RESUMO
This study addresses the environmental problem of PET plastic through in silico bioprospecting for the identification and experimental validation of novel PET degrading eukaryotes through the in silico bioprospectingI of PETases, employing a methodology that combines Hidden Markov Models (HMMs), clustering techniques, molecular docking, and dynamic simulations. A total of 424 putative PETase sequences were identified from 219 eukaryotic organisms, highlighting six sequences with low affinity energies. The Aspergillus luchuensis sequence showed the lowest Gibbs free energy and exhibited stability at different temperatures in molecular dynamics assays. Experimental validation, through a plate clearance assay and HPLC, confirmed PETase activity in three wild-type fungal strains, with A. luchuensis showing the highest efficiency. The results obtained demonstrate the effectiveness of combining computational and experimental approaches as proof of concept to discover and validate eukaryotes with PET-degrading capabilities opening new perspectives for the sustainable management of this type of waste and contributing to its environmental mitigation.
Assuntos
Biodegradação Ambiental , Bioprospecção , Eucariotos , Simulação por Computador , Aspergillus/enzimologiaRESUMO
Lignin, a complex heteropolymer present in plant cell walls, is now recognized as a valuable renewable resource with potential applications in various industries. The lignin biorefinery concept, which aims to convert lignin into value-added products, has gained significant attention in recent years. ß-etherases, enzymes that selectively cleave ß-O-4 aryl ether bonds in lignin, have shown promise in lignin depolymerization. In this study, the ß-etherase LigF from Altererythrobacter sp. B11 was cloned, expressed, purified, and biochemically characterized. The LigF-AB11 enzyme exhibited optimal activity at 32 °C and pH 8.5 when catalyzing the substrate PNP-AV. The enzyme displayed mesophilic behavior and demonstrated higher activity at moderate temperatures. Stability analysis revealed that LigF-AB11 was not thermostable, with a complete loss of activity at 60 °C within an hour. Moreover, LigF-AB11 exhibited excellent pH stability, retaining over 50 % of its activity after 1 h under pH conditions ranging from 3.0 to 11.0. Metal ions and surface impregnation agents were found to affect the enzyme's activity, highlighting the importance of considering these factors in enzymatic processes for lignin depolymerization. This study provides valuable insights into the biochemical properties of LigF-AB11 and contributes to the development of efficient enzymatic processes for lignin biorefineries. Further optimization and understanding of ß-etherases will facilitate their practical application in the valorization of lignin.
RESUMO
OBJECTIVES: Ustilago maydis lipase A (UMLA) expressed in Pichia pastoris was compared with Candida antarctica lipase A (CALA) to study its biochemical properties such as thermostability and selectivity. RESULTS: UMLA had similar behavior to its homologue CALA regarding the effect of pH and temperature on enzymatic activity, substrate preference and selectivity. Both lipases were active on insoluble triglycerides as well as natural oils and hydrolyzed preferably esters with short and medium acyl and alkyl chains. Both enzymes were slightly selective for the (S)-glycidyl butyrate enantiomer and had a remarkable preference for the sn-2 position of triglycerides. The optimal activity was 40 and 50 °C for UMLA and CALA, respectively. However, temperature had a greater effect on the stability of UMLA compared to CALA, observing a half-life at 50 °C of 2.07 h and 12.83 h, respectively. CONCLUSIONS: UMLA shares some biochemical properties with CALA such as the sn-2 preference on triglyceride hydrolysis and transesterification. However, the high thermostability attributed to CALA was not observed in UMLA; this can be due to the lack of stabilization via AXXXA motifs in helices and fewer proline residues at the surface.