Poly(ethylene terephthalate) (PET) is one of the main synthetic plastics produced worldwide. The extensive use of this polymer causes several problems due to its low degradability. In this scenario, biocatalysts dawn as an alternative to enhance PET recycling. The enzymatic hydrolysis of PET results in a mixture of terephthalic acid (TPA), ethylene glycol (EG), mono-(2-hydroxyethyl) terephthalate (MHET) and bis-(2-hydroxyethyl) terephthalate (BHET) as main products. This work developed a new methodology to quantify the hydrolytic activity of biocatalysts, using BHET as a model substrate. The protocol can be used in screening enzymes for PET depolymerization reactions, amongst other applications. The very good fitting (R² = 0.993) between experimental data and the mathematical model confirmed the feasibility of the Michaelis-Menten equation to analyze the effect of BHET concentration (8–200 mmol L⁻¹) on initial hydrolysis rate catalyzed by Humicola insolens cutinase (HiC). In addition to evaluating the effects of enzyme and substrate concentration on the enzymatic hydrolysis of BHET, a novel and straightforward method for MHET synthesis was developed using an enzyme load of 0.025 g_protein g_BHET⁻¹ and BHET concentration of 60 mmol L⁻¹ at 40 °C. MHET was synthesized with high selectivity (97 %) and yield (82 %). The synthesized MHET properties were studied using differential scanning calorimetry (DSC), thermogravimetry (TGA), and proton nuclear magnetic resonance (¹H NMR), observing the high purity of the final product (86.7 %). As MHET is not available commercially, this synthesis using substrate and enzyme from open suppliers adds new perspectives to monitoring PET hydrolysis reactions.

聚对苯二甲酸乙二醇酯 (PET) 是全球生产的主要合成塑料之一。由于其低降解性，这种聚合物的广泛使用引起了几个问题。在这种情况下，生物催化剂开始成为增强 PET 回收利用的替代品。PET的酶水解产生对苯二甲酸(TPA)、乙二醇(EG)、对苯二甲酸单(2-羟乙基)酯(MHET)和对苯二甲酸双(2-羟乙基)酯(BHET)的混合物作为主要产品。这项工作开发了一种新的方法来量化生物催化剂的水解活性，使用 BHET 作为模型底物。该协议可用于筛选用于 PET 解聚反应的酶，以及其他应用。非常好的拟合（R ²= 0.993) 实验数据和数学模型之间证实了 Michaelis-Menten 方程分析 BHET 浓度 (8-200 mmol L ^{-1 ) 对}特异腐质霉角质酶 (HiC)催化的初始水解速率影响的可行性。除了评估酶和底物浓度对 BHET 酶水解的影响外，还开发了一种新的和直接的 MHET 合成方法，使用 0.025 g_蛋白质g _BHET ^{-1的酶负荷和 60 mmol L -1}的 BHET 浓度在 40 °C。MHET 的合成具有高选择性 (97%) 和产率 (82%)。使用差示扫描量热法 (DSC)、热重分析法 (TGA) 和质子核磁共振 ( ¹ H NMR) 研究合成的 MHET 性能，观察到最终产物的高纯度 (86.7%)。由于 MHET 无法在商业上获得，这种使用来自开放供应商的底物和酶的合成为监测 PET 水解反应增加了新的视角。