The recent discovery of IsPETase, a hydrolytic enzyme that can deconstruct poly(ethylene terephthalate) (PET), has sparked great interest in biocatalytic approaches to recycle plastics. Realization of commercial use will require the development of robust engineered enzymes that meet the demands of industrial processes. Although rationally engineered PETases have been described, enzymes that have been experimentally optimized via directed evolution have not previously been reported. Here, we describe an automated, high-throughput directed evolution platform for engineering polymer degrading enzymes. Applying catalytic activity at elevated temperatures as a primary selection pressure, a thermostable IsPETase variant (HotPETase, T_m = 82.5 °C) was engineered that can operate at the glass transition temperature of PET. HotPETase can depolymerize semicrystalline PET more rapidly than previously reported PETases and can selectively deconstruct the PET component of a laminated multimaterial. Structural analysis of HotPETase reveals interesting features that have emerged to improve thermotolerance and catalytic performance. Our study establishes laboratory evolution as a platform for engineering useful plastic degrading enzymes.

最近发现的Is PETase 是一种可以解构聚对苯二甲酸乙二醇酯 (PET) 的水解酶，引起了人们对回收塑料的生物催化方法的极大兴趣。商业用途的实现将需要开发满足工业过程需求的强大工程酶。尽管已经描述了合理工程化的 PETase，但以前没有报道过通过定向进化实验优化的酶。在这里，我们描述了一种用于工程聚合物降解酶的自动化、高通量定向进化平台。在高温下应用催化活性作为主要选择压力，热稳定的PETase变体（HotPETase，T _m = 82.5 °C）被设计成可以在 PET 的玻璃化转变温度下运行。HotPETase 可以比以前报道的 PETase 更快地解聚半结晶 PET，并且可以选择性地解构层压多材料的 PET 组分。HotPETase 的结构分析揭示了一些有趣的特征，这些特征已经出现以提高耐热性和催化性能。我们的研究将实验室进化确立为设计有用的塑料降解酶的平台。