RESUMO
As genomic databases expand and artificial intelligence tools advance, there is a growing demand for efficient characterization of large numbers of proteins. To this end, here we describe a generalizable pipeline for high-throughput protein purification using small-scale expression in E. coli and an affordable liquid-handling robot. This low-cost platform enables the purification of 96 proteins in parallel with minimal waste and is scalable for processing hundreds of proteins weekly per user. We demonstrate the performance of this method with the expression and purification of the leading poly(ethylene terephthalate) hydrolases reported in the literature. Replicate experiments demonstrated reproducibility and enzyme purity and yields (up to 400 µg) sufficient for comprehensive analyses of both thermostability and activity, generating a standardized benchmark dataset for comparing these plastic-degrading enzymes. The cost-effectiveness and ease of implementation of this platform render it broadly applicable to diverse protein characterization challenges in the biological sciences.
Assuntos
Escherichia coli , Robótica , Robótica/métodos , Escherichia coli/genética , Engenharia de Proteínas/métodos , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/economia , Hidrolases/metabolismo , Hidrolases/química , Hidrolases/genética , Polietilenotereftalatos/química , Reprodutibilidade dos TestesRESUMO
Enzymatic degradation of poly(ethylene terephthalate) (PET) has emerged as a promising route for ecofriendly biodegradation of plastic waste. Several discontinuous activity assays have been developed for assessing PET hydrolyzing enzymes, usually involving manual sampling at different time points during the course of the enzymatic reaction. In this work, we present a novel, compartmentalized UV absorbance assay for continuous detection of soluble hydrolysis products released during enzymatic degradation of PET. The methodology is based on removal of the walls separating two diagonally adjacent wells in UV-transparent microplates, to ensure passage of soluble enzymatic hydrolysis products between the two adjacent wells: One well holds an insoluble PET disk of defined dimensions and the other is used for continuous reading of the enzymatic product formation (at 240 nm). The assay was validated by quantifying the rate of mixing of the soluble PET degradation product BHET (bis(2-hydroxyethyl) terephthalate) between the two adjacent wells. The assay validation also involved a simple adjustment for water evaporation during prolonged assays. With this new assay, we determined the kinetic parameters for two PET hydrolases, DuraPETase and LCCICCG, and verified the underlying assumption of steady-state reaction rates. This new continuous assay enables fast exploration and robust kinetic characterization of PET degrading enzymes.
Assuntos
Ácidos Ftálicos , Polietilenotereftalatos , Polietilenotereftalatos/metabolismo , Ácidos Ftálicos/metabolismo , Hidrolases/metabolismo , EtilenosRESUMO
Recently, a bacterium strain of Ideonella sakaiensis was identified with the uncommon ability to degrade the poly(ethylene terephthalate) (PET). The PETase from I. sakaiensis strain 201-F6 (IsPETase) catalyzes the hydrolysis of PET converting it to mono(2-hydroxyethyl) terephthalic acid (MHET), bis(2-hydroxyethyl)-TPA (BHET), and terephthalic acid (TPA). Despite the potential of this enzyme for mitigation or elimination of environmental contaminants, one of the limitations of the use of IsPETase for PET degradation is the fact that it acts only at moderate temperature due to its low thermal stability. Besides, molecular details of the main interactions of PET in the active site of IsPETase remain unclear. Herein, molecular docking and molecular dynamics (MD) simulations were applied to analyze structural changes of IsPETase induced by PET binding. Results from the essential dynamics revealed that the ß1-ß2 connecting loop is very flexible. This loop is located far from the active site of IsPETase and we suggest that it can be considered for mutagenesis to increase the thermal stability of IsPETase. The free energy landscape (FEL) demonstrates that the main change in the transition between the unbound to the bound state is associated with the ß7-α5 connecting loop, where the catalytic residue Asp206 is located. Overall, the present study provides insights into the molecular binding mechanism of PET into the IsPETase structure and a computational strategy for mapping flexible regions of this enzyme, which can be useful for the engineering of more efficient enzymes for recycling plastic polymers using biological systems.
