Search Results (76)

Plastic pollution is a global environmental crisis, and microbial degradation represents a promising remediation strategy. While bacteria have been widely studied, yeasts offer unique advantages for plastic degradation due to their metabolic versatility, stress tolerance, and enzymatic capabilities. However, plastic degradative yeasts have not been reviewed comprehensively. Although several yeasts capable of degrading polyethylene terephthalate (PET) or polyethylene (PE) have been reported (e.g., Moesziomyces antarcticus, Candida tropicalis, Yarrowia lipolytica and Rhodotorula mucilaginosa), degraders of other plastic types are less studied. Although some yeasts can assimilate carbon from plastics, the diversity of yeasts capable of participating in plastic mineralization remains vastly underexplored. In recent years, yeast cell surface display systems for bacterial PETase and fungal cutinase have been developed, demonstrating promising PET degradation efficiency. However, PETase is feedback-inhibited by the intermediate product mono(2-hydroxyethyl)terephthalate (MHET). Systems synergizing PETase with MHETase have shown superior stability during long-term PET degradation and enable large-scale depolymerization of PET waste. For high-crystallinity PET, fungal hydrophobins can be used to modify the surface hydrophobicity of PETase-displaying yeast cells, facilitating their attachment to hydrophobic PET surfaces and ultimately enhancing the degradation efficiency of the whole-cell biocatalyst. Limitations of current research and future directions are also discussed. Full article

Background/Objectives: Microbial infections represent a major challenge in the food processing chain. Postharvest fungal control has historically relied on chemical control; however, their use is increasingly restricted due to environmental and health risks. Therefore, the aim of this study was to evaluate the antifungal potential of essential oils obtained from high-yield plant species and characterize the potential mechanisms of action of their major volatiles, with the goal of proposing a prospective formulation for the control of postharvest fungi. Methods: Cinnamon, rosemary, allspice, and Peruvian pepper essential oils were extracted by hydrodistillation, tested against Botrytis cinerea and Colletotrichum sp., and analyzed by gas chromatography-mass spectrometry. Finally, in silico bioactivity analyses were performed on the most abundant volatiles. Results: Cinnamon and rosemary produced the most effective oils against both fungal species. Cinnamaldehyde, cinnamyl acetate, eugenol, methyleugenol, (+)-2-bornanone, eucalyptol, α-phellandrene, and β-myrcene were some of the most abundant volatiles in the analyzed oils. In silico analyses predicted 56 antifungal mechanisms, including inhibition of cell membrane and wall synthesis, affectation of primary metabolism, inhibition of molecular processes, redox homeostasis, and protein degradation and cutinase inhibition. The last one is a specific mechanism mediating in vivo plant-fungal interactions found exclusively in β-terpinene and β-ocimene. Conclusions: Compounds with cutinase inhibition activity such as β-terpinene and β-ocimene are of great potential to complement the activity of other bioactive compounds. According to literature and in silico analyses the mixture of cinnamaldehyde, eugenol, β-terpinene and β-ocimene could be a potential formulation for the management of postharvest fungi. Full article

Microbial cutinases are promising biocatalysts for polymer recycling. Here, we investigated the structural basis of catalytic activation in a thermophilic cutinase from Chaetomium thermophilum (CtCut). Differential scanning calorimetry revealed a three-state thermal unfolding pathway (Tm = 66.4 °C and 69.5 °C), indicating hierarchical stability. To capture distinct conformational states while avoiding affinity-tag artifacts, we employed both tag-free and tagged constructs. We determined apo-structures of wild-type and S136A mutant CtCut at 1.7 Å resolution and a complementary inhibitor complex at 2.65 Å. In the apo-state, a chloride ion coordinated the electrostatically pre-organized active site, while the catalytic H204 adopted a solvent-exposed, inactive loop conformation. In the inhibitor complex, p-nitrophenol displaced the chloride, establishing a characteristic oxyanion hole network. Concomitantly, the “lid” loop transitioned to an open state, with H204 exhibiting pronounced conformational heterogeneity across eight independent molecules. These complementary structures provide structural evidence for conformational dynamics of the catalytic lid loop, consistent with the conformational cycling model previously proposed for a mesophilic homolog. Full article

