Bioprocessing of poly(ethylene terephthalate) (PET) waste has emerged as a promising approach to a circular economy. The key to this development has been the discovery of enzymes that effectively depolymerize PET to its constituent monomers, and hitherto, the most promising candidates are quite closely related, bacterial cutinases. Fungal enzymes have been less investigated, although a few examples of hydrolytic activity against PET have been reported. To further assess this, we have produced 24 fungal cutinases that are homologues to known PET hydrolases and identified three previously uncharacterized enzymes. The activity of the newly discovered enzymes was comparable to or higher than that of the fungal cutinase from Humicola insolens (HiC). One enzyme from Thermocarpiscus australiensis (TaC) appeared particularly proficient on PET, with up to four times higher activity than that of HiC, but required high salt concentrations (about 0.5 M NaCl) to reach maximal activity. Biochemical and biophysical measurements suggested that this salt dependence could be ascribed to electrostatic repulsion between TaC and the PET, and to mitigate this, we designed and produced 40 TaC variants with a reduced (negative) net charge. Several variants performed up to 2.5-fold better in salt-free buffer compared to wild-type TaC, and a few of them entirely escaped the dependence of salt but retained the maximal catalytic performance of the wild type. Overall, we expanded the diversity of fungal PET hydrolases, and learnings from this study may be of more general relevance for engineering balanced enzyme–substrate interaction strength in PET hydrolases.

中文翻译：

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用于增强聚对苯二甲酸乙二醇酯活性的真菌角质酶的发现和表面电荷工程

聚对苯二甲酸乙二醇酯 (PET) 废物的生物处理已成为循环经济的一种有前途的方法。这一发展的关键是发现了能够有效地将 PET 解聚为其组成单体的酶，迄今为止，最有希望的候选者是与之密切相关的细菌角质酶。尽管已经报道了一些针对 PET 水解活性的例子，但对真菌酶的研究较少。为了进一步评估这一点，我们生产了 24 种真菌角质酶，它们与已知的 PET 水解酶同源，并鉴定了三种以前未表征的酶。新发现的酶的活性与来自特异腐质霉（HiC）的真菌角质酶的活性相当或更高。来自澳大利亚Thermocarpiscus australiensis (TaC)的一种酶似乎对 PET 特别有效，其活性比 HiC 高出四倍，但需要高盐浓度（约 0.5 M NaCl）才能达到最大活性。生化和生物物理测量表明，这种盐依赖性可能归因于 TaC 和 PET 之间的静电排斥，为了减轻这种排斥，我们设计并生产了 40 种具有减少（负）净电荷的 TaC 变体。与野生型 TaC 相比，几种变体在无盐缓冲液中的表现提高了 2.5 倍，其中一些变体完全摆脱了盐的依赖性，但保留了野生型的最大催化性能。总体而言，我们扩大了真菌 PET 水解酶的多样性，并且从这项研究中获得的经验可能对于在 PET 水解酶中设计平衡的酶-底物相互作用强度具有更普遍的相关性。