Poly(butylene adipate-co-terephthalate) (PBAT), a polyester made of terephthalic acid (TPA), 1,4-butanediol, and adipic acid, is extensively utilized in plastic production and has accumulated globally as environmental waste. Biodegradation is an attractive strategy to manage PBAT, but an effective PBAT-degrading enzyme is required. Here, we demonstrate that cutinases are highly potent enzymes that can completely decompose PBAT films in 48 h. We further show that the engineered cutinases, by applying a double mutation strategy to render a more flexible substrate-binding pocket exhibit higher decomposition rates. Notably, these variants produce TPA as a major end-product, which is beneficial feature for the future recycling economy. The crystal structures of wild type and double mutation of a cutinase from Thermobifida fusca in complex with a substrate analogue are also solved, elucidating their substrate-binding modes. These structural and biochemical analyses enable us to propose the mechanism of cutinase-mediated PBAT degradation.
Cutinases can play a significant role in a biotechnology-based circular economy. However, relatively little is known about the structure-function relationship of these enzymes, knowledge that is vital to advance optimized, engineered enzyme candidates. Here, two almost identical cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) with only 18 amino acids difference were used for a rigorous biochemical characterization of their ability to hydrolyze PET, PET-model substrates, and cutin-model substrates. Kinetic parameters were compared with detailed in-silico docking studies of enzyme-ligand interactions. The two enzymes interacted with, and hydrolyzed PET differently, with Thc_Cut1 generating smaller PET-degradation products. Thc_Cut1 also showed higher catalytic efficiency on long-chain aliphatic substrates, an effect likely caused by small changes in the binding architecture. Thc_Cut2, in contrast, showed improved binding and catalytic efficiency when approaching the glass transition temperature of PET, an effect likely caused by longer amino acid residues in one area at the enzyme's surface. Finally, the position of the single residue Q93 close to the active site, rotated out in Thc_Cut2, influenced the ligand position of a trimeric PET-model substrate. In conclusion, we illustrate that even minor sequence differences in cutinases can affect their substrate binding, substrate specificity, and catalytic efficiency drastically. This article is protected by copyright. All rights reserved.
Present estimates suggest that of the 359 million tons of plastics produced annually worldwide(1), 150-200 million tons accumulate in landfill or in the natural environment(2). Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic, with almost 70 million tons manufactured annually worldwide for use in textiles and packaging(3). The main recycling process for PET, via thermomechanical means, results in a loss of mechanical properties(4). Consequently, de novo synthesis is preferred and PET waste continues to accumulate. With a high ratio of aromatic terephthalate units-which reduce chain mobility-PET is a polyester that is extremely difficult to hydrolyse(5). Several PET hydrolase enzymes have been reported, but show limited productivity(6,7). Here we describe an improved PET hydrolase that ultimately achieves, over 10 hours, a minimum of 90 per cent PET depolymerization into monomers, with a productivity of 16.7 grams of terephthalate per litre per hour (200 grams per kilogram of PET suspension, with an enzyme concentration of 3 milligrams per gram of PET). This highly efficient, optimized enzyme outperforms all PET hydrolases reported so far, including an enzyme(8,9) from the bacterium Ideonella sakaiensis strain 201-F6 (even assisted by a secondary enzyme(10)) and related improved variants(11-14) that have attracted recent interest. We also show that biologically recycled PET exhibiting the same properties as petrochemical PET can be produced from enzymatically depolymerized PET waste, before being processed into bottles, thereby contributing towards the concept of a circular PET economy.