Nigrospora musae ST1 is a newly identified pathogen responsible for leaf spot disease in Idesia polycarpa. In order to further advance our understanding of this strain and improve management strategies for the leaf spot disease, the PacBio Sequel II platform was used to perform whole-genome sequencing of N. musae ST1. The assembled genome comprised 42 contigs, with a total length of 49,259,803 bp and an average GC content of 56.23%. Functional annotation identified 12,063 protein-coding genes, including 125 Transporter Classification Database (TCDB)-related genes, 3600 pathogen host interaction (PHI) genes, 2503 Virulence Factor Database (DFVF)-related genes, and 722 genes encoding carbohydrate-active enzymes (CAZymes). Integrated analyses of the secretome, PHI, and DFVF databases revealed six secreted carbohydrate-active enzymes implicated in plant pathogenicity, including three glycoside hydrolases, two pectinate lyases, and one cutinase, potentially playing important roles in pathogenicity. A total of 77 secondary metabolite gene clusters were predicted. Comparative genomic analysis between N. musae ST1 and other Nigrospora species revealed differences in genome rearrangements in Nigrospora fungi. In conclusion, this study has clarified the whole-genome structural characteristics and evolutionary relationships of the newly reported pathogenic fungus, N. musae ST1. It provides a theoretical foundation for future investigations into the pathogenic mechanisms of N. musae ST1 infection in I. polycarpa, as well as potential targets for disease control. Full article

PET biodegradation remains limited due to its intrinsic properties—high crystallinity, hydrophobicity, and strong chemical stability. These characteristics lead to extremely slow degradation rates and contribute to PET’s persistence in the environment. Understanding how microorganisms respond at the molecular level when exposed to such a recalcitrant polymer is therefore essential. Living organisms express genes in response to their needs during development. When microbes are under critical conditions, such as when contaminants are present, they express genes encoding specific enzymes that attack the pollutant. In this study, a fungus isolated from the infected fruit of the plant Randia monantha was identified as Aspergillus terreus. It was tested for polyethylene terephthalate (PET) degradation, and the fungus Aspergillus nidulans was evaluated due to its previously reported recombinant cutinases for PET degradation. A microplastic polyethylene terephthalate (PET-MP) particle size of <355 μm for degradation was established, and a PET weight loss of 1.62% for A. nidulans and 1.01% for A. terreus was found. Additionally, the degradation of PET was confirmed by FTIR and SEM. This study also compares the transcriptomic profiles of Aspergillus nidulans and Aspergillus terreus during cultivation with PET-MP residues, which serve as a replacement for the carbon source. We present the first evidence of chitinase overexpression during direct exposure of PET to Aspergillus fungi. Interestingly, chitinase expression was detected in the crude extracts of A. nidulans and A. terreus during culture in the presence of PET residues, which replaced the carbon source. The chitinase produced by each fungus has a similar molecular weight of approximately 44 kDa. Chitinase activity was monitored over a 14-day cultivation period; from day 2, chitinase activity was detected in both cultures and continued to increase until day 14, when the highest values reported in this work were 24.88 ± 4.17 U mg⁻¹ and 10.41 ± 0.47 U mg⁻¹ for A. nidulans and A. terreus, respectively. Finally, we proposed a pathway for PET degradation by Aspergillus fungi that involves mycelial adherence and the secretion of hydrophobins, followed by the production of intermediates and monomers via esterase hydrolysis, and ultimately, the entry of monomers to the ethylene glycol (EG) and terephthalic acid (TPA) pathways, further suggesting these Aspergillus as candidates to produce valuable compounds under these conditions, such as muconic acid, gallic acid, and vanillic acid. Full article

Esterases are ubiquitous enzymes found in all living organisms, including animals, plants, and microorganisms. They are involved in several biological processes, including the synthesis and breakdown of biomolecules, such as nucleic acids, lipids, and esters; phosphorus metabolism; detoxification of natural and artificial toxicants; polymer breakdown and synthesis; remodeling; and cell signaling. The present review focuses on the most industrially important esterases, namely lipases, phospholipases, cutinases, and polyethylene terephthalate hydrolases (PETases). Esterases are widely used in industrial and biotechnological applications. Notably, the biotechnological production of esters, including methyl acetate, ethyl acetate, vinyl acetate, polyvinyl acetate, and ethyl lactate, as an alternative to chemical production, represents a multi-billion-dollar industry. Currently, most enzymes (>75%) used in industrial processes are hydrolytic. Among them, lipases and phospholipases are primarily used for lipid modification. Lipases are the third most commercialized enzymes after proteases and carboxyhydrases, and their production is steadily increasing, currently representing over one-fifth of the global enzyme market. Esterases, particularly lipases, phospholipases, and cutinases, are employed in cosmetics, food, lubricants, pharmaceuticals, paints, detergents, paper, and biodiesel, among other industries. Overall, biotechnological production using enzymes is gaining global traction owing to its environmental benefits, high yields, and efficiency, aligning with green economy principles. Full article

