Enzymatic and biophysical characterization of a novel modular cellulosomal GH5 endoglucanase multifunctional from the anaerobic gut fungus Piromyces finnis.

Cellulases from anaerobic fungi are enzymes less-studied biochemically and structurally than cellulases from bacteria and aerobic fungi. Currently, only thirteen GH5 cellulases from anaerobic fungi were biochemically characterized and two crystal structures were reported. In this context, here, we report the functional and biophysical characterization of a novel multi-modular cellulosomal GH5 endoglucanase from the anaerobic gut fungus Piromyces finnis (named here PfGH5). Multiple sequences alignments indicate that PfGH5 is composed of a GH5 catalytic domain and a CBM1 carbohydrate-binding module connected through a CBM10 dockerin module. Our results showed that PfGH5 is an endoglucanase from anaerobic fungus with a large spectrum of activity. PfGH5 exhibited preference for hydrolysis of oat ß-glucan, followed by galactomannan, carboxymethyl cellulose, mannan, lichenan and barley ß-glucan, therefore displaying multi-functionality. For oat ß-glucan, PfGH5 reaches its optimum enzymatic activity at 40 °C and pH 5.5, with Km of 7.1 µM. Ion exchange chromatography analyzes revealed the production of oligosaccharides with a wide degree of polymerization indicated that PfGH5 has endoglucanase activity. The ability to bind and cleave different types of carbohydrates evidence the potential of PfGH5 for use in biotechnology and provide a useful basis for future investigation and application of new anaerobic fungi enzymes.

Unveiling the crystal structure of thermostable dienelactone hydrolase exhibiting activity on terephthalate esters.

Dienelactone hydrolase (DLH) is one of numerous hydrolytic enzymes with an α/ß-hydrolase fold, which catalyze the hydrolysis of dienelactone to maleylacetate. The DLHs share remarkably similar tertiary structures and a conserved arrangement of catalytic residues. This study presents the crystal structure and comprehensive functional characterization of a novel thermostable DLH from the bacterium Hydrogenobacter thermophilus (HtDLH). The crystal structure of the HtDLH, solved at a resolution of about 1.67 Å, exhibits a canonical α/ß-hydrolase fold formed by eight ß-sheet strands in the core, with one buried α-helix and six others exposed to the solvent. The structure also confirmed the conserved catalytic triad of DHLs formed by Cys121, Asp170, and His202 residues. The HtDLH forms stable homodimers in solution. Functional studies showed that HtDLH has the expected esterase activity over esters with short carbon chains, such as p-nitrophenyl acetate, reaching optimal activity at pH 7.5 and 70 °C. Furthermore, HtDLH maintains more than 50 % of its activity even after incubation at 90 °C for 16 h. Interestingly, HtDLH exhibits catalytic activity towards polyethylene terephthalate (PET) monomers, including bis-1,2-hydroxyethyl terephthalate (BHET) and 1-(2-hydroxyethyl) 4-methyl terephthalate, as well as other aliphatic and aromatic esters. These findings associated with the lack of activity on amorphous PET indicate that HtDLH has characteristic of a BHET-degrading enzyme. This work expands our understanding of enzyme families involved in PET degradation, providing novel insights for plastic biorecycling through protein engineering, which could lead to eco-friendly solutions to reduce the accumulation of plastic in landfills and natural environments.

Plastic-degrading microbial communities reveal novel microorganisms, pathways, and biocatalysts for polymer degradation and bioplastic production.

Plastics derived from fossil fuels are used ubiquitously owing to their exceptional physicochemical characteristics. However, the extensive and short-term use of plastics has caused environmental challenges. The biotechnological plastic conversion can help address the challenges related to plastic pollution, offering sustainable alternatives that can operate using bioeconomic concepts and promote socioeconomic benefits. In this context, using soil from a plastic-contaminated landfill, two consortia were established (ConsPlastic-A and -B) displaying versatility in developing and consuming polyethylene or polyethylene terephthalate as the carbon source of nutrition. The ConsPlastic-A and -B metagenomic sequencing, taxonomic profiling, and the reconstruction of 79 draft bacterial genomes significantly expanded the knowledge of plastic-degrading microorganisms and enzymes, disclosing novel taxonomic groups associated with polymer degradation. The microbial consortium was utilized to obtain a novel Pseudomonas putida strain (BR4), presenting a striking metabolic arsenal for aromatic compound degradation and assimilation, confirmed by genomic analyses. The BR4 displays the inherent capacity to degrade polyethylene terephthalate (PET) and produce polyhydroxybutyrate (PHB) containing hydroxyvalerate (HV) units that contribute to enhanced copolymer properties, such as increased flexibility and resistance to breakage, compared with pure PHB. Therefore, BR4 is a promising strain for developing a bioconsolidated plastic depolymerization and upcycling process. Collectively, our study provides insights that may extend beyond the artificial ecosystems established during our experiments and supports future strategies for effectively decomposing and valorizing plastic waste. Furthermore, the functional genomic analysis described herein serves as a valuable guide for elucidating the genetic potential of microbial communities and microorganisms in plastic deconstruction and upcycling.