Boros Krisztina, Nagy Blanka Eszter, Tomoiagă Raluca Bianca, Tőtős Róbert, Toșa Monica Ioana, Paizs Csaba, Bencze László Csaba
Enzymology and Applied Biocatalysis Research Center, Faculty of Chemistry and Chemical Engineering, Babeș-Bolyai University, Arany János Street 11, 400028, Cluj-Napoca, Romania.
Sci Rep. 2025 Jan 13;15(1):1877. doi: 10.1038/s41598-024-84177-7.
Efficient monitoring of the enzymatic PET-hydrolysis is crucial for developing novel plastic-degrading biocatalysts. Herein, we aimed to upgrade in terms of accuracy the analytical methods useful for monitoring enzymatic PET-degradation. For the HPLC-based assessment, the incorporation of an internal standard within the analytic procedure enabled a more accurate quantification of the overall TPA content and the assessment of molar distributions and relative content of each aromatic degradation product. The provided calibration curves cover a broad concentration range, from µM to low mM scale, facilitating assessment of both lower and higher PETase activities, with a limit of detection positioned below the reported PET-degrading activities. The increased reproducibility and accuracy of the improved HPLC method, compared to the previous methods, was supported by lower dispersion of product concentrations and their lower deviation from theoretical values over multiple measurements. The other predominantly employed UV-spectroscopy assay was also improved in terms of employed wavelength and medium extinction coefficient of the three aromatic degradation products, while being cross-validated by the improved HPLC method. Finally, both methods were used for monitoring the product formation within the leaf-branch compost cutinase (LCC)-mediated PET-hydrolysis and provided individual time-productivity profiles for each aromatic degradation product.
高效监测酶促PET水解对于开发新型塑料降解生物催化剂至关重要。在此，我们旨在提高用于监测酶促PET降解的分析方法的准确性。对于基于HPLC的评估，在分析过程中加入内标能够更准确地定量总TPA含量，并评估每种芳香族降解产物的摩尔分布和相对含量。所提供的校准曲线涵盖了从微摩尔到低毫摩尔范围的宽浓度区间，便于评估较低和较高的PETase活性，检测限低于报道的PET降解活性。与先前方法相比，改进后的HPLC方法具有更高的重现性和准确性，这体现在多次测量中产物浓度的离散度更低以及与理论值的偏差更小。另一种主要使用的紫外光谱法在所用波长和三种芳香族降解产物的介质消光系数方面也得到了改进，同时通过改进后的HPLC方法进行了交叉验证。最后，两种方法都用于监测叶枝堆肥角质酶（LCC）介导的PET水解过程中的产物形成，并为每种芳香族降解产物提供了各自的时间-生产率曲线。