Assuntos
Proteínas de Bactérias/metabolismo , Burkholderiales/metabolismo , Hidrolases/metabolismo , Polietilenotereftalatos/metabolismo , Biocatálise , HidróliseRESUMO
Bifidogenic effect is a main target for the assessment of prebiotic activity. pH-controlled batch processes of bifidobacteria and fecal microbiota are herein presented. Growth of bifidobacteria, carbohydrate breakdown and consumption, organic acid production, and activity of specific glycosyl hydrolases involved in the hydrolysis of di-, oligo-, or polysaccharides are exploited to study and compare substrate preference of bifidobacteria for candidate prebiotics.
Assuntos
Bifidobacterium/metabolismo , Polissacarídeos Bacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Bifidobacterium/química , Bifidobacterium/crescimento & desenvolvimento , Reatores Biológicos , Metabolismo dos Carboidratos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Cromatografia em Camada Fina/métodos , Ensaios Enzimáticos/métodos , Fermentação , Microbioma Gastrointestinal , Humanos , Hidrolases/metabolismo , Hidrólise , Polissacarídeos Bacterianos/análiseRESUMO
Directed evolution has become an important method to unleash the latent potential of enzymes to make them uniquely suited for human purposes. However, the need for a large reagent volume and sophisticated instrumentation hampers its broad implementation. In an attempt to address this problem, here we report a paper-based high-throughput screening approach that should find broad application in generating desired enzymes. As an example case, the dehalogenation reaction of the halohydrin dehalogenase was adopted for assay development. In addition to visual detection, quantitative measurements were performed by measuring the color intensity of an image that was photographed by a smartphone and processed using ImageJ free software. The proposed method was first validated using a gold standard method and then applied to mutagenesis library screening with reduced consumption of reagents (i.e., ≤ 10 µl per assay) and a shorter assay time. We identified two active mutants (P135A and G137A) with improved activities toward four tested substrates. The assay not only consumes less reagents but also eliminates the need for expensive instrumentation. The proposed method demonstrates the potential of paper-based whole-cell screening coupled with digital image colorimetry as a promising approach for the discovery of industrially important enzymes.Key Points⢠A frugal method was developed for directed enzyme evolution.⢠Mutagenesis libraries were successfully screened on a paper platform.⢠Smartphone imaging was efficiently used to measure enzyme activities.
Assuntos
Evolução Molecular Direcionada/métodos , Ensaios de Triagem em Larga Escala/métodos , Papel , Catálise , Colorimetria , Evolução Molecular Direcionada/economia , Evolução Molecular Direcionada/normas , Escherichia coli/genética , Escherichia coli/metabolismo , Biblioteca Gênica , Ensaios de Triagem em Larga Escala/economia , Ensaios de Triagem em Larga Escala/normas , Hidrolases/genética , Hidrolases/metabolismo , Mutagênese , Mutação , Reprodutibilidade dos Testes , SmartphoneRESUMO
The aim of this study was to apply the enzymatic treatment and fermentation by Pediococcus acidilactici BaltBio01 strain for industrial cereal by-products conversion to food/feed bioproducts with high amount of probiotic lactic acid bacteria (LAB). LAB propagated in potato media and spray-dried remained viable during 12 months (7.0 log10 cfu/g) of storage and was used as a starter for cereal by-products fermentation. The changes of microbial profile, biogenic amines (BAs), mycotoxins, lactic acid (L+/D-), lignans and alkylresorcinols (ARs) contents in fermented cereal by-product were analysed. Cereal by-products enzymatic hydrolysis before fermentation allows to obtain a higher count of LAB during fermentation. Fermentation with P. acidilactici reduce mycotoxins content in fermented cereal by-products. According to our results, P. acidilactici multiplied in potato juice could be used for cereal by-products fermentation, as a potential source to produce safer food/feed bioproduct with high amount of probiotic LAB for industrial production.
