L2455 dad detector

The PET degradation rates for the original DuraPETase and its variants were tested using amorphous PET (2.4% crystallinity, determined by differential scanning calorimetry) with a thickness of 0.25 mm purchased from Goodfellow Cambridge Limited (Huntingdon, England). Substrate cut-outs with a diameter of 6 mm were incubated in 300 µl of 50 mM bicine NaOH buffer pH 9 containing 100 nM of the respective enzyme in microvolume reaction tubes. All reactions were performed as triplicates and were incubated at 50 °C or 52 °C and 300 rpm for 3 days. The reactions were stopped by removing the substrate. Three volumes of acetonitrile were added, and a centrifugation step (20,000 × g, 10 min) was performed. For each sample, 10 µl of the supernatant was injected into a LaChrom Elite HPLC system equipped with a L-2455 DAD detector (Hitachi, Chiyoda, Japan) and a Nucleodur C18 HTec column (Macherey–Nagel, Düren, Germany). The separation was performed isocratically at 40 °C with a flow rate of 0.5 ml/min for 8 min using 70% ddH₂O, 20% acetonitrile, and 10% formic acid. The signal was detected at 254 nm, and standards of the reaction products terephthalic acid (TCI, Tokyo, Japan) and 2-hydroxyethyl terephthalic acid (Activate Scientific, Prien am Chiemsee, Germany) were used to quantify the degradation rate.