The rapid accumulation of plastic and textile waste, particularly polyethylene terephthalate (PET), has emerged as a global challenge for sustainable resource management. Conventional recycling methods, including mechanical and chemical routes, recover limited value and often degrade material quality while consuming substantial energy. Biocatalytic recycling, by contrast, offers a resource-efficient alternative that transforms post-consumer PET into high-purity monomers under mild and environmentally benign conditions. This review examines advances in enzymatic PET depolymerization, focusing on hydrolases such as cutinases, PETases, MHETases, and lipases. The discussion highlights enzyme engineering, reactor design, and process integration that improve kinetics, thermostability, and yield. From a resource perspective, biocatalytic recycling redefines PET waste as a renewable carbon feedstock capable of re-entering industrial cycles, thereby reducing reliance on virgin petrochemicals and mitigating greenhouse gas emissions. Ultimately, this review positions biocatalytic PET recycling as a cornerstone technology for achieving circularity and advancing global resource sustainability. Full article

The accumulation of polyethylene terephthalate (PET) in the environment demands efficient microbial strategies for its degradation. This study evaluates the biodegradation potential of Schizophyllum commune BNT39 toward bis(2-hydroxyethyl) terephthalate (BHET), a major PET intermediate, and PET itself. Clear halos on BHET-agar plates indicated extracellular hydrolytic activity. In liquid culture, thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analyses revealed a three-phase degradation profile characterized by rapid BHET hydrolysis, transient dimer accumulation, and subsequent conversion to terephthalic acid (TPA). BHET was reduced by approximately 96% within seven days, while TPA accumulation reached 0.8 mg/mL after 30 days of incubation. Although PET degradation was limited, TPA was consistently detected as the principal product, with no BHET or MHET intermediates. To explore strategies for enhancing enzymatic activity, apple-derived cutin, PET, BHET, and polycaprolactone (PCL) were tested as inducers. Cutin markedly stimulated extracellular enzyme production, suggesting activation of cutinase-like enzymes. Overall, S. commune BNT39 demonstrates the ability to transform PET-related substrates, with cutin emerging as a promising natural stimulant to enhance enzymatic depolymerization. Future studies should focus on enzyme purification, activity profiling, and reaction optimization near PET’s glass transition temperature, where the polymer becomes more accessible for enzymatic attack. Full article

The Pichia system has been exploited for decades as a host for recombinant protein production, but there is still an information gap regarding problems that may arise with its use. The application of strains based on the methanol-induced alcohol oxidase 1 (AOX1) promoter may represent a safety issue, and its performance varies among strains. In this study, the ability of a Komagataella phaffii Mut^S KM71H strain to produce recombinant cutinases was evaluated and compared to that of the more widely used Mut⁺ X-33 strain. The effects of the nature of the cutinase (ANCUT1 and ANCUT3, from Aspergillus nidulans), methanol level, and inoculum concentrations were evaluated in shake flasks containing a complex medium. Higher activities and volumetric cutinase productivity were observed at lower induction cell densities (0.5%) for the Mut^S KM71H aox1::pPICZα-A-ANCUT1 strain, while a higher one (2%) yielded better results in KM71H aox1::pPICZα-A-ANCUT3. The best inoculum and inducer conditions for both strains yielded similar results. The behavior of the different cutinases in the Mut^S or Mut⁺ genetic background was opposed: strain KM71H aox1::pPICZα-A-ANCUT3 produced 19% more activity than strain X-33 aox1::pPICZα-A-ANCUT3, while the ANCUT1 containing strain produced significantly higher activity in the X-33 Mut⁺ strain. These results indicate that Mut^S strains are viable host options without the complications of rapidly growing methanol strains. The effect of the gene structure being expressed is a phenomenon that needs further exploration. Full article