Assuntos
Ração Animal/microbiologia , Grão Comestível/metabolismo , Alimentos Fermentados/microbiologia , Aditivos Alimentares/metabolismo , Hidrolases/metabolismo , Pediococcus acidilactici/metabolismo , Probióticos/metabolismo , Alquilação , Ração Animal/efeitos adversos , Ração Animal/análise , Ração Animal/economia , Animais , Aminas Biogênicas/efeitos adversos , Aminas Biogênicas/análise , Aminas Biogênicas/metabolismo , Grão Comestível/efeitos adversos , Grão Comestível/química , Grão Comestível/economia , Fermentação , Alimentos Fermentados/efeitos adversos , Alimentos Fermentados/análise , Alimentos Fermentados/economia , Aditivos Alimentares/efeitos adversos , Aditivos Alimentares/química , Aditivos Alimentares/economia , Contaminação de Alimentos/prevenção & controle , Manipulação de Alimentos , Indústria de Processamento de Alimentos/economia , Humanos , Hidrolases/efeitos adversos , Hidrólise , Resíduos Industriais/economia , Letônia , Lignanas/efeitos adversos , Lignanas/análise , Lignanas/metabolismo , Viabilidade Microbiana , Micotoxinas/isolamento & purificação , Micotoxinas/metabolismo , Micotoxinas/toxicidade , Pediococcus acidilactici/crescimento & desenvolvimento , Probióticos/efeitos adversos , Resorcinóis/efeitos adversos , Resorcinóis/análise , Resorcinóis/metabolismoRESUMO
This study reports enzymatic hydrolysis of the biomass of the giant reed (Arundo donax L.) after ammonia fibre expansion (AFEX) pretreatment. In particular, the capacity of the arabinofuranosidase from the fungus Pleurotus ostreatus recombinantly expressed in Pichia pastoris rPoAbf, its evolved mutant rPoAbf F435Y/Y446F and the endo-cellulase from Streptomyces sp. G12 CelStrep recombinantly expressed in Escherichia coli to enhance the hydrolysis of AFEX-treated A. donax was investigated, using the corn stover as reference feedstock. The investigated enzymes were assayed using a mixture of purified cellulases (CBHI, CBHII, EGI and ßG), endoxylanases (LX3, LX4) and accessory hemicellulases (LarbF and LßX) as reference enzyme mixture and substituting EGI with rCelStrep and LarbF with rPoAbf or rPoAbf F435Y/Y446F. The use of rPoAbf F435Y/Y446F in the substitution of LarbF led to improvements in sugar conversion, giving a glucan, xylan and arabinan conversion after 72 h of around 62, 63 and 80 %, respectively, similar or higher than those (44, 66 and 55 %) achieved by 72 h hydrolysis with commercial enzymes Novozymes Cellic®, Ctec3 and Htec3. The enzymes rPoAbf, rPoAbf F435Y/Y446F and rCelStrep were also investigated for their effect on hydrolysis of AFEX-pretreated A. donax by addition to commercial enzyme mixture Novozymes Cellic®, Ctec3 and Htec3, and it was shown that the addition of rPoAbf and its evolved mutant rPoAbf F435Y/Y446F enhanced both xylan and arabinan conversions, which achieved 80 % after 6 days of saccharification with rPoAbf F435Y/Y446F.
Assuntos
Amônia/metabolismo , Bactérias/enzimologia , Celulose/metabolismo , Fungos/enzimologia , Hidrolases/metabolismo , Poaceae/química , Poaceae/efeitos dos fármacos , Bactérias/genética , Biotransformação , Fungos/genética , Expressão Gênica , Hidrolases/genética , Hidrólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Objectives To assess the relationships among alkali production, diet, oral health behaviors, and oral hygiene. Methods Data from 52 subjects including demographics, diet, and oral hygiene scores were analyzed against the level of arginine and urea enzymes in plaque and saliva samples. An oral habit survey was completed that included: use of tobacco (TB), alcohol (AH), sugary drinks (SD), and diet. Alkali production through arginine deiminase (ADS) and urease activities were measured in smooth-surface supragingival dental plaque and un stimulated saliva samples from all subjects. ADS and urease activities were measured by quantification of the ammonia generated from the incubation of plaque or saliva samples. Spearman correlations were used to compute all associations. Results Participants in the lowest SES (Socio-economic status) group had the habit of consuming sugary drinks the most and had the highest rate of tobacco use. Males consumed significantly more alcohol than females. No significant relationship was found between age or gender and alkali production. Higher rates of sugary drink consumption and tobacco use were significantly related to lower alkali production. Conclusion The study showed a relationship between alkali production and oral hygiene, diet, and certain oral health behaviors. Poor oral hygiene was significantly associated with age, lower SES, tobacco use, and alcohol, and sugary drinks consumption. Clinical relevance Certain oral health behaviors have an impact on oral hygiene and on alkali production; it is important to address these factors with patients as a strategy for caries control. .
