| Summary: | Psychrophilic alkaline phosphatase from the Antarctic strain TAB5 is a typical cold adapted enzyme exhibiting higher catalytic efficiencies at low and moderate temperatures, along with remarkable thermolability. In an effort to investigate the evolutionary process and the molecular basis of cold adaptation, we have used directed evolution to engineer the cold adapted enzyme. A round of error-prone PCR and screening 13000 clones yielded three thermostable and three thermolabile mutants. Three libraries were produced by in vitro recombination of the selected mutations in different combinations. After screening, three thermolabile double mutants were selected. The combination of mutations S86A and G87A was achieved through site-directed mutagenesis in order to see if there was any additive effect concerning thermostability. Selected mutants exhibiting different temperature adaptations than the wt were characterized by the calculation of activation parameters. Destabilized mutants H135E and H135E/G149D exhibited increased kcat values at 30oC by 2 and 3 fold respectively when compared to native enzyme. Mutants S42G, S338T, S42G/S338T and S42G/H135E were also destabilized but appeared less active. This observation demonstrates that several structural constraints should be satisfied to maintain optimal activity. All stabilized variants (S86A, G87A, S86A/G87A and G149D) appeared less active compared to wt protein. As demonstrated by differential scanning calorimetry the engineered mutations exhibited a complete disappearance of unfolding transitions and large shifts of the Tm values. Stabilized mutants were characterized by increased Tm1 and Tm2 as compared to that of the wild type AP. On the contrary, destabilized mutants appeared to have completely lost the unfolding intermediates around 60 oC. Except from S86A/G87A the rest of the mutants with higher Tm1 and Tm2 have no significant variation of ΔHcal. This value is drastically decreased for the mutants with lower Tm values. Comparison of the mutated and the wild type ...
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