RESUMO
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 1.67â¯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 50â¯% of its activity even after incubation at 90⯰C for 16â¯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.
Assuntos
Hidrolases de Éster Carboxílico , Estabilidade Enzimática , Especificidade por Substrato , Cristalografia por Raios X , Hidrolases de Éster Carboxílico/metabolismo , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Ácidos Ftálicos/metabolismo , Ácidos Ftálicos/química , Ésteres/metabolismo , Ésteres/química , Modelos Moleculares , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Conformação Proteica , Concentração de Íons de Hidrogênio , Cinética , Hidrólise , Domínio Catalítico , TemperaturaRESUMO
BACKGROUND: Esterases (EC 3.1.1.X) are enzymes that catalyze the hydrolysis ester bonds. These enzymes have large potential for diverse applications in fine industries, particularly in pharmaceuticals, cosmetics, and bioethanol production. METHODS AND RESULTS: In this study, a gene encoding an esterase from Thermobifida fusca YX (TfEst) was successfully cloned, and its product was overexpressed in Escherichia coli and purified using affinity chromatography. The TfEst kinetic assay revealed catalytic efficiencies of 0.58 s-1 mM-1, 1.09 s-1 mM-1, and 0.062 s-1 mM-1 against p-Nitrophenyl acetate, p-Nitrophenyl butyrate, and 1-naphthyl acetate substrates, respectively. Furthermore, TfEst also exhibited activity in a pH range from 6.0 to 10.0, with maximum activity at pH 8.0. The enzyme demonstrated a half-life of 20 min at 70 °C. Notably, TfEst displayed acetyl xylan esterase activity as evidenced by the acetylated xylan assay. The structural prediction of TfEst using AlphaFold indicated that has an α/ß-hydrolase fold, which is consistent with other esterases. CONCLUSIONS: The enzyme stability over a broad pH range and its activity at elevated temperatures make it an appealing candidate for industrial processes. Overall, TfEst emerges as a promising enzymatic tool with significant implications for the advancement of biotechnology and biofuels industries.
Assuntos
Acetilesterase , Esterases , Thermobifida , Acetilesterase/metabolismo , Acetilesterase/genética , Acetilesterase/química , Concentração de Íons de Hidrogênio , Cinética , Especificidade por Substrato , Thermobifida/enzimologia , Thermobifida/genética , Esterases/metabolismo , Esterases/genética , Esterases/química , Estabilidade Enzimática , Temperatura , Escherichia coli/genética , Escherichia coli/metabolismo , Clonagem Molecular/métodos , Hidrólise , Xilanos/metabolismo , Butiratos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , NitrofenóisRESUMO
Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer that is used worldwide and raises concerns because of its prevalence in the environment and potential toxicity. Herein, the capability of Fusarium culmorum to degrade a high concentration (3 g/L) of DEHP as the sole carbon and energy source in solid-state fermentation (SSF) was studied. Cultures grown on glucose were used as controls. The biodegradation of DEHP by F. culmorum reached 96.9% within 312 h. This fungus produced a 3-fold higher esterase activity in DEHP-supplemented cultures than in control cultures (1288.9 and 443.2 U/L, respectively). In DEHP-supplemented cultures, nine bands with esterase activity (24.6, 31.2, 34.2, 39.5, 42.8, 62.1, 74.5, 134.5, and 214.5 kDa) were observed by zymography, which were different from those in control cultures and from those previously reported for cultures grown in submerged fermentation. This is the first study to report the DEHP biodegradation pathway by a microorganism grown in SSF. The study findings uncovered a novel biodegradation strategy by which high concentrations of DEHP could be biodegraded using two alternative pathways simultaneously. F. culmorum has an outstanding capability to efficiently degrade DEHP by inducing esterase production, representing an ecologically promising alternative for the development of environmental biotechnologies, which might help mitigate the negative impacts of environmental contamination by this phthalate. KEY POINTS: ⢠F. culmorum has potential to tolerate and remove di(2-ethylhexyl) phthalate (DEHP) ⢠Solid-state fermentation is an efficient system for DEHP degradation by F. culmorum ⢠High concentrations of DEHP induce high levels of esterase production by F. culmorum.