Assuntos
Humanos , Masculino , Feminino , Adulto , Adulto Jovem , Álcalis/análise , Comportamento Alimentar , Boca/química , Higiene Bucal , Fatores Etários , Consumo de Bebidas Alcoólicas/efeitos adversos , Álcalis/metabolismo , Carboidratos/efeitos adversos , Cárie Dentária/etiologia , Cárie Dentária/prevenção & controle , Hidrolases/análise , Hidrolases/metabolismo , Fatores de Risco , Saliva/química , Fumar/efeitos adversos , Fatores Socioeconômicos , Estatísticas não Paramétricas , Urease/análise , Urease/metabolismoRESUMO
Structural characterization of membrane proteins and other large proteins with NMR relies increasingly on perdeuteration combined with incorporation of specifically protonated amino acid moieties, such as methyl groups of isoleucines, valines, or leucines. The resulting proton dilution reduces dipolar broadening producing sharper resonance lines, ameliorates spectral crowding, and enables measuring of crucial distances between and to methyl groups. While incorporation of specific methyl labeling is now well established for bacterial expression using suitable precursors, corresponding methods are still lacking for cell-free expression, which is often the only choice for producing labeled eukaryotic membrane proteins in mg quantities. Here we show that we can express methyl-labeled human integral membrane proteins cost-effectively by cell-free expression based of crude hydrolyzed ILV-labeled OmpX inclusion bodies. These are obtained in Escherichia coli with very high quantity and represent an optimal intermediate to channel ILV precursors into the eukaryotic proteins.
Assuntos
Sistema Livre de Células , Proteínas de Membrana/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Isótopos de Carbono/química , Análise Custo-Benefício , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Humanos , Hidrogênio/química , Hidrolases/metabolismo , Hidrólise , Corpos de Inclusão/metabolismo , Isoleucina/química , Leucina/química , Espectroscopia de Ressonância Magnética , Micelas , Valina/químicaRESUMO
OBJECTIVES: To assess the relationships among alkali production, diet, oral health behaviors, and oral hygiene. METHODS: Data from 52 subjects including demographics, diet, and oral hygiene scores were analyzed against the level of arginine and urea enzymes in plaque and saliva samples. An oral habit survey was completed that included: use of tobacco (TB), alcohol (AH), sugary drinks (SD), and diet. Alkali production through arginine deiminase (ADS) and urease activities were measured in smooth-surface supragingival dental plaque and un stimulated saliva samples from all subjects. ADS and urease activities were measured by quantification of the ammonia generated from the incubation of plaque or saliva samples. Spearman correlations were used to compute all associations. RESULTS: Participants in the lowest SES (Socio-economic status) group had the habit of consuming sugary drinks the most and had the highest rate of tobacco use. Males consumed significantly more alcohol than females. No significant relationship was found between age or gender and alkali production. Higher rates of sugary drink consumption and tobacco use were significantly related to lower alkali production. CONCLUSION: The study showed a relationship between alkali production and oral hygiene, diet, and certain oral health behaviors. Poor oral hygiene was significantly associated with age, lower SES, tobacco use, and alcohol, and sugary drinks consumption. Clinical relevance Certain oral health behaviors have an impact on oral hygiene and on alkali production; it is important to address these factors with patients as a strategy for caries control.
