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Complete Depolymerization of PET Wastes by an Evolved PET Hydrolase from Directed Evolution |
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| Authors: | Lixia Shi Dr Pi Liu Zijian Tan Wei Zhao Junfei Gao Prof Dr Qun Gu Prof Dr Hongwu Ma Prof Dr Haifeng Liu Prof Dr Leilei Zhu |
| Institution: | 1. 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 National Technology Innovation Center of Synthetic Biology, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin, 300308 P. R. China These authors contributed equally to this work.;2. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin, 300308 P. R. China National Technology Innovation Center of Synthetic Biology, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin, 300308 P. R. China These authors contributed equally to this work.;3. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin, 300308 P. R. China Jiangsu Collaborative Innovation Centre of Chinese Medicinal Resources Industrialization, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023 China;4. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin, 300308 P. R. China National Technology Innovation Center of Synthetic Biology, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin, 300308 P. R. China;5. Jiangsu Collaborative Innovation Centre of Chinese Medicinal Resources Industrialization, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023 China;6. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin, 300308 P. R. China |
| Abstract: | 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 Tm value than the PETase WT. DepoPETase enabled complete depolymerization of seven untreated PET wastes and 19.1 g PET waste (0.4 % Wenzyme/WPET) 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. |
| Keywords: | Directed Evolution High-Throughput Screening PET Depolymerization PETase Thermal Stability |