Assuntos
Dietilexilftalato , Fusarium , Ácidos Ftálicos , Dietilexilftalato/metabolismo , Biodegradação Ambiental , Esterases/metabolismoRESUMO
Abstract Earias vittellaFabricius, 1794 (Noctuidae: Lepidoptera) is deliberated to be one of the most destructive pests of cotton and okra vegetation in the world including Asia. The pest has established resistance to various synthetic insecticides. The use of bio-pesticide is one of the unconventional approaches to develop a vigorous ecosystem without harming non- target pests and beneficial natural insect fauna. In the present study, the toxicity levels of Citrullus colocynthis seed extract have been evaluated against the populations of E. vittellaunder standardized laboratory conditions. The toxic effects of C. colocynthis on development periods, protein contents and esterase activity of the life stages of E. vittella were also evaluated. The toxicity levels of methanol, ethanol, hexane, water and profenofos were evaluated on the 1st instar larvae of E. vittella. LC30 and LC80 concentrations exhibited the effectiveness of methanol-based C. colocynthis seed extract against 1st instar larvae of E. vitella. The enhanced larval and pupal periods were revealed in treated samples during the comparison with untreated samples. The intrinsic rate of increase, net reproductive rate in the LC30 and LC80 concentrations exposed larvae remained less than the control treatment. Fecundity, the esterase activity and protein contents were declined in LC30 and LC80 treated samples as compared to the control. The present findings suggest that C. colosynthis extracts based botanical insecticides are beneficial, ecosystem sustainable and can be integrated with insect management programs from environment safety perspective.
Resumo Earias vittella Fabricius, 1794 (Noctuidae: Lepidoptera) é considerada uma das pragas mais destrutivas de algodão e quiabo no mundo, incluindo a Ásia. Essa praga estabeleceu resistência a vários inseticidas sintéticos. O uso de biopesticidas é uma das abordagens não convencionais para desenvolver um ecossistema saudável sem prejudicar as pragas não alvo e a fauna natural benéfica de insetos. No presente estudo, os níveis de toxicidade do extrato de semente de Citrullus colocynthis foram avaliados nas populações de E. vittella em condições de laboratório padronizadas. Os efeitos tóxicos de C. colocynthis nos períodos de desenvolvimento, conteúdo de proteína e atividade esterase das fases de vida de E. vittella também foram avaliados. Os níveis de toxicidade de metanol, etanol, hexano, água e profenofós foram avaliados em larvas de 1º instar de E. vittella. As concentrações de LC30 e LC80 apresentaram eficácia do extrato de sementes de C. colocynthis à base de metanol contra larvas de 1º instar de E. vittella. Os períodos larval e pupal aumentados foram revelados nas amostras tratadas durante a comparação com as amostras não tratadas. A taxa intrínseca de aumento e a taxa reprodutiva líquida nas concentrações de larvas expostas LC30 e LC80 permaneceram menores do que o tratamento controle. A fecundidade, a atividade da esterase e o conteúdo de proteína diminuíram nas amostras tratadas com LC30 e LC80 em comparação com o controle. As presentes descobertas sugerem que os extratos de C. colocynthis à base de inseticidas botânicos são benéficos, sustentáveis para o ecossistema e podem ser integrados com programas de manejo de insetos do ponto de vista da segurança ambiental.