Assuntos
Álcalis/análise , Comportamento Alimentar , Boca/química , Higiene Bucal , Adulto , Fatores Etários , Consumo de Bebidas Alcoólicas/efeitos adversos , Álcalis/metabolismo , Carboidratos/efeitos adversos , Cárie Dentária/etiologia , Cárie Dentária/prevenção & controle , Feminino , Humanos , Hidrolases/análise , Hidrolases/metabolismo , Masculino , Fatores de Risco , Saliva/química , Fumar/efeitos adversos , Fatores Socioeconômicos , Estatísticas não Paramétricas , Urease/análise , Urease/metabolismo , Adulto JovemRESUMO
Arginine deiminase is a promising anticancer drug active against melanoma, hepatocarcinoma and other tumors. Recombinant strains of Escherichia coli that express arginine deiminase from pathogenic bacteria Mycoplasma have been developed. However, production costs of heterologous arginine deiminase are high due to use of an expensive inducer and extraction buffer, as well as using diluted culture for enzyme induction. We report on a new advanced protocol for Mycoplasma hominis arginine deiminase expression, extraction and renaturation. The main improvements include manipulation with dense suspensions of E. coli, use of lactose instead of isopropyl ß-D-1-thiogalactopyranoside as an inducer and a cheaper but not less efficient buffer for solubilization of arginine deiminase inclusion bodies. In addition, supplementation of the storage culture medium with glucose and substrate (arginine) significantly stabilized the recombinant arginine deiminase producer. Homogenous preparations of recombinant arginine deiminase were obtained using anion-exchange and hydrophobic chromatography. The purified enzyme retained a specific activity of 30-34 U/mg for 12 months when stored at 4°C in 20 mM sodium phosphate buffer pH 7.2 containing 1 M NaCl.
Assuntos
Clonagem Molecular , Escherichia coli/enzimologia , Hidrolases/isolamento & purificação , Hidrolases/metabolismo , Mycoplasma hominis/enzimologia , Antineoplásicos/farmacologia , Biotecnologia , Meios de Cultura , Escherichia coli/genética , Hidrolases/economia , Hidrolases/genética , Corpos de Inclusão , Lactose/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The biological synthesis methods have been emerging as a promising new approach for production of nanoparticles due to their simplicity and non-toxicity. In the present study, spores of Bacillus athrophaeus were used to achieve the objective of developing a green synthesis method of silver nanoparticles. Enzyme assay revealed that the spores and their heat inactivated forms (microcapsules) were highly active and their enzymatic contents differed from the vegetative cells. Laccase, glucose oxidase, and alkaline phosphatase activities were detected in the dormant forms, but not in the vegetative cells. Although no nanoparticle was produced by active cells of B. athrophaeus, both spores and microcapsules were efficiently capable of reducing the silver ions (Agâº) to elemental silver (Ag°) leading to the formation of nanoparticles from silver nitrate (AgNO3). The presence of biologically synthesized silver nanoparticles was determined by obtaining broad spectra with maximum absorbance at 400 nm in UV-visible spectroscopy. The X-ray diffraction analysis pattern revealed that the nanoscale particles have crystalline nature with various topologies, as confirmed by transmission electron microscopy (TEM). The TEM micrograph showed the nanocrystal structures with dimensions ranging from 5 to 30 nm. Accordingly, the spore mixture could be employed as a factory for detoxification of heavy metals and subsequent production of nanoparticles. This research introduces an environmental friendly and cost effective biotechnological process for the extracellular synthesis of silver nanoparticles using the bacterial spores.
Assuntos
Bacillus/fisiologia , Proteínas de Bactérias/metabolismo , Nanopartículas Metálicas/química , Água do Mar/microbiologia , Bacillus/classificação , Bacillus/enzimologia , Biotecnologia/economia , Biotecnologia/métodos , Cápsulas , Ecossistema , Química Verde , Hidrolases/metabolismo , Microscopia Eletrônica de Transmissão , Oxirredutases/metabolismo , Prata , Nitrato de Prata/química , Espectrofotometria Ultravioleta , Esporos Bacterianos/fisiologia , Difração de Raios XRESUMO
The post-translational modifications of histone proteins are highly diverse and dynamic processes. It is becoming increasingly evident that modifying histone proteins can have a direct influence on both cellular homeostasis and disease states. Protein arginine deiminase 4 (PAD4) is an enzyme that converts peptidyl-arginine to citrulline. The overexpression of PAD4 has been found in numerous types of human cancer and autoimmune diseases. We report a new, facile, fluorescence-based assay for the detection of PAD4 activity that exploits the substrate specificity of trypsin to monitor the citrullination reaction carried out by PAD4 based on the fact that, upon citrullination, the positively charged arginine side chain is converted to the neutral citrulline. We show that the assay can be performed rapidly with readily available reagents and that it responds accordingly to a known PAD4 inhibitor.
