Shi Lixia, Liu Pi, Tan Zijian, Zhao Wei, Gao Junfei, Gu Qun, Ma Hongwu, Liu Haifeng, Zhu Leilei
University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing, 100049, China.
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin, 300308, P. R. China.
Angew Chem Int Ed Engl. 2023 Mar 27;62(14):e202218390. doi: 10.1002/anie.202218390. Epub 2023 Feb 21.
PETase displays great potential in PET depolymerization. Directed evolution has been limited to engineer PETase due to the lack of high-throughput screening assay. In this study, a novel fluorescence-based high-throughput screening assay employing a newly designed substrate, bis (2-hydroxyethyl) 2-hydroxyterephthalate (termed BHET-OH), was developed for PET hydrolases. The best variant DepoPETase produced 1407-fold more products towards amorphous PET film at 50 °C and showed a 23.3 °C higher T value than the PETase WT. DepoPETase enabled complete depolymerization of seven untreated PET wastes and 19.1 g PET waste (0.4 % W /W ) in liter-scale reactor, suggesting that it is a potential candidate for industrial PET depolymerization processes. The molecular dynamic simulations revealed that the distal substitutions stabilized the loops around the active sites and transmitted the stabilization effect to the active sites through enhancing inter-loop interactions network.
聚对苯二甲酸乙二酯酶（PETase）在聚对苯二甲酸乙二酯（PET）解聚方面显示出巨大潜力。由于缺乏高通量筛选测定方法，定向进化在改造PETase方面受到限制。在本研究中，开发了一种基于荧光的新型高通量筛选测定方法，该方法采用新设计的底物双（2-羟乙基）2-羟基对苯二甲酸酯（称为BHET-OH）用于PET水解酶。最佳变体DepoPETase在50°C下对无定形PET薄膜产生的产物比PETase野生型（WT）多1407倍，并且其熔点（Tm）值比PETase WT高23.3°C。DepoPETase能够使七种未处理的PET废料完全解聚，并且在升规模反应器中能解聚19.1 g PET废料（0.4％W / W），这表明它是工业PET解聚过程的潜在候选者。分子动力学模拟表明，远端取代稳定了活性位点周围的环，并通过增强环间相互作用网络将稳定作用传递到活性位点。
