:pmid:18411226 |
op_rights |
open access http://purl.org/coar/access_right/c_abf2 info:eu-repo/semantics/openAccess |
op_doi |
https://doi.org/10.1093/protein/gzn009 |
container_title |
Protein Engineering Design and Selection |
container_volume |
21 |
container_issue |
5 |
container_start_page |
319 |
op_container_end_page |
327 |
_version_ |
1812819686543327232 |