RESUMO
Glucuronoyl esterases (GEs) are carbohydrate active enzymes in carbohydrate esterase family 15 which are involved in the hydrolysis of lignin-carbohydrate complexes. They are encoded by a wide range of aerobic and anaerobic fungi and bacteria inhabiting diverse environments. The rumen microbiome is a complex microbial community with a wide array of enzymes that specialize in deconstructing plant cell wall carbohydrates. Enzymes from the rumen tend to show low similarity to homologues found in other environments, making the rumen microbiome a promising source for the discovery of novel enzymes. Using a combination of phylogenetic and structural analysis, we investigated the structure-function relationship of GEs from the rumen bacteria Fibrobacter succinogenes and Ruminococcus flavefaciens, and from the rumen fungus, Piromyces rhizinflata. All adopt a canonical α/ß hydrolase fold and possess a structurally conserved Ser-His-Glu/Asp catalytic triad. Structural variations in the enzymes are localized to loops surrounding the active site. Analysis of the active site structures in these enzymes emphasized the importance of structural plasticity in GEs with non-canonical active site conformations. We hypothesize that interkingdom HGT events may have contributed to the diversity of GEs in the rumen, and this is demonstrated by the phylogenetic and structural similarity observed between rumen bacterial and fungal GEs. This study advances our understanding of the structure-function relationship in glucuronoyl esterases and illuminates the evolutionary dynamics that contribute to enzyme diversity in the rumen microbiome.
Assuntos
Proteínas de Bactérias , Filogenia , Piromyces , Rúmen , Rúmen/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Piromyces/enzimologia , Piromyces/genética , Esterases/genética , Esterases/química , Esterases/metabolismo , Esterases/classificação , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fibrobacter/enzimologia , Fibrobacter/genética , Fibrobacter/classificação , Domínio Catalítico , Ruminococcus/enzimologia , Ruminococcus/genética , Ruminococcus/classificação , Modelos MolecularesRESUMO
This study examined the pattern of resistance to widely applied synthetic pyrethroids, i.e., cypermethrin and deltamethrin, against larvae of Rhipicephalus microplus ticks sampled from Marathwada region in Maharashtra, India. The study also examined the role of α- and ß-esterases and glutathione-S-transferase (GST) in resistance development. All eight R. microplus isolates tested were resistant to deltamethrin (RL IV), having RR50 values from 6.88 to 131.26. LPT analysis exhibited the resistance level II deltamethrin resistance in Beed and Hingoli, III in Dharashiv, and IV in Sambhajinagar, Parbhani, Latur, Jalna, and Nanded isolates. The LIT analysis showed that Dharashiv field isolates had the lowest LC50 value of 229.09 ppm against cypermethrin, while Sambhajinagar field isolates had the highest at 489.78 ppm. The RR50 ranged from 1145.45 to 2448.9. Seven isolates were level I resistant to cypermethrin while the Jalna isolate was level II resistant. In larvae treated with deltamethrin and cypermethrin, the activity of α- and ß-esterase enzymes increased significantly compared to control groups. The enzyme ratios in treated larvae ranged from 0.7533 to 1.7023 for α-esterase and 0.7434 to 3.2054 for ß-esterase. The Hingoli isolate treated with cypermethrin exhibited the highest α-esterase activity (903.261), whereas Sambhajinagar isolate had the highest GST enzyme ratio (2.8224) after deltamethrin exposure. When exposed to cypermethrin, the Hingoli isolate showed the highest GST enzyme ratio, 2.0832. The present study provides the current resistance status in tick populations from Marathwada region indicating deltamethrin and cypermethrin to be ineffective for tick control. The results also suggest that SP compounds should be regulated in this region and alternative control strategies should be introduced.