Plastic waste, particularly poly(ethylene terephthalate) (PET), negatively impacts the environment and human health. Biotechnology could become an alternative to managing PET waste if enzymes ensure the recovery of terephthalic acid with efficiencies comparable to those of chemical treatments. Recent research has highlighted the potential of fungal cutinases, such as wild-type ANCUT1 (ANCUT1^wt) from Aspergillus nidulans, in achieving PET depolymerization. Fungal cutinases’ structures differ from those of bacterial cutinases, while their PET depolymerization mechanism has not been well studied. Here, a reliable model of the ANCUT1^wt was obtained using AlphaFold 2.0. Computational chemistry revealed potential cation-binding sites, which had not been described regarding enzymatic activation in fungal cutinases. Moreover, it allowed the prediction of residues with the ability to interact with a PET trimer that were mutation candidates to engineer the substrate binding cleft, seeking enhancements of PET hydrolysis. Enzyme kinetics revealed that both ANCUT1^wt and ANCUT1^N73V/L171Q (DM) were activated by MgCl₂, increasing the dissociation constant of the substrate and maximal reaction rate. We found that in the presence of MgCl₂, DM hydrolyzed different PET samples and released 9.1-fold more products than ANCUT1^wt. Scanning Electron Microscopy revealed a different hydrolysis mode of these enzymes, influenced by the polymer’s crystallinity and structure. Full article

Plastic waste accumulation presents critical environmental challenges demanding innovative circular economy solutions. This study developed a comprehensive machine learning framework to systematically identify optimal enzyme combinations for polyester depolymerization. We integrated kinetic parameters from the BRENDA database with sequence-derived features and network topology metrics to train ensemble classifiers predicting enzyme-substrate relationships. A multi-objective optimization algorithm evaluated enzyme combinations across four criteria: prediction confidence, substrate coverage, operational compatibility, and functional diversity. The ensemble classifier achieved 86.3% accuracy across six polymer families, significantly outperforming individual models. Network analysis revealed a modular organization with hub enzymes exhibiting broad substrate specificity. Multi-objective optimization identified 156 Pareto-optimal enzyme combinations, with top-ranked pairs achieving composite scores exceeding 0.89. The Cutinase–PETase combination demonstrated exceptional complementarity (score: $0.875\pm 0.008$), combining complete substrate coverage with high catalytic efficiency. Validation against experimental benchmarks confirmed enhanced depolymerization rates for recommended enzyme cocktails. This framework provides a systematic approach for enzyme prioritization in plastic valorization, advancing biological recycling technologies through data-driven biocatalyst selection while identifying key economic barriers requiring technological innovation. Full article

Poly(ε-caprolactone) (PCL) is a synthetic plastic known for its excellent physicochemical properties and a wide range of applications in packaging, coatings, foaming, and agriculture. In medicine, its versatility allows it to function as a scaffold for drug delivery, sutures, implants, tissue engineering, and 3D printing. In addition to its biocompatibility, PCL’s most notable characteristic is its biodegradability. However, this property is affected by temperature, microbial activity, and environmental conditions, which means PCL can sometimes remain in nature for long periods. This review shows that various types of microorganisms can efficiently degrade PCL, including different strains of Pseudomonas spp., Streptomyces spp., Alcaligenes faecalis, and fungi like Aspergillus oryzae, Fusarium spp., Rhizopus delemar, and Thermomyces lanuginosus. These microorganisms produce enzymes such as lipases, esterases, and cutinases that break down PCL into smaller molecules that act as substrates. The review also examines the phylogenetic diversity of organisms capable of biodegrading PCL, the biochemical pathways involved in this process, and specific aspects of the genetic framework responsible for the expression of the enzymes that facilitate degradation. Targeted research on microbial PCL biodegradation and its practical applications could significantly aid in reducing and managing plastic waste on a global ecological scale. Full article

Industrial biotechnology offers a potential ecological solution for PET recycling under relatively mild reaction conditions via enzymatic degradation, particularly using the leaf branch compost cutinase (LCC) quadruple mutant ICCG. To improve the efficient downstream processing of this biocatalyst after heterologous gene expression with a suitable production host, protein crystallization can serve as an effective purification/capture step. Enhancing protein crystallization was achieved in recent studies by introducing electrostatic (and aromatic) interactions in two homologous alcohol dehydrogenases (Lb/LkADH) and an ene reductase (NspER1-L1,5) produced with Escherichia coli. In this study, ICCG, which is difficult to crystallize, was utilized for the application of crystal contact engineering strategies, resulting in ICCG mutant L50Y (ICCGY). Previously focused on the Lys-Glu interaction for the introduction of electrostatic interactions at crystal contacts, the applicability of the engineering strategy was extended here to an Arg-Glu interaction to increase crystallizability, as shown for ICCGY T110E. Furthermore, the rationale of the engineering approach is demonstrated by introducing Lys and Glu at non-crystal contacts or sites without potential interaction partners as negative controls. These resulting mutants crystallized comparably but not superior to the wild-type protein. As demonstrated by this study, crystal contact engineering emerges as a promising approach for rationally enhancing protein crystallization. This advancement could significantly streamline biotechnological downstream processing, offering a more efficient pathway for research and industry. Full article

