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Plastic waste poses an ecological challenge. While current plastic waste management largely relies on unsustainable, energy-intensive, or even hazardous physicochemical and mechanical processes, enzymatic degradation offers a green and sustainable route for plastic waste recycling. Poly(ethylene terephthalate) (PET) has been extensively used in packaging and for the manufacture of fabrics and single-used containers, accounting for 12% of global solid waste. The practical application of PET hydrolases has been hampered by their lack of robustness and the requirement for high processing temperatures. Here, we use a structure-based, deep learning algorithm to engineer an extremely robust and highly active PET hydrolase. Our best resulting mutant (FAST-PETase: Functional, Active, Stable, and Tolerant PETase) exhibits superior PET-hydrolytic activity relative to both wild-type and engineered alternatives, (including a leaf-branch compost cutinase and its mutant) and possesses enhanced thermostability and pH tolerance. We demonstrate that whole, untreated, post-consumer PET from 51 different plastic products can all be completely degraded by FAST-PETase within one week, and in as little as 24 hours at 50 C. Finally, we demonstrate two paths for closed-loop PET recycling and valorization. First, we re-synthesize virgin PET from the monomers recovered after enzymatic depolymerization. Second, we enable in situ microbially-enabled valorization using a Pseudomonas strain together with FAST-PETase to degrade PET and utilize the evolved monomers as a carbon source for growth and polyhydroxyalkanoate production. Collectively, our results demonstrate the substantial improvements enabled by deep learning and a viable route for enzymatic plastic recycling at the industrial scale.

compounds. This investigation was undertaken to examine This investigation was undertaken to determine prolonged ad-mechanisms of prolonged oculotoxicity of model comverse effects of benzalkonium chloride (BzCl), a cationic surfac-pounds with primary cultures of rabbit corneal epithelial tant, and sodium dodecyl sulfate (SDS), an anionic surfactant, (CE) 1 cells.after an initial treatment of and subsequent removal from a priBenzalkonium chloride (BzCl), a cationic surfactant, and mary culture system of rabbit corneal epithelial cells. Metabolic sodium dodecyl sulfate (SDS), an anionic surfactant, were chointegrity and cell growth were evaluated at specified periods after sen as model compounds for this study because rabbit eyes a 1-hr treatment with the surfactants because of their importance exposed to BzCl and SDS during the Draize test experience (Kennah et al., 1989). Draize scores of initial pH (pH i ) were also measured because of their importance in cellutoxicity and recovery are readily available for these surfactants lar homeostasis. ATP/ADP ratios were used to assess metabolic and their toxic actions have been described by many researchers integrity, and propidium iodide staining of cells was used to mea- (Grant, 1986;Kennah et al., 1989;Bartnik, 1992). These sur- ] i with fura-PE3 and pH i with factants are widely used commercially in soaps, shampoos, 2, 7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). BzCl cosmetics, and other consumer products and can be obtained induced a concentration-dependent decrease in ATP/ADP ratios in purified form without preservatives. Cationic and anionic 24 hr after its removal from the cultures, whereas SDS had mini-surfactants are major classes of surfactants, of which cationic mal effects on metabolic integrity throughout the 48-hr postexpo-surfactants are generally more toxic than anionic surfactants sure measurement period. The proliferative response of cultures (North-Root et al., 1982;Borenfreund and Borrero, 1984). Both treated with SDS, however, was decreased when compared with cationic and anionic surfactants are generally more toxic than transiently increased pH i 1 hr after its removal and decreased pH i at the 48-hr post-treatment period. In conclusion, the two Surfactants are rapidly and extensively incorporated into surfactants in vitro had distinctly different prolonged effects on lipid cellular structures because they contain both long-chain corneal epithelial cells, which may suggest that BzCl and SDS lipophilic and charged moieties. They have structures similar differentially affect cellular recovery in vivo. ᭧ 1996 Society of Toxicology to that of the native constituents of plasma membrane lipids that contain carbon chain lengths of between 12 and 25. The uptake and loss of BzCl and SDS from ocular tissues were The in vivo test for ocular irritancy, the Draize rabbit eye test (Draize et al., 1944), is based mainly on observations tives that provide a quantitative and mechanistic evaluation 71

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