Assuntos
Acaricidas , Glutationa Transferase , Larva , Nitrilas , Piretrinas , Rhipicephalus , Animais , Piretrinas/farmacologia , Índia , Rhipicephalus/efeitos dos fármacos , Rhipicephalus/enzimologia , Nitrilas/farmacologia , Larva/efeitos dos fármacos , Glutationa Transferase/metabolismo , Acaricidas/farmacologia , Esterases/metabolismo , Resistência a Inseticidas , Resistência a MedicamentosRESUMO
GDSL-type esterase/lipase protein (GELP) genes are crucial in the specialized lipid metabolism, in the responses to abiotic stresses, and in the regulation of plant homeostasis. R. communis is an important oilseed crop species that can sustain growth and productivity when exposed to harsh environmental conditions. Herein, we raised the question of whether the GELP gene family could be involved in the acquisition of R. communis tolerance to abiotic stresses during seed germination and seedling establishment. Thus, we used bioinformatics and transcriptomics to characterize the R. communis GELP gene family. R. communis genome possesses 96 GELP genes that were characterized by extensive bioinformatics, including phylogenetic analysis, subcellular localization, exon-intron distribution, the analysis of regulatory cis-elements, tandem duplication, and physicochemical properties. Transcriptomics indicated that numerous RcGELP genes are readily responsive to high-temperature and salt stresses and might be potential candidates for genome editing techniques to develop abiotic stress-tolerant crops.
Assuntos
Regulação da Expressão Gênica de Plantas , Germinação , Proteínas de Plantas , Ricinus , Plântula , Estresse Fisiológico , Plântula/genética , Plântula/crescimento & desenvolvimento , Estresse Fisiológico/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Germinação/genética , Ricinus/genética , Ricinus/metabolismo , Esterases/genética , Esterases/metabolismo , Filogenia , Lipase/genética , Lipase/metabolismo , Família Multigênica , Genoma de Planta/genéticaRESUMO
Esterases are crucial for aryloxyphenoxypropionate herbicide (AOPP) biodegradation. However, the underlying molecular mechanisms of AOPP biodegradation by esterases are poorly understood. In the current work, Corynebacterium sp. Z-1 was isolated and found to degrade multiple AOPPs, including quizalofop-p-ethyl (QPE), haloxyfop-p-methyl (HPM), fenoxaprop-p-ethyl (FPE), cyhalofop-butyl (CYB), and clodinafop-propargyl (CFP). A novel esterase, QfeH, which catalyzes the cleavage of ester bonds in AOPPs to form AOPP acids, was identified from strain Z-1. The catalytic activities of QfeH toward AOPPs decreased in the following order: CFP > FPE > CYB > QPE > HPM. Molecular docking, computational analyses, and site-directed mutagenesis indicated the catalytic mechanisms of QfeH-mediated degradation of different AOPPs. Notably, the key residue S159 is essential for the activity of QfeH. Moreover, V222Y, T227M, T227A, A271R, and M275K mutants, exhibiting 2.9-5.0 times greater activity than QfeH, were constructed. This study facilitates the mechanistic understanding of AOPPs bioremediation by esterases.
Assuntos
Biodegradação Ambiental , Corynebacterium , Esterases , Herbicidas , Herbicidas/metabolismo , Herbicidas/química , Esterases/metabolismo , Esterases/genética , Esterases/química , Corynebacterium/metabolismo , Corynebacterium/genética , Corynebacterium/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Simulação de Acoplamento Molecular , Propionatos/metabolismoRESUMO
The promoter is an essential component of an expression system since it regulates the transcriptional beginning of related genes. The optimal expression level can be achieved by employing a promoter engineering approach. Typically, creating a library of T7 promoters allows for titratable protein expression. In the process of making ß-amino acid (sitagliptin intermediate) from ß-keto ester, esterase from Pseudomonas stutzeri (Est PS) is used to convert the ß-keto ester to ß-keto acid. Subsequently, transaminase from Ilumatobacter coccineus (TAIC) transforms the ß-keto acid to its corresponding ß-amino acid. Here, we describe the optimization of the expression levels of Est PS for the maximum production of sitagliptin intermediate. The different promoter strengths for Est PS were built into the T7 promoters of the pET15b vector. With the help of these new co-expressing entire cells, the expressed enzyme ratio for each enzyme was determined. As the strength of the promoter of Est PS decreases, the expression level also decreases (from 100% to 10%). Conversely, the TAIC expression level is increased. This developed system produced a higher sitagliptin intermediate than enzymes' unoptimized expression level.