Assuntos
Animais , Citrullus colocynthis , Inseticidas , Mariposas , Extratos Vegetais/farmacologia , Ecossistema , LarvaRESUMO
Abstract Earias vittellaFabricius, 1794 (Noctuidae: Lepidoptera) is deliberated to be one of the most destructive pests of cotton and okra vegetation in the world including Asia. The pest has established resistance to various synthetic insecticides. The use of bio-pesticide is one of the unconventional approaches to develop a vigorous ecosystem without harming non- target pests and beneficial natural insect fauna. In the present study, the toxicity levels of Citrullus colocynthis seed extract have been evaluated against the populations of E. vittellaunder standardized laboratory conditions. The toxic effects of C. colocynthis on development periods, protein contents and esterase activity of the life stages of E. vittella were also evaluated. The toxicity levels of methanol, ethanol, hexane, water and profenofos were evaluated on the 1st instar larvae of E. vittella. LC30 and LC80 concentrations exhibited the effectiveness of methanol-based C. colocynthis seed extract against 1st instar larvae of E. vitella. The enhanced larval and pupal periods were revealed in treated samples during the comparison with untreated samples. The intrinsic rate of increase, net reproductive rate in the LC30 and LC80 concentrations exposed larvae remained less than the control treatment. Fecundity, the esterase activity and protein contents were declined in LC30 and LC80 treated samples as compared to the control. The present findings suggest that C. colosynthis extracts based botanical insecticides are beneficial, ecosystem sustainable and can be integrated with insect management programs from environment safety perspective.
Resumo Earias vittella Fabricius, 1794 (Noctuidae: Lepidoptera) é considerada uma das pragas mais destrutivas de algodão e quiabo no mundo, incluindo a Ásia. Essa praga estabeleceu resistência a vários inseticidas sintéticos. O uso de biopesticidas é uma das abordagens não convencionais para desenvolver um ecossistema saudável sem prejudicar as pragas não alvo e a fauna natural benéfica de insetos. No presente estudo, os níveis de toxicidade do extrato de semente de Citrullus colocynthis foram avaliados nas populações de E. vittella em condições de laboratório padronizadas. Os efeitos tóxicos de C. colocynthis nos períodos de desenvolvimento, conteúdo de proteína e atividade esterase das fases de vida de E. vittella também foram avaliados. Os níveis de toxicidade de metanol, etanol, hexano, água e profenofós foram avaliados em larvas de 1º instar de E. vittella. As concentrações de LC30 e LC80 apresentaram eficácia do extrato de sementes de C. colocynthis à base de metanol contra larvas de 1º instar de E. vittella. Os períodos larval e pupal aumentados foram revelados nas amostras tratadas durante a comparação com as amostras não tratadas. A taxa intrínseca de aumento e a taxa reprodutiva líquida nas concentrações de larvas expostas LC30 e LC80 permaneceram menores do que o tratamento controle. A fecundidade, a atividade da esterase e o conteúdo de proteína diminuíram nas amostras tratadas com LC30 e LC80 em comparação com o controle. As presentes descobertas sugerem que os extratos de C. colocynthis à base de inseticidas botânicos são benéficos, sustentáveis para o ecossistema e podem ser integrados com programas de manejo de insetos do ponto de vista da segurança ambiental.
RESUMO
Background Esterases (EC 3.1.1.X) are enzymes that catalyze the hydrolysis ester bonds. These enzymes have large potential for diverse applications in fne industries, particularly in pharmaceuticals, cosmetics, and bioethanol production. Methods and results In this study, a gene encoding an esterase from Thermobifda fusca YX (TfEst) was successfully cloned, and its product was overexpressed in Escherichia coli and purifed using afnity chromatography. The TfEst kinetic assay revealed catalytic efciencies of 0.58 s −1 mM−1, 1.09 s−1 mM−1, and 0.062 s−1 mM−1 against p-Nitrophenyl acetate, p-Nitrophenyl butyrate, and 1-naphthyl acetate substrates, respectively. Furthermore, TfEst also exhibited activity in a pH range from 6.0 to 10.0, with maximum activity at pH 8.0. The enzyme demonstrated a half-life of 20 min at 70 °C. Notably, TfEst displayed acetyl xylan esterase activity as evidenced by the acetylated xylan assay. The structural prediction of TfEst using AlphaFold indicated that has an α/β-hydrolase fold, which is consistent with other esterases. Conclusions The enzyme stability over a broad pH range and its activity at elevated temperatures make it an appealing candidate for industrial processes. Overall, TfEst emerges as a promising enzymatic tool with signifcant implications for the advancement of biotechnology and biofuels industries.