Assuntos
Arginina/metabolismo , Citrulina/metabolismo , Hidrolases/metabolismo , Ensaios Enzimáticos/economia , Ensaios Enzimáticos/métodos , Humanos , Modelos Moleculares , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Espectrometria de Fluorescência/métodos , Especificidade por Substrato , Tripsina/metabolismoRESUMO
Due to the growing demand of enantiomerically pure compounds, as well as the increasing strict safety, quality and environmentally requirements of industrial synthetic processes, the development of more sustainable, healthy and economically attractive strategies for the synthesis of chiral biologically active molecules is still an open challenge in the pharmaceutical industry. In this context, the biotransformations field has emerged as a real alternative to traditional synthetic routes, because of the exquisite chemo-, regio- and enantioselectivities commonly displayed by enzymes; thus, biocatalysis is becoming a widespread methodology for the synthesis of chiral compounds, not only at laboratory scale, but also at industrial scale. As hydrolases and oxido-reductases are the most employed enzymes, this review is focused on describing several industrial processes based on the use of these enzymes for obtaining chiral compounds useful for the pharmaceutical industry.
Assuntos
Indústria Farmacêutica/métodos , Preparações Farmacêuticas/química , Preparações Farmacêuticas/síntese química , Biotransformação , Hidrolases/metabolismo , Oxirredução , EstereoisomerismoRESUMO
BACKGROUND: A few common spices are known to stimulate secretion of bile with higher amount of bile acids which play a major role in digestion and absorption of dietary lipids. It would be appropriate to verify if these spices enable efficient digestion and absorption during high-fat intake. In this context, dietary ginger (0.05%), piperine (0.02%), capsaicin (0.015%), and curcumin (0.5%) were examined for their influence on bile secretion, digestive enzymes of pancreas and absorption of dietary fat in high-fat (30%) fed Wistar rats for 8 weeks. RESULTS: These spices enhanced the activity of pancreatic lipase, amylase, trypsin and chymotrypsin by 22-57%, 32-51%, 63-81% and 12-38%, respectively. Dietary intake of spices along with high-fat enhanced fat absorption. These dietary spices increased bile secretion with higher bile acid content. Stimulation of lipid mobilisation from adipose tissue was suggested by the decrease in perirenal adipose tissue weight by dietary capsaicin and piperine. This was also accompanied by prevention of the accumulation of triglyceride in liver and serum in high-fat fed rats. Activities of key lipogenic enzymes in liver were reduced which was accompanied by an increased activity of hormone-sensitive lipase. CONCLUSION: Thus, dietary ginger and other spice compounds enhance fat digestion and absorption in high-fat fed situation through enhanced secretion of bile salts and a stimulation of the activity pancreatic lipase. At the same time, the energy expenditure is facilitated by these spices to prevent the accumulation of absorbed fat.
Assuntos
Bile/metabolismo , Gorduras na Dieta/metabolismo , Digestão , Absorção Intestinal , Especiarias , Regulação para Cima , Zingiber officinale , Adiposidade , Alcaloides/administração & dosagem , Animais , Benzodioxóis/administração & dosagem , Bile/química , Ácidos e Sais Biliares/análise , Ácidos e Sais Biliares/metabolismo , Capsaicina/administração & dosagem , Curcumina/administração & dosagem , Dieta Hiperlipídica/efeitos adversos , Zingiber officinale/química , Hidrolases/química , Hidrolases/metabolismo , Índia , Metabolismo dos Lipídeos , Fígado/metabolismo , Masculino , Pâncreas/enzimologia , Pâncreas/metabolismo , Piperidinas/administração & dosagem , Alcamidas Poli-Insaturadas/administração & dosagem , Ratos Wistar , Rizoma/química , Especiarias/análise , Especiarias/economiaRESUMO
The relationship between the expression of extracellular enzymatic system and a metal stress is scarce in fungi, hence limiting the possible use of secretion profiles as tools for metal ecotoxicity assessment. In the present study, we investigated the effect of Zn, Cu, Pb and Cd, tested alone or in equimolar cocktail, on the secretion profiles at enzymatic and protein levels in Trametesversicolor. For that purpose, extracellular hydrolases (acid phosphatase, ß-glucosidase, ß-galactosidase and N-acetyl-ß-glucosaminidase) and ligninolytic oxidases (laccase, Mn-peroxidase) were monitored in liquid cultures. Fungal secretome was analyzed by electrophoresis and laccase secretion was characterized by western-blot and mass spectrometry analyses. Our results showed that all hydrolase activities were inhibited by the metals tested alone or in cocktail, whereas oxidase activities were specifically stimulated by Cu, Cd and metal cocktail. At protein level, metal exposure modified the electrophoretic profiles of fungal secretome and affected the diversity of secreted proteins. Two laccase isoenzymes, LacA and LacB, identified by mass spectrometry were differentially glycosylated according to the metal exposure. The amount of secreted LacA and LacB was strongly correlated with the stimulation of laccase activity by Cu, Cd and metal cocktail. These modifications of extracellular enzymatic system suggest that fungal oxidases could be used as biomarkers of metal exposure.