Assuntos
Regiões Promotoras Genéticas , Fosfato de Sitagliptina , Esterases/genética , Esterases/metabolismo , Pseudomonas stutzeri/genética , Pseudomonas stutzeri/metabolismo , Transaminases/genética , Transaminases/metabolismo , Vetores Genéticos/genética , Expressão Gênica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Environmental concerns arising from the increasing use of polluting plastics highlight polylactic acid (PLA) as a promising eco-friendly alternative. PLA is a biodegradable polyester that can be produced through the fermentation of renewable resources. Together with its excellent properties, suitable for a wide range of applications, the use of PLA has increased significantly over the years and is expected to further grow. However, insufficient degradability under natural conditions emphasizes the need for the exploration of biodegradation mechanisms, intending to develop more efficient techniques for waste disposal and recycling or upcycling. Biodegradation occurs through the secretion of depolymerizing enzymes, mainly proteases, lipases, cutinases, and esterases, by various microorganisms. This review focuses on the enzymatic degradation of PLA and presents different enzymes that were isolated and purified from natural PLA-degrading microorganisms, or recombinantly expressed. The review depicts the main characteristics of the enzymes, including recent advances and analytical methods used to evaluate enantiopurity and depolymerizing activity. While complete degradation of solid PLA particles is still difficult to achieve, future research and improvement of enzyme properties may provide an avenue for the development of advanced procedures for PLA degradation and upcycling, utilizing its building blocks for further applications as envisaged by circular economy principles. KEY POINTS: ⢠Enzymes can be promisingly utilized for PLA upcycling. ⢠Natural and recombinant PLA depolymerases and methods for activity evaluation are summarized. ⢠Approaches to improve enzymatic degradation of PLA are discussed.
Assuntos
Biodegradação Ambiental , Poliésteres , Poliésteres/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Hidrolases de Éster Carboxílico/genética , Lipase/metabolismo , Esterases/metabolismo , Bactérias/enzimologia , Bactérias/metabolismo , Peptídeo Hidrolases/metabolismoRESUMO
The primary control methods for the African malaria mosquito, Anopheles gambiae, are based on insecticidal interventions. Emerging resistance to these compounds is therefore of major concern to malaria control programs. The organophosphate (OP), pirimiphos-methyl, is a relatively new chemical in the vector control armory but is now widely used in indoor-residual spray campaigns. While generally effective, phenotypic resistance has developed in some areas in malaria vectors. Here, we used a population genomic approach to identify novel mechanisms of resistance to pirimiphos-methyl in A. gambiae s.l mosquitoes. In multiple populations, we found large and repeated signals of selection at a locus containing a cluster of detoxification enzymes, some of whose orthologs are known to confer resistance to OPs in Culex pipiens. Close examination revealed a pair of alpha-esterases, Coeae1f and Coeae2f, and a complex and diverse pattern of haplotypes under selection in A. gambiae, A. coluzzii and A. arabiensis. As in C. pipiens, copy number variants have arisen at this locus. We used diplotype clustering to examine whether these signals arise from parallel evolution or adaptive introgression. Using whole-genome sequenced phenotyped samples, we found that in West Africa, a copy number variant in A. gambiae is associated with resistance to pirimiphos-methyl. Overall, we demonstrate a striking example of contemporary parallel evolution which has important implications for malaria control programs.
Assuntos
Anopheles , Esterases , Resistência a Inseticidas , Inseticidas , Mosquitos Vetores , Compostos Organotiofosforados , Animais , Anopheles/genética , Resistência a Inseticidas/genética , Mosquitos Vetores/genética , Inseticidas/farmacologia , Esterases/genética , Evolução MolecularRESUMO
Multitarget strategies are essential in addressing complex diseases, yet developing multitarget-directed ligands (MTDLs) is particularly challenging when aiming to engage multiple therapeutic targets across different tissues. Here, we present a molecular transformer strategy, enhancing traditional MTDLs. By utilizing esterase-driven hydrolysis, this approach mimics the adaptive nature of transformers for enabling molecules to modify their pharmacological effects in response to the biological milieu. By virtual screening and biological evaluation, we identified KGP-25, a novel compound initially targeting the voltage-gated sodium channel 1.8 (Nav1.8) in the peripheral nervous system (PNS) for analgesia, and later the γ-aminobutyric acid subtype A receptor (GABAA) in the central nervous system (CNS) for general anesthesia. Our findings confirm KGP-25's dual efficacy in cellular and animal models, effectively reducing opioid-related side effects. This study validates the molecular transformer approach in drug design and highlights its potential to overcome the limitations of conventional MTDLs, paving new avenues in innovative therapeutic strategies.
