While the plastic industry brings convenience to the life and production of human society, it also leads to the production of large amounts of waste plastic waste. Due to its inherent hardness, strength, durability and stability requirements, waste plastic products cannot be automatically degraded, and long-term exposure causes serious harm to the environment. At present, the disposal methods of plastic waste are usually landfill and incineration. This one-way process of "production-disposal-treatment" does not conform to the concept of circular economy and cannot solve the problem of "white pollution" from the source.
In 2004, Professor Richard C. Thompson of the University of Plymouth in the United Kingdom and others published a short article on Science, proposing the concept of "microplastics". A large number of non-degradable tiny plastic fragments (~20 μm) are gradually transferred to the soil and ocean in the environment, and finally enter the ecosystem and food chain, and are ingested by organisms including humans, threatening human health and the survival of animals and plants, and developing green Biodegradation strategies have become an urgent need in the fields of ecology and environment. In 2016, the Kohei Oda team of Kyoto University of Technology, Japan, reported in Science the first IsPETase degrading enzyme that can effectively degrade PET plastic with low crystallinity at 30°C. However, the enzyme has poor stability and cannot meet the needs of practical applications for biodegradation.
The team of Wu Bian, a researcher at the Institute of Microbiology, Chinese Academy of Sciences, proposed a new protein stability calculation design strategy (greedy accumulated strategy for protein engineering, GRAPE) (Figure 1). Based on computer protein design, the stability of IsPETase was modified, and the The redesigned enzyme with significantly enhanced stickiness provides new ideas for broadening the application scenarios of biodegradable plastics. Researchers adopted a fusion strategy, combined with four different single-point prediction algorithms supplemented by structural defect analysis, and predicted 85 potential beneficial mutations. After experimental tests on predicted mutations, 21 beneficial single point mutations (ΔTm ≥ 1.5°C) were obtained. Through the K-means clustering algorithm, researchers divided 21 beneficial single point mutations into 3 clusters, and performed iterative stacking of each cluster according to the greedy algorithm (Figure 2). After 10 rounds of iterative stacking, the researchers obtained the IsPETase mutant (named DuraPETase) whose melting temperature was increased by 31°C. Under mild conditions, the degradation efficiency of DuraPETase on 30% crystallinity PET film is 300 times higher than that of the wild type (Figure 3B). It can be observed by scanning electron microscope that the internal structure of the PET film after DuraPETase treatment has undergone significant corrosion changes (Figure 3D). Researchers analyzed the crystal structure of DuraPETase protein (Figure 4), verified the synergistic interaction between amino acids in the active site region of the mutant, and explored the molecular mechanism of DuraPETase performance improvement. The study achieved the complete degradation of 2g/L microplastics under mild conditions, and provided new treatment ideas for the pretreatment of microplastics in wastewater.
The advantage of the GRAPE strategy is to use clustering algorithm and greedy algorithm to perform systematic clustering analysis on the beneficial mutants obtained by calculation, and then combine the greedy algorithm to perform network iterative superposition, which greatly avoids the negative synergistic interaction between different mutation sites. Explore the superposition path of sequence space to the maximum in a short time. This research provides new ideas for computer-aided protein transformation, and provides a valuable tool for further understanding and promoting polyester hydrolysis in the natural environment.
related research results were published on ACS Catalysis and were selected as the cover article of the current issue. Assistant researcher Cui Yinglu and PhD student Yanchun Chen are the co-first authors of the paper, and Wu Bian is the corresponding author of the paper. Researchers of the Institute of Microbiology Xiang Hua, Tang Shuangyan, Du Wenbin, Tianjin Institute of Industrial Biotechnology Researcher Liu Weidong's team, University of Science and Technology of China Professor Liu Haiyan's team, Nanjing University Professor Liang Yong's team, and University of California Professor Houk provided important guidance and assistance for the research work. The research work was obtained by the National Natural Science Foundation of China and the European Union's cooperation project "Synthetic Plastic Degradation and Transformation of Microbial Flora", the National Key Research and Development Program, the National Natural Science Foundation of China Outstanding Youth Project and General Program, and the Chinese Academy of Sciences Strategic Biological Resources Service Network Program Biological Resources Library project, and the support of the Beijing Natural Science Foundation project.