Plastic, a ubiquitous part of our daily lives, has become a global necessity, with annual production exceeding 300 million tons. However, the accumulation of synthetic polymers in our environment poses a pressing global challenge. To address this urgent issue, fungi have emerged as potential agents for plastic degradation. In our previous manuscript, ‘A Review of the Fungi That Degrade Plastic’, we explored the taxonomic placement of plastic-degrading fungi across three main phyla: Ascomycota, Basidiomycota, and Mucoromycota. In this review, we built upon that foundation and aimed to further explore the taxonomic relationships of these fungi in a comprehensive and detailed manner, leaving no stone unturned. Moreover, we linked metabolic activity and enzyme production of plastic-degrading fungi to their taxonomy and summarized a phylogenetic tree and a detailed table on enzyme production of plastic-degrading fungi presented here. Microbial enzymes are key players in polymer degradation, operating intra-cellularly and extra-cellularly. Fungi, one of the well-studied groups of microbes with respect to plastic degradation, are at the forefront of addressing the global issue of plastic accumulation. Their unique ability to hydrolyze synthetic plastic polymers and produce a wide range of specific enzymes is a testament to their potential. In this review, we gather and synthesize information concerning the metabolic pathways of fungi involved in the degradation of plastics. The manuscript explores the diverse range of specific enzymes that fungi can produce for plastic degradation and the major pathways of plastic metabolism. We provide a listing of 14 fungal enzymes (Esterase, Cutinase, Laccase, Peroxidases, Manganese peroxidase, Lignin peroxidase, Oxidoreductases, Urease, Protease, Lipase, Polyesterase, Dehydrogenase, Serine hydrolase, and PETase) involved in pathways for plastic degradation alongside the relevant fungi known to produce these enzymes. Furthermore, we integrate the fungi’s enzyme-producing capabilities with their taxonomy and phylogeny. Taxonomic and phylogenetic investigations have pinpointed three primary fungal classes (Eurotiomycetes, Sordariomycetes (Ascomycota), and Agaricomycetes (Basidiomycota)) as significant plastic degraders that produce the vital enzymes mentioned earlier. This paper provides a foundational resource for recognizing fungal involvement in the biodegradation of synthetic polymers. It will ultimately advance fungal biotechnology efforts to address the global issue of plastic accumulation in natural environments. Full article

Two recombinant cutinases, AfCutA and AfCutB, derived from Aspergillus fumigatus, were heterologously expressed in Pichia pastoris and systematically characterized for their biochemical properties and polyester-degrading capabilities. AfCutA demonstrated superior catalytic performance compared with AfCutB, displaying higher optimal pH (8.0–9.0 vs. 7.0–8.0), higher optimal temperature (60 °C vs. 50 °C), and greater thermostability. AfCutA exhibited increased hydrolytic activity toward p-nitrophenyl esters (C4–C16) and synthetic polyesters. Additionally, AfCutA released approximately 3.2-fold more acetic acid from polyvinyl acetate (PVAc) hydrolysis than AfCutB. Quartz crystal microbalance with dissipation monitoring (QCM-D) revealed rapid adsorption of both enzymes onto polyester films. However, their adsorption capacity on poly (ε-caprolactone) (PCL) films was significantly higher than on polybutylene succinate (PBS) films, and was influenced by pH. Comparative modeling of catalytic domains identified distinct structural differences between the two cutinases. AfCutA possesses a shallower substrate-binding cleft, fewer acidic residues, and more extensive hydrophobic regions around the active site, potentially explaining its enhanced interfacial activation and catalytic efficiency toward synthetic polyester substrates. The notably superior performance of AfCutA suggests its potential as a biocatalyst in industrial applications, particularly in polyester waste bioremediation and sustainable polymer processing. Full article