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.
Submitter: Shu-Ching Ou
Submission Date: Dec. 6, 2018, 2:35 p.m.
|Number of data points||161|
|Proteins||TAB5 alkaline phosphatase|
|Assays/Quantities/Protocols||Experimental Assay: Thermal Stability: Tm2 ; Experimental Assay: Thermal Stability: Tm3 ; Experimental Assay: Thermal Stability: Tm1 ; Experimental Assay: Kinetic Constant: Activation Energy (Eα) ; Experimental Assay: Kinetic Constant: kcat ; Experimental Assay: Remaining Activity: 60 °C for 60 min ; Experimental Assay: Remaining Activity: 60 °C for 5 min ; Experimental Assay: Remaining Activity: 55 °C for 120 min ; Experimental Assay: Remaining Activity: 55 °C for 5 min ; Experimental Assay: Thermal Stability: ΔHcal ; Derived Quantity: TΔΔS# ; Derived Quantity: ΔΔH# ; Derived Quantity: ΔΔG# ; Derived Quantity: Entropy of Activation (TΔS#) ; Derived Quantity: Enthalpy of Activation (ΔH#) ; Derived Quantity: Free Energy of Activation (ΔG#)|
|Libraries||Variants for TAB5 ; Variants for TAB5_pNPP|