Chemo-Enzymatic Depolymerization of Functionalized Low-Molecular-Weight Polyethylene.

Polyethylene (PE) is the most commonly used plastic type in the world, contributing significantly to the plastic waste crisis. Microbial degradation of PE in natural environments is unlikely due to its inert saturated carbon-carbon backbones, which are difficult to break down by enzymes, challenging the development of a biocatalytic recycling method for PE waste. Here, we demonstrated the depolymerization of low-molecular-weight (LMW) PE using an enzyme cascade that included a catalase-peroxidase, an alcohol dehydrogenase, a Baeyer Villiger monooxygenase, and a lipase after the polymer was chemically pretreated with m-chloroperoxybenzoic acid (mCPBA) and ultrasonication. In a preparative experiment with gram-scale pretreated polymers, GC-MS and weight loss determinations confirmed ~27% polymer conversion including the formation of medium-size functionalized molecules such as ω-hydroxy acids and α,ω-carboxylic acids. Additional polymer property analyses using AFM showed that enzymatic depolymerization reduced the particle sizes of this mCPBA- and enzyme-treated LMWPE. This multi-enzyme catalytic concept with distinct chemical steps represents a unique starting point for future development of bio-based recycling methods for polyolefin waste.

Structural Elucidation of a Metagenomic Urethanase and Its Engineering Towards Enhanced Hydrolysis Profiles.

While plastics like polyethylene terephthalate can already be degraded efficiently by the activity of hydrolases, other synthetic polymers like polyurethanes (PUs) and polyamides (PAs) largely resist biodegradation. In this study, we solved the first crystal structure of the metagenomic urethanase UMG-SP-1, identified highly flexible loop regions to comprise active site residues, and targeted a total of 20 potential hot spots by site-saturation mutagenesis. Engineering campaigns yielded variants with single mutations, exhibiting almost 3- and 8-fold improved activity against highly stable N-aryl urethane and amide bonds, respectively. Furthermore, we demonstrated the release of the corresponding monomers from a thermoplastic polyester-PU and a PA (nylon 6) by the activity of a single, metagenome-derived urethanase after short incubation times. Thereby, we expanded the hydrolysis profile of UMG-SP-1 beyond the reported low-molecular weight carbamates. Together, these findings promise advanced strategies for the bio-based degradation and recycling of plastic materials and waste, aiding efforts to establish a circular economy for synthetic polymers.

An Extremely Sensitive Ultra-High Throughput Growth Selection Assay for the Identification of Amidase Activity.

In the last decades, biocatalysis has offered new perspectives for the synthesis of (chiral) amines, which are essential building blocks for pharmaceuticals, fine and bulk chemicals. In this regard, amidases have been employed due to their broad substrate scope and their independence from expensive cofactors. To expand the repertoire of amidases, tools for their rapid identification and characterization are greatly demanded. In this work an ultra-high throughput growth selection assay based on the production of the folate precursor p-aminobenzoic acid (PABA) is introduced to identify amidase activity. PABA-derived amides structurally mimic the broad class of commonly used chromogenic substrates derived from p-nitroaniline. This suggests that the assay should be broadly applicable for the identification of amidases. Unlike conventional growth selection assays that rely on substrates as nitrogen or carbon source, our approach requires PABA in sub-nanomolar concentrations, making it exceptionally sensitive and ideal for engineering campaigns that aim at enhancing amidase activities from minimally active starting points, for example. The presented assay offers flexibility in the adjustment of sensitivity to suit project-specific needs using different expression systems and fine-tuning with the antimetabolite sulfathiazole. Application of this PABA-based assay facilitates the screening of millions of enzyme variants on a single agar plate within two days, without the need for laborious sample preparation or expensive instruments, with transformation efficiency being the only limiting factor. KEY POINTS: • Ultra-high throughput assay (tens of millions on one agar plate) for amidase screening • High sensitivity by coupling selection to folate instead of carbon or nitrogen source • Highly adjustable in terms of sensitivity and expression of the engineering target.

Urethanases for the Enzymatic Hydrolysis of Low Molecular Weight Carbamates and the Recycling of Polyurethanes.

Enzymatic degradation and recycling can reduce the environmental impact of plastics. Despite decades of research, no enzymes for the efficient hydrolysis of polyurethanes have been reported. Whereas the hydrolysis of the ester bonds in polyester-polyurethanes by cutinases is known, the urethane bonds in polyether-polyurethanes have remained inaccessible to biocatalytic hydrolysis. Here we report the discovery of urethanases from a metagenome library constructed from soil that had been exposed to polyurethane waste for many years. We then demonstrate the use of a urethanase in a chemoenzymatic process for polyurethane foam recycling. The urethanase hydrolyses low molecular weight dicarbamates resulting from chemical glycolysis of polyether-polyurethane foam, making this strategy broadly applicable to diverse polyether-polyurethane wastes.

High-Throughput Screening for Thermostable Polyester Hydrolases.

Due to the promise of more sustainable recycling of plastics through biocatalytic degradation, the search for and engineering of polyester hydrolases have become a thriving field of research. Furthermore, among other methods, halo formation assays have become popular for the detection of polyester-hydrolase activity. However, established halo-formation assays are limited in their ability to screen for thermostable enzymes, which are particularly important for efficient plastic degradation. The incubation of screening plates at temperatures above 50 °C leads to cell lysis and death. Therefore, equivalent master plates are commonly required to maintain and identify the active strains found on the screening plates. This replica plating procedure necessitates 20- to 60-fold more plates than our method, assuming the screened library is transferred to 384-well microtiter plates or 96-well microtiter plates, respectively, to organize the colonies in a retraceable manner, thus significantly lowering throughput. Here, we describe a halo formation assay that is designed to screen thermostable polyesterases independent of master plates and colony replication, thereby markedly reducing the workload and increasing the throughput.

Biosensor and chemo-enzymatic one-pot cascade applications to detect and transform PET-derived terephthalic acid in living cells.

Plastic waste imposes a serious problem to the environment and society. Hence, strategies for a circular plastic economy are demanded. One strategy is the engineering of polyester hydrolases toward higher activity for the biotechnological recycling of polyethylene terephthalate (PET). To provide tools for the rapid characterization of PET hydrolases and the detection of degradation products like terephthalic acid (TPA), we coupled a carboxylic acid reductase (CAR) and the luciferase LuxAB. CAR converted TPA into the corresponding aldehydes in Escherichia coli, which yielded bioluminescence that not only semiquantitatively reflected amounts of TPA in hydrolysis samples but is suitable as a high-throughput screening assay to assess PET hydrolase activity. Furthermore, the CAR-catalyzed synthesis of terephthalaldehyde was combined with a reductive amination cascade in a one-pot setup yielding the corresponding diamine, suggesting a new strategy for the transformation of TPA as a product obtained from PET biodegradation.