RESUMO
Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer, which is considered an endocrine disrupting pollutant. Growth kinetics and esterases activity by biochemical tests and polyacrylamide gel electrophoresis were characterized for Fusarium culmorum grown in DEHP-supplemented (1000â¯mg/L) medium as the only carbon source and in control medium with glucose. Intermediate compounds of biodegraded DEHP were identified by GC-MS. F. culmorum degraded 92% of DEHP within 36â¯h. DEHP was degraded to butanol, hexanal, catechol and acetic acid. It is suggested that the first two compounds would transform into butanediol and the last two would enter into the Krebs cycle and would be mineralized to CO2 and H2O. DEHP induced eight esterase isoforms, which were different to those constitutive isoforms produced in the control medium. It is suggested that five enzymes (25.7, 29.5, 31.8, 97.6 and 144.5â¯kDa) detected during the first 36â¯h be involved in the primary biodegradation of DEHP. The rest of the enzymes (45.9, 66.6 and 202.9â¯kDa) might be involved in the final steps for DEHP metabolism. F. culmorum has a promising practical application in the treatment of DEHP-contaminated environments because it can secrete specific esterase to breakdown high concentrations of DEHP in a short period of time. This research represents the first approach for the study of esterase involved in the DEHP degradation by fungi using this phthalate as the sole source of carbon and energy.
Assuntos
Dietilexilftalato/análise , Disruptores Endócrinos/análise , Poluentes Ambientais/análise , Fusarium/crescimento & desenvolvimento , Plastificantes/análise , Biodegradação Ambiental , Esterases/metabolismo , Fusarium/enzimologia , CinéticaRESUMO
Cinnamoyl esterases (CE) are microbial and mammalian intestinal enzymes able to release antioxidant hydroxycinnamic acids from their non-digestible ester-linked forms naturally present in vegetable foods. Previous findings showed that oral administration of Lactobacillus fermentum CRL1446 increased intestinal CE activity and improved oxidative status in mice. The aim of this work was to evaluate the in vitro CE activity of L. fermentum CRL1446 and the effect of bile on this activity, as well as strain resistance to simulated gastrointestinal tract (GIT) conditions and its ability to adhere to intestinal epithelium and influence its basal CE activity. L. fermentum CRL1446 and L. fermentum ATCC14932 (positive control for CE activity) were able to hydrolyse different synthetic hydroxycinnamates, with higher specificity toward methyl ferulate (3,853.73 and 899.19 U/g, respectively). Feruloyl esterase (FE) activity was mainly intracellular in L. fermentum CRL1446 and cell-surface associated in L. fermentum ATCC14932. Both strains tolerated simulated GIT conditions and were able to adhere ex vivo to intestinal epithelium. Pre-incubation of L. fermentum strains with bile increased FE activity in both whole cells and supernatants (~2-fold), compared to controls, suggesting that cells were permeabilised by bile, allowing more substrate to enter the cell and/or leakage of FE enzymes. Three-fold higher FE activities were detected in intestinal tissue fragments with adhered L. fermentum CRL1446 cells compared to control fragments (without bacteria), indicating that this strain provides exogenous FE activity and could stimulate esterase activity in the intestinal mucosa. Finally, we found that milk fat had a negative effect on FE activity of intestinal tissue, in absence or presence of adhered L. fermentum. These results help explaining the increase in intestinal FE activity previously observed in mice fed with L. fermentum CRL1446, and support the potential use of this strain for the development of new functional foods directed to oxidative stress-related ailments.