Assuntos
Analgésicos , Esterases , Ligantes , Analgésicos/farmacologia , Analgésicos/química , Animais , Humanos , Esterases/metabolismo , Anestésicos/farmacologia , Anestésicos/química , Receptores de GABA-A/metabolismo , Receptores de GABA-A/química , Desenho de Fármacos , Camundongos , MasculinoRESUMO
Genetic code expansion has emerged as a powerful tool for precisely introducing unnatural chemical structures into proteins to improve their catalytic functions. Given the high catalytic propensity of histidine in the enzyme pocket, increasing the chemical diversity of catalytic histidine could result in new characteristics of biocatalysts. Herein, we report the genetically encoded Nδ-Vinyl Histidine (δVin-H) and achieve the wild-type-like incorporation efficiency by the evolution of pyrrolysyl tRNA synthetase. As histidine usually acts as the nucleophile or the metal ligand in the catalytic center, we replace these two types of catalytic histidine to δVin-H to improve the performance of the histidine-involved catalytic center. Additionally, we further demonstrate the improvements of the hydrolysis activity of a previously reported organocatalytic esterase (the OE1.3 variant) in the acidic condition and myoglobin (Mb) catalyzed carbene transfer reactions under the aerobic condition. As histidine is one of the most frequently used residues in the enzyme catalytic center, the derivatization of the catalytic histidine by δVin-H holds a great potential to promote the performance of biocatalysts.
Assuntos
Domínio Catalítico , Histidina , Histidina/metabolismo , Histidina/química , Histidina/genética , Mioglobina/genética , Mioglobina/química , Mioglobina/metabolismo , Biocatálise , Catálise , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Aminoacil-tRNA Sintetases/química , Esterases/genética , Esterases/metabolismo , Esterases/química , Hidrólise , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
With an increase in the commercialization of bioplastics, the importance of screening for plastic-degrading strains and microbes has emerged. Conventional methods for screening such strains are time-consuming and labor-intensive. Therefore, we suggest a method for quickly and effectively screening plastic-degrading microbial strains through dual esterase assays for soil and isolated strains, using p-nitrophenyl alkanoates as substrates. To select microbe-abundant soil, the total amount of phospholipid fatty acids (PLFAs) included in each soil sample was analyzed, and esterase assays were performed for each soil sample to compare the esterase activity of each soil. In addition, by analyzing the correlation coefficients and sensitivity between the amount of PLFAs and the degree of esterase activity according to the substrate, it was confirmed that substrate pNP-C2 is the most useful index for soil containing several microbes having esterase activity. In addition, esterase assays of the isolated strains allowed us to select the most active strain as the degrading strain, and 16S rRNA results confirmed that it was Bacillus sp. N04 showed the highest degradation activity for polybutylene succinate (PBS) as measured in liquid culture for 7 days, with a degradation yield of 99%. Furthermore, Bacillus sp. N04 showed degradation activity against various bioplastics. We propose the dual application of p-nitrophenyl alkanoates as an efficient method to first select the appropriate soil and then to screen for plastic-degrading strains in it, and conclude that pNP-C2 in particular, is a useful indicator.
Assuntos
Biodegradação Ambiental , Esterases , Nitrofenóis , Microbiologia do Solo , Nitrofenóis/metabolismo , Esterases/metabolismo , Solo/química , Bactérias/metabolismo , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/classificação , RNA Ribossômico 16S/genética , Ácidos Graxos/metabolismo , Bacillus/metabolismo , Bacillus/genética , Bacillus/isolamento & purificação , Fosfolipídeos/metabolismo , Plásticos Biodegradáveis/metabolismoRESUMO
In recent years, numerous attempts have been made to develop a low-cost adsorbent for selectively recovering industrially important products from fermentation broth or complex mixtures. The current study is a novel attempt to selectively adsorb esterase from Trichoderma harzianum using cheap adsorbents like bentonite (BT), activated charcoal (AC), silicon dioxide (SiO2), and titanium dioxide (TiO2). AC had the highest esterase adsorption of 97.58% due to its larger surface area of 594.45 m3/g. SiO2 was found to have the highest selectivity over esterase, with an estimated purification fold of 7.2. Interestingly, the purification fold of 5.5 was found in the BT-extracted fermentation broth. The functional (FT-IR) and morphological analysis (SEM-EDX) were used to characterize the adsorption of esterase. Esterase adsorption on AC, SiO2, and TiO2 was well fitted by Freundlich isotherm, demonstrating multilayer adsorption of esterase. A pseudo-second-order kinetic model was developed for esterase adsorption in various adsorbents. Thermodynamic analysis revealed that adsorption is an endothermic process. AC has the lowest Gibbs free energy of -10.96 kJ/mol, which supports the spontaneous maximum adsorption of both esterase and protein. In the desorption study, the maximum recovery of esterase from TiO2 using sodium chloride was 41.34 %. Unlike other adsorbents, the AC-adsorbed esterase maintained its catalytic activity and stability, implying that it could be used as an immobilization system for commercial applications. According to the kinetic analysis, the overall rate of the reaction was controlled by reaction kinetics rather than external mass transfer resistance, as indicated by the Damkohler number.
