Effect of pH and temperature on activity and stability of wild-type BceGO and evolved variant B3S1.

<p><b>A</b>. The optimal pH. Enzyme activity was determined with 100 mM glyphosate at 25 °C and within a pH gradient range of 4.0~11.0 with the following buffers: 0.2 mM Na₂HPO₄-0.1 mM citric acid buffer for pH 4.0~8.0, and 50 mM sodium pyrophosphate buffer for pH 8.0~11.0. The maximum activity observed was taken as 100%. <b>B</b>. The pH stability. Enzymes were incubated at 0 °C for 6 h over a pH buffer range of 4.0~11.0, then the enzyme activity was determined with 100 mM glyphosate at 25 °C and the optimal pH. The maximum activity observed was taken as 100%. <b>C</b>. The optimal temperature. The enzymes were added to the reaction mixture and the reaction was carried out at an indicated temperature from 0 to 70 °C. Then the enzyme activity was determined with 100 mM glyphosate at 25 °C and the optimal pH. The maximum activity observed was taken as 100%. <b>D</b>. The temperature stability. Enzymes were incubated for 1h at indicated temperature from 0 to 70 °C and then the enzyme activity was determined with 100 mM glyphosate at 25 °C and in the optimal pH. The activity without treatment was taken as 100%. Error bars represent the SD of the mean calculated for three replicates. <i>Solid </i><i>dots</i> represent wild-type BceGO, <i>solid </i><i>blocks</i> represent variant B3S1.