Figure 2.

Validation of the absorbance method to measure IsPETase activity. (A) Absorbance profile of TPA and MHET as measured by NanoDrop 1000 (based on path length of 1 cm). Each concentration was measured in triplicate and error bars show standard error of mean (SEM). (B) HPLC-determined product concentrations compared to bulk absorbance estimation using the MHET extinction coefficient for the wild-type (WT) and TS-PETase. Reactions were performed at 30 °C for six days. Bars represent HPLC results, pink triangles are product concentrations estimated from bulk absorbance at A₂₆₀. All points represent mean (n ≥ 3) and error bars show SEM. Percentage of additional product estimated by bulk absorbance is indicated above each enzyme concentration (in terms of total estimated product by bulk absorbance). (C) Fold-difference in total products for data presented in panel B between concentrations and between IsPETase variants as measured by HPLC or bulk A₂₆₀. “Condition 1” to “Condition 2” indicates that fold-difference was calculated as mean of “Condition 1” divided by mean of “Condition 2”. Standard deviation was calculated using ratio distribution. (D) Initial rates of IsPETase variants incubated with PET film as measured by HPLC and A₂₆₀ (values based on path length of 1 cm) at 30 °C. Triplicate enzyme reactions were performed for each time point and variant; error bars show standard error of the mean. At each time point, the products in the supernatant of the same reaction were measured by both HPLC and bulk absorbance. The interpolated rates from four time points based on HPLC and A₂₆₀ are presented for (i) 40 and 60 nM of wild-type IsPETase (WT) and (ii) 40 nM of WT and TS-PETase. Fold-difference between reaction rates determined by HPLC or A₂₆₀ are included for each panel, calculated as “rate of condition 1” divided by “rate of condition 2” for each method. All enzyme reactions were performed at 30 °C. Amounts of TPA, MHET, and BHET can be found in Supplementary Table 1.