Polyethylene terephthalate (PET) is one of the most widely produced man-made polymers and is a significant contributor to microplastics pollution. The environmental and human health impacts of microplastics pollution have motivated a concerted effort to develop microbe- and enzyme-based strategies to degrade PET and similar plastics. A PETase derived from the bacteria Ideonella sakaiensis was previously shown to enzymatically degrade PET, triggering multidisciplinary efforts to improve the robustness and activity of this and other PETases. However, because these enzymes only erode the surface of the insoluble PET substrate, it is difficult to measure standard kinetic parameters, such as kon, koff, and kcat, complicating interpretation of the activity of mutants using traditional enzyme kinetics frameworks. To address this challenge, we developed a single-molecule microscopy assay that quantifies the landing rate and binding duration of quantum dot-labeled PETase enzymes interacting with a surface-immobilized PET film. Wild-type PETase binding durations were well fit by a biexponential with a fast population having a 2.7 s time constant, interpreted as active binding events, and a slow population interpreted as nonspecific binding interactions that last tens of seconds. A previously described hyperactive mutant, S238F/W159H had both a faster apparent on-rate and a slower off-rate than wild-type PETase, potentially explaining its enhanced activity. Because this single-molecule approach provides a more detailed mechanistic picture of PETase enzymatic activity than standard bulk assays, it should aid future efforts to engineer more robust and active PETases to combat global microplastics pollution.

中文翻译：

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通过单分子显微镜测量 PETase 酶动力学


聚对苯二甲酸乙二醇酯 （PET） 是生产最广泛的人造聚合物之一，是造成微塑料污染的重要因素。微塑料污染对环境和人类健康的影响促使人们齐心协力开发基于微生物和酶的策略来降解 PET 和类似塑料。源自 Ideonella sakaiensis 细菌的 PETase 先前已被证明可以酶促降解 PET，从而引发多学科努力提高该 PETase 和其他 PETase 的稳健性和活性。然而，由于这些酶仅腐蚀不溶性 PET 底物的表面，因此很难测量标准动力学参数，例如 kon、koff 和 kcat，这使得使用传统酶动力学框架解释突变体的活性变得复杂。为了应对这一挑战，我们开发了一种单分子显微镜检测法，可量化量子点标记的 PETase 酶与表面固定化 PET 薄膜相互作用的着陆速率和结合持续时间。野生型 PETase 结合持续时间与双指数非常拟合，其中快速种群具有 2.7 s 的时间常数，被解释为活性结合事件，而慢速种群被解释为持续数十秒的非特异性结合相互作用。先前描述的过度活跃突变体 S238F/W159H 比野生型 PETase 具有更快的表观导通速率和更慢的关速率，这可能解释了其增强的活性。由于这种单分子方法提供了比标准批量分析更详细的 PETase 酶活性机理图，因此它应该有助于未来设计更强大、更活跃的 PETase 来对抗全球微塑料污染。