Assuntos
Esterases , Adsorção , Cinética , Esterases/metabolismo , Esterases/química , Esterases/isolamento & purificação , Carvão Vegetal/química , Titânio/química , Termodinâmica , Dióxido de Silício/química , Hypocreales/enzimologia , Biocatálise , Bentonita/químicaRESUMO
The global landscape of Candida infections has seen a significant shift. Previously, Candida albicans was the predominant species. However, there has been an emergence of non-albicans Candida species, which are often less susceptible to antifungal treatment. Candida kefyr, in particular, has been increasingly associated with infections. This study aimed to investigate the profiles of enzymatic activity and biofilm formation in both clinical and non-clinical isolates of C. kefyr. A total of 66 C. kefyr isolates were analysed. The activities of proteinase and phospholipase were assessed using bovine serum albumin and egg yolk agar, respectively. Haemolysin, caseinolytic and esterase activities were evaluated using specific methods. Biofilm formation was investigated using crystal violet staining. The findings indicated that biofilm and proteinase activity were detected in 81.8% and 93.9% of all the isolates, respectively. Haemolysin activity was observed with the highest occurrence (95.5%) among normal microbiota isolates. Esterase activity was predominantly identified in dairy samples and was absent in hospital samples. Caseinase production was found with the highest occurrence (18.2%) in normal microbiota and hospital samples. Phospholipase activity was limited, found in only 3% of all the isolates. These findings reveal variations in enzyme activity between clinical and non-clinical C. kefyr isolates. This sheds light on their pathogenic potential and has implications for therapeutic strategies.
Assuntos
Biofilmes , Candida , Candidíase , Fosfolipases , Biofilmes/crescimento & desenvolvimento , Candida/isolamento & purificação , Candida/enzimologia , Candida/fisiologia , Candida/classificação , Humanos , Candidíase/microbiologia , Fosfolipases/metabolismo , Esterases/metabolismo , Proteínas Hemolisinas/metabolismo , Peptídeo Hidrolases/metabolismo , Microbiologia AmbientalRESUMO
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
The rising use of plastic results in an appalling amount of waste which is scattered into the environment. One of these plastics is PET which is mainly used for bottles. We have identified and characterized an esterase from Streptomyces, annotated as LipA, which can efficiently degrade the PET-derived oligomer BHET. The Streptomyces coelicolor ScLipA enzyme exhibits varying sequence similarity to several BHETase/PETase enzymes, including IsPETase, TfCut2, LCC, PET40 and PET46. Of 96 Streptomyces strains, 18% were able to degrade BHET via one of three variants of LipA, named ScLipA, S2LipA and S92LipA. SclipA was deleted from S. coelicolor resulting in reduced BHET degradation. Overexpression of all LipA variants significantly enhanced BHET degradation. All variants were expressed in E. coli for purification and biochemical analysis. The optimum conditions were determined as pH 7 and 25 °C for all variants. The activity on BHET and amorphous PET film was investigated. S2LipA efficiently degraded BHET and caused roughening and indents on the surface of PET films, comparable to the activity of previously described TfCut2 under the same conditions. The abundance of the S2LipA variant in Streptomyces suggests an environmental advantage towards the degradation of more polar substrates including these polluting plastics.
Assuntos
Streptomyces , Streptomyces/enzimologia , Streptomyces/genética , Microbiologia do Solo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Biodegradação Ambiental , Streptomyces coelicolor/enzimologia , Streptomyces coelicolor/genética , Esterases/metabolismo , Esterases/genética , Esterases/química , Polietilenotereftalatos/metabolismoRESUMO
Early detection of insecticide resistance is essential to develop resistance countermeasures and depends on accurate and rapid biological and biochemical tests to monitor resistance and detect associated mechanisms. Many such studies have measured activities of esterases, enzymes associated with resistance to ester- containing insecticides, using the model substrate, α-naphthyl acetate (α-NA). However, in the field, pests are exposed to ester-containing insecticides such as malathion, that are structurally distinct from α-NA. In the current study, malathion resistance in C. quinquefasciatus (3.2- to 10.4-fold) was highly associated with esterase activity measured with either α-NA (R2 = 0.92) or malathion (R2 = 0.90). In addition, genes encoding two esterases (i.e., EST-2 and EST-3) were over-expressed in field- collected strains, but only one (EST-3) was correlated with malathion hydrolysis (R2 = 0.94) and resistance (Rs = 0.96). These results suggest that, in the strains studied, α-NA is a valid surrogate for measuring malathion hydrolysis, and that heightened expression of an esterase gene is not necessarily associated with metabolic resistance to insecticidal esters.
Assuntos
Culex , Esterases , Resistência a Inseticidas , Inseticidas , Malation , Malation/farmacologia , Animais , Esterases/metabolismo , Esterases/genética , Culex/efeitos dos fármacos , Culex/genética , Culex/enzimologia , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Naftalenos/farmacologia , Hidrólise , Biomarcadores/metabolismo , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , NaftóisRESUMO
Evolutionary engineering involves repeated mutations and screening and is widely used to modify protein functions. However, it is important to diversify evolutionary pathways to eliminate the bias and limitations of the variants by using traditionally unselected variants. In this study, we focused on low-stability variants that are commonly excluded from evolutionary processes and tested a method that included an additional restabilization step. The esterase from the thermophilic bacterium Alicyclobacillus acidocaldarius was used as a model protein, and its activity at its optimum temperature of 65 °C was improved by evolutionary experiments using random mutations by error-prone PCR. After restabilization using low-stability variants with low-temperature (37 °C) activity, several re-stabilizing variants were obtained from a large number of variant libraries. Some of the restabilized variants achieved by removing the destabilizing mutations showed higher activity than that of the wild-type protein. This implies that low-stability variants with low-temperature activity can be re-evolved for future use. This method will enable further diversification of evolutionary pathways.
Assuntos
Mutação , Engenharia de Proteínas , Engenharia de Proteínas/métodos , Estabilidade Enzimática , Esterases/genética , Esterases/metabolismo , Esterases/química , Evolução Molecular Direcionada , Alicyclobacillus/genética , Alicyclobacillus/enzimologia , Temperatura , Evolução Molecular , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismoRESUMO
Macrolide antibiotics, pivotal in clinical therapeutics, are confronting resistance challenges mediated by enzymes like macrolide esterases, which are classified into Ere-type and the less studied Est-type. In this study, we provide the biochemical confirmation of EstX, an Est-type macrolide esterase that initially identified as unknown protein in the 1980s. EstX is capable of hydrolyzing four 16-membered ring macrolides, encompassing both veterinary (tylosin, tidipirosin, and tilmicosin) and human-use (leucomycin A5) antibiotics. It uses typical catalytic triad (Asp233-His261-Ser102) from alpha/beta hydrolase superfamily for ester bond hydrolysis. Further genomic context analysis suggests that the dissemination of estX is likely facilitated by mobile genetic elements such as integrons and transposons. The global distribution study indicates that bacteria harboring the estX gene, predominantly pathogenic species like Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae, are prevalent in 74 countries across 6 continents. Additionally, the emergence timeline of the estX gene suggests its proliferation may be linked to the overuse of macrolide antibiotics. The widespread prevalence and dissemination of Est-type macrolide esterase highlight an urgent need for enhanced monitoring and in-depth research, underlining its significance as an escalating public health issue.
Assuntos
Esterases , Esterases/genética , Esterases/metabolismo , Esterases/química , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Macrolídeos/metabolismo , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Filogenia , Hidrolases/genética , Hidrolases/metabolismo , Hidrolases/químicaRESUMO
Phthalate esters (PAEs) are widely used as plasticizers and cause serious complex pollution problem in environment. Thus, strains with efficient ability to simultaneously degrade various PAEs are required. In this study, a newly isolated strain Rhodococcus sp. AH-ZY2 can degrade 500 mg/L Di-n-octyl phthalate completely within 16 h and other 500 mg/L PAEs almost completely within 48 h at 37 °C, 180 rpm, and 2 % (v/v) inoculum size of cultures with a OD600 of 0.8. OD600 = 0.8, 2 % (v/v). Twenty genes in its genome were annotated as potential esterase and four of them (3963, 4547, 5294 and 5359) were heterogeneously expressed and characterized. Esterase 3963 and 4547 is a type I PAEs esterase that hydrolyzes PAEs to phthalate monoesters. Esterase 5294 is a type II PAEs esterase that hydrolyzes phthalate monoesters to phthalate acid (PA). Esterase 5359 is a type III PAEs esterase that simultaneously degrades various PAEs to PA. Molecular docking results of 5359 suggested that the size and indiscriminate binding feature of spacious substrate binding pocket may contribute to its substrate versatility. AH-ZY2 is a potential strain for efficient remediation of PAEs complex pollution in environment. It is first to report an esterase that can efficiently degrade mixed various PAEs.
Assuntos
Biodegradação Ambiental , Esterases , Ésteres , Simulação de Acoplamento Molecular , Ácidos Ftálicos , Rhodococcus , Rhodococcus/metabolismo , Rhodococcus/genética , Rhodococcus/enzimologia , Ácidos Ftálicos/metabolismo , Ácidos Ftálicos/química , Esterases/metabolismo , Esterases/genética , Ésteres/metabolismo , Ésteres/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Plastificantes/metabolismoRESUMO
Human serum albumin (HSA) effectively binds to compounds having different molecular weight and thus facilitates their distribution in the living organisms. Thus, the binding interactions between a potential antibacterial drug (levofloxacin) and synthesized choline based levofloxacinate conjugates with HSA have been explored. The binding efficacy and mechanism were explored by utilizing different spectroscopic techniques; UV-Visible, steady state fluorescence, time resolved fluorescence and esterase-like activity. The interactions between the ligands and protein were electrostatic as well as hydrophobic in nature. The influence of different ligands having different alkyl chain shows quenching of the fluorescence emission of HSA. The spontaneous binding/quenching of HSA with ligands was static in nature, validated by steady state and time resolved fluorescence spectroscopy. Also, the impact of these ligands on the conformation of the native HSA structure was evaluated by using circular dichroism spectroscopy. In combination to the structural change study, the native protein functionality was observed (in terms of 'esterase-like activity') which has been found to be on lower side due to ligand binding. Further, we have performed the reverse study to check the impact of HSA on the fluorescent fluoroquinolone drug. The current study may prove helpful in elucidating the chemico-biological interactions which may prove useful in the pharmaceuticals, pharmacology, and different biochemistry fields.
Assuntos
Colina , Esterases , Levofloxacino , Ligação Proteica , Albumina Sérica Humana , Humanos , Levofloxacino/química , Esterases/metabolismo , Esterases/química , Albumina Sérica Humana/química , Albumina Sérica Humana/metabolismo , Colina/química , Ligantes , Espectrometria de Fluorescência , Interações Hidrofóbicas e Hidrofílicas , Dicroísmo CircularRESUMO
As a marine antifouling biocide, 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) exhibited high toxicity to marine organisms. This study investigated the interaction between DCOIT and human serum albumin (HSA) using several spectroscopic techniques combined with computer prediction methods. The UV-vis absorption spectra, Stern-Volmer constant (KSV) and fluorescence resonance energy transfer (FRET) results indicated that DCOIT caused static quenching of HSA fluorescence. The ΔG°, ΔH° and ΔS° values were -31.03 ± 0.17 kJ·mol-1, -133.54 ± 0.88 kJ·mol-1 and -348.46 ± 2.86 J.mol-1·K-1, respectively, suggesting that van der Waals forces and hydrogen bonds governed the spontaneous formation of the complex. Synchronous fluorescence and circular dichroism (CD) spectroscopy observed the burial of Trp residues within HSA and the unfolding of HSA secondary structure induced by DCOIT. Three-dimensional (3D) fluorescence and Atomic Force Microscopy (AFM) further detected DCOIT-induced loosening of HSA peptide chain structure. Site displacement experiments indicated that DCOIT binding at site I of HSA. Computational predictions indicated that hydrophobic interactions were also essential in the complex. The increased RMSD, Rg, SASA, and RMSF confirmed that DCOIT weakened the stability and compactness of HSA, rendering residues more flexible. Lastly, esterase activity assays demonstrated that DCOIT inhibited esterase activity and interfered with the human detoxification process.