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Protein Engineering Design and Selection 2008 21(5):319-327; doi:10.1093/protein/gzn009
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Directed evolution on the cold adapted properties of TAB5 alkaline phosphatase

Dimitris Koutsioulis1, Ellen Wang2, Maria Tzanodaskalaki3, Dimitra Nikiforaki1, Alexandra Deli1, Georges Feller4, Pirkko Heikinheimo2,5 and Vassilis Bouriotis1,3,6

1Department of Biology, Enzyme Biotechnology Group, University of Crete, PO Box 2208, Vasilika Vouton, 71409 Heraklion, Crete, Greece 2NORSTRUCT, Institute of Chemistry, University of Tromsø, N-9037 Tromsø, Norway 3 Institute of Molecular Biology and Biotechnology, PO Box 1527, Vasilika Vouton 71110, Heraklion, Crete, Greece 4Laboratory of Biochemistry, University of Liège, Institute of Chemistry B6a, B-4000 Liège-Sart Tilman, Belgium 5 Institute of Biotechnology, University of Helsinki, PO Box 65, FIN-00014 Helsinki, Finland

6 To whom correspondence should be addressed. E-mail: bouriotis{at}imbb.forth.gr (V.B.); dkoutsioulis{at}hotmail.com or dkoutsioulis{at}gmail.com (D.K.)

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.

Keywords: catalysis/directed evolution/mutagenesis/psychrophilic enzymes/thermostability

Received November 15, 2007; revised February 10, 2008; accepted February 20, 2008.


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