Assuntos
Monitoramento Ambiental/métodos , Metais/toxicidade , Poluentes do Solo/toxicidade , Trametes/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Crescimento e Desenvolvimento/efeitos dos fármacos , Hidrolases/metabolismo , Lacase/metabolismo , Peroxidases/metabolismo , Trametes/enzimologia , Trametes/metabolismoRESUMO
The objective of this study was to utilize extrusion cooking and hydrothermal treatment to produce resistant starch (RS) as an economical alternative to a batch-cooking process. A hydrothermal treatment (110 degrees C, 3 d) of batch-cooked and extruded starch samples facilitated propagation of heat-stable starch crystallites and increased the RS contents from 2.1% to 7.7% up to 17.4% determined using AOAC Method 991.43 for total dietary fiber. When starch samples were batch cooked and hydrothermally treated at a moisture content below 70%, acid-modified normal-maize starch (AMMS) produced a greater RS content than did native normal-maize starch (NMS). This was attributed to the partially hydrolyzed, smaller molecules in the AMMS, which had greater mobility and freedom than the larger molecules in the NMS. The RS contents of the batch-cooked and extruded AMMS products after the hydrothermal treatment were similar. A freezing treatment of the AMMS samples at -20 degrees C prior to the hydrothermal treatment did not increase the RS content. The DSC thermograms and the X-ray diffractograms showed that retrograded amylose and crystalline starch-lipid complex, which had melting temperatures above 100 degrees C, accounted for the RS contents.
Assuntos
Culinária/métodos , Hidrolases/metabolismo , Amido/química , Amido/metabolismo , Temperatura , Amilose/análise , Amilose/síntese química , Amilose/metabolismo , Varredura Diferencial de Calorimetria , Cromatografia em Gel , Cristalização , Hidrólise , Lipídeos/análise , Peso Molecular , Prebióticos/economia , Amido/classificação , Água/análise , Difração de Raios XRESUMO
In bacteria, the transcription of virulence genes is usually controlled by a cell density-dependent process known as "quorum sensing" (QS). QS relies on small diffusible signaling molecules that cross the bacterial cell wall and activate target transcription factors after a threshold concentration has been reached. Besides two hierarchical QS circuits based on N-acylhomoserine lactones, the human opportunistic pathogen Pseudomonas aeruginosa integrates a signaling system that depends on 2-heptyl-3-hydroxy-4-quinolone, termed "Pseudomonas quinolone signal" (PQS). PQS is produced from genes encoded in the pqs operon, which in addition to the biosynthetic enzymes PqsA-D contains a fifth gene, pqsE, that is not required for production of PQS but whose disruption leads to loss of signal transduction in several but not all pqs operon-dependent processes. PqsE was hence termed "PQS response protein", but its exact mechanism of action is unknown. We have determined the crystal structure of recombinant PqsE and show that it possesses a metallo-beta-lactamase fold with an Fe(II)Fe(III) center in the active site. A copurified ligand was assigned as benzoate and may indicate that PqsE exerts its regulatory effect by converting a chorismate-derived molecule. Further, PqsE was found to slowly hydrolyze phosphodiesters including single- and double-stranded DNA as well as mRNA and also the thioester S-(4-nitrobenzoyl)mercaptoethane. Higher activity was observed after incubation with Co(2+) and, to lesser entent, Mn(2+), suggesting that the Fe(II)Fe(III) center of recombinant PqsE may be an artifact of heterologous expression. A crystal complex of the E182A mutant with bis-pNPP was obtained and suggests a catalytic mechