Poly(ethylene terephthalate) (PET), a widely used plastic around the world, causes various environmental and health problems. Several groups have been extensively conducting research to solve these problems through enzymatic degradation of PET at high temperatures around 70 °C. Recently, Ideonella sakaiensis, a bacterium that degrades PET at mild temperatures, has been newly identified, and further protein engineering studies on the PET degrading enzyme from the organism (IsPETase) have also been conducted to overcome the low thermal stability of the enzyme. In this study, we performed structural bioinformatics-based protein engineering of IsPETase to optimize the substrate binding site of the enzyme and developed two variants, IsPETaseS242T and IsPETaseN246D, with higher enzymatic activity at both 25 and 37 °C compared with IsPETaseWT. We also developed the IsPETaseS121E/D186H/S242T/N246D variant by integrating the S242 T and N246D mutations into the previously reported IsPETaseS121E/D186H/R208A variant. At the 37 °C incubation, the quadruple variant maintained the PET degradation activity for 20 days, unlike IsPETaseWT that lost its activity within a day. Consequently, this study exhibited 58-fold increase in the activity compared with IsPETaseWT.

中文翻译：

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基于结构生物信息学的 Ideonella sakaiensis 热稳定 PETase 蛋白质工程

聚对苯二甲酸乙二醇酯 (PET) 是一种在世界范围内广泛使用的塑料，会导致各种环境和健康问题。几个小组一直在进行广泛的研究，通过在 70 °C 左右的高温下对 PET 进行酶促降解来解决这些问题。最近，新鉴定了一种在温和温度下降解 PET 的细菌 Ideonella sakaiensis，并且还对来自生物体的 PET 降解酶 (IsPETase) 进行了进一步的蛋白质工程研究，以克服酶的低热稳定性。在这项研究中，我们对 IsPETase 进行了基于结构生物信息学的蛋白质工程，以优化酶的底物结合位点，并开发了两种变体 IsPETaseS242T 和 IsPETaseN246D，与 IsPETaseWT 相比，它们在 25 和 37°C 下的酶活性更高。我们还通过将 S242 T 和 N246D 突变整合到先前报道的 IsPETaseS121E/D186H/R208A 变体中，开发了 IsPETaseS121E/D186H/S242T/N246D 变体。在 37 °C 孵育时，四重变体保持了 20 天的 PET 降解活性，这与 IsPETaseWT 在一天内失去活性不同。因此，与 IsPETaseWT 相比，本研究的活性增加了 58 倍。