Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic and a major contributor to plastic pollution. IsPETase, from the PET-assimilating bacterium Ideonella sakaiensis, is a unique PET-hydrolytic enzyme that shares high sequence identity to canonical cutinases, but shows substrate preference towards PET and exhibits higher PET-hydrolytic activity at ambient temperature. Structural analyses suggest that IsPETase harbours a substrate-binding residue, W185, with a wobbling conformation and a highly flexible W185-locating β6-β7 loop. Here, we show that these features result from the presence of S214 and I218 in IsPETase, whose equivalents are strictly His and Phe, respectively, in all other homologous enzymes. We found that mutating His/Phe residues to Ser/Ile could enhance the PET-hydrolytic activity of several IsPETase-like enzymes. In conclusion, the Ser/Ile mutations should provide an important strategy to improve the activity of potential PET-hydrolytic enzymes with properties that may be useful for various applications.

聚对苯二甲酸乙二醇酯 (PET) 是最丰富的聚酯塑料，也是造成塑料污染的主要原因。Is PETase，来自 PET 同化细菌Ideonella sakaiensis，是一种独特的 PET 水解酶，与经典角质酶具有高度序列同一性，但对 PET 表现出底物偏好，并在环境温度下表现出更高的 PET 水解活性。结构分析表明，Is PETase 含有一个底物结合残基 W185，具有摇摆不定的构象和高度灵活的 W185 定位 β6-β7 环。在这里，我们表明这些特征是由于Is中存在 S214 和 I218PETase，在所有其他同源酶中，其等价物分别是严格的 His 和 Phe。我们发现将 His/Phe 残基突变为 Ser/Ile 可以增强几种Is PETase 样酶的 PET 水解活性。总之，Ser/Ile 突变应该提供一个重要的策略来提高潜在的 PET 水解酶的活性，这些酶具有可能对各种应用有用的特性。