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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Bibliographic Details
Published in:Protein Engineering Design and Selection
Main Authors: Koutsioulis, D., Wang, E., Tzanodaskalaki, M., Nikiforaki, D., Deli, A., Feller, Georges, Heikinheimo, P., Bouriotis, V.
Format: Article in Journal/Newspaper
Language:English
Published: Oxford University Press 2008
Subjects:
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
Antarc*
Antarctic
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
Description
Summary: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.

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