SusChEM: Collaborative proposal: Engineering increased activity of cutinase toward poly(ethyleneterephthalate) for recycling of plastic

塑料固体废物的环境后果是严重的。随着塑料使用和应用的多样化和扩大，这个问题继续加剧。这个问题最显著的例子是塑料在海洋、湖泊和河流中积聚的速度。使用最广泛的塑料是聚对苯二甲酸乙二醇酯（PET）。PET用于制造瓶子、聚酯织物和食品托盘。生产水平估计每年超过5000万吨。本项目将尝试开发一种高效的降解PET的酶。高中学生将有机会从事暑期研究。本科生和研究生也将参与该项目。这些机会将有助于培养STEM劳动力。最近，几个研究小组发现了在温和条件下催化PET水解的酶。不幸的是，这些酶太慢，太不稳定，无法实际使用。通过蛋白质工程来提高它们的催化效率是一种潜在的解决方案。该项目测试了一种工程酶作用于不溶性底物的新方法。这种方法认识到酶和不溶性底物之间的相互作用是复杂的，并且涉及延伸的结合位点。酶必须从大块聚合物中拉出链，定位酯基进行水解，并允许聚合物底物在结合位点内滑动以重复水解链。以前试图增加酶对不溶性底物的活性，但收效甚微。本项目提出的大面积诱变（LAM）方法是一种针对不溶性合成聚合物底物的工程酶的系统策略。该方法将：1)确定角质酶中影响其对PET催化活性的结合区大小；2)优化PET延伸结合区的形状，3)提高酶的热稳定性，使其在PET玻璃化转变温度以上使用，此时链迁移率更高，使酶能够更好地接近聚合物底物。该项目的结果将是一种高效、稳定的酶的工程设计，其催化活性至少提高了100倍，可能适用于商业PET回收，这是减少塑料污染的重要一步。本项目由CBET/ENG的细胞与生化工程专业和CHE/MPS的生命过程化学专业联合支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The environmental consequences of plastic solid waste are serious. The problem continues to increase as we diversify and expand plastic use and applications. The most significant example of this problem is the rate that plastics are accumulating in oceans, lakes and rivers. The most widely used plastic is polyethylene terephthalate, or PET. PET is used in the manufacture of bottles, polyester fabrics, and food trays. Production levels are estimated to be over 50 million tons per year. This project will attempt to develop an efficient enzyme for the degradation of PET. High school students will be engaged in summer research opportunities. Undergraduate and graduate students will also be involved in the project. These opportunities will help develop a STEM workforce.Recently, several groups discovered enzymes that catalyze PET hydrolysis under mild conditions. Unfortunately, these enzymes are too slow and too unstable for practical use. Protein engineering to increase their catalytic efficiency is a potential solution. The project tests a new approach for engineering enzymes to act on insoluble substrates. This approach recognizes that the interaction between an enzyme and an insoluble substrate is complex and involves an extended binding site. The enzyme must pull the chain from the bulk polymer, position the ester group for hydrolysis, and allow the polymer substrate to slide within the binding site for repeated hydrolysis of the chain. Previous attempts to increase the activity of enzymes toward insoluble substrates have met with limited success. The proposed large-area-mutagenesis (LAM) methodology to be implemented in this program is a systematic strategy for engineering enzymes for insoluble synthetic polymer substrates. The approach will: 1) establish the size of the binding region in cutinase that influences its catalytic activity toward PET; 2) optimize the shape of the extended binding region for PET, 3) increase the thermal stability of the enzyme for use above the glass transition temperature of PET, where chain mobility is higher, allowing better access of the enzyme to the polymer substrate. The results of this program will be the engineering of an efficient, stable enzyme with increased catalytic activity of at least 100-fold that may be suitable for commercial PET recycling, which is an essential step to reducing plastic pollution. This project is supported jointly by the Cellular and Biochemical Engineering Program in CBET/ENG and the Chemistry of Life Processes Program in CHE/MPS.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.