High Throughput Screening for New Fungal Polyester Hydrolyzing Enzymes

Abstract

There is a strong need for novel and more efficient polyester hydrolyzing enzymes in order to enable the development of more environmentally friendly plastics recycling processes allowing the closure of the carbon cycle. In this work, a high throughput system on microplate scale was used to screen a high number of fungi for their ability to produce polyester-hydrolyzing enzymes. For induction of responsible enzymes, the fungi were cultivated in presence of aliphatic and aromatic polyesters [poly(1,4butylene adipate co terephthalate) (PBAT), poly(lactic acid) (PLA) and poly(1,4-butylene succinate) (PBS)], and the esterase activity in the culture supernatants was compared to the culture supernatants of fungi grown without polymers. The results indicate that the esterase activity of the culture supernatants was induced in about 10% of the tested fungi when grown with polyesters in the medium, as indicated by increased activity (to >50 mU/mL) toward the small model substrate para-nitrophenylbutyrate (pNPB). Incubation of these 50 active culture supernatants with different polyesters (PBAT, PLA, PBS) led to hydrolysis of at least one of the polymers according to liquid chromatography-based quantification of the hydrolysis products terephthalic acid, lactic acid and succinic acid, respectively. Interestingly, the specificities for the investigated polyesters varied among the supernatants of the different fungi.

Together Is Better: The Rumen Microbial Community as Biological Toolbox for Degradation of Synthetic Polyesters

“…All short-chain p-NP-esters were hydrolyzed into p-nitrophenol, with slightly higher activity on those esters based on C 4 , C 6 , and C 8 acids (6.5, 5.8, and 5.5 U ml −1 , respectively) (Figure 1). Therefore, rumen content demonstrated considerably higher esterase activity in comparison with polyester-induced fungal supernatants, where esterase activity accounted for approximately 0.4 U ml −1 with p-NPB (C 4 ) as a model substrate (Weinberger et al, 2020).…”

“…Regarding fungi, different hydrolases (lipase and esterase) from Aspergillus, Penicillium, and Candida species are already exploited at industrial level and found applications in different sectors like detergents, textile industry, diary, and food preparation (Weinberger et al, 2020). Hydrolysis of polyesters and other synthetic polymers was previously described for Penicillium and Beauveria species (Heumann et al, 2006;Liebminger et al, 2007;Almansa et al, 2009).…”

Enzyme discovery and engineering for sustainable plastic recycling

“…Traditional agar plate assays have limited capability in screening large-sized metagenomic libraries. Recent studies in developing high-throughput screening approaches might accelerate the discovery of new plastic-degrading microbes and enzymes [19,20]. When using a functional screening approach, it is important to select a proper host cell for constructing a heterologous gene expression library, with desirable expression level and library representativeness.…”

“…Figure 1C shows the commonly used workflow of the proteomic approach for mining plastic-degrading enzymes. First, the pure or environmental microbial consortia are grown with and without the plastic substrate, as the presence of plastics could differentially induce the functional microorganisms to express enzymes with plastic hydrolytic activity [19]. Proteins produced by the microbial cultures are extracted and digested into small peptides, which are subjected to sequencing, followed by protein identification via bioinformatic analysis.…”

Biotechnology of Plastic Waste Degradation, Recycling, and Valorization: Current Advances and Future Perspectives

“…Polyester hydrolases from fungi and bacteria exhibit excellent hydrolytic activity in converting PET into two environmentally friendly monomers, terephthalic acid and ethylene glycol. [95][96][97][98] The polyester hydrolase from Thermobifida fusca DSM43793 was first reported to degrade PET at 55°C, albeit slowly. In subsequent years, various thermophilic PET hydrolases from thermophilic host microorganisms, both eukaryotic and prokaryotic, have been identified and characterized for PET degradation, with thermal stability at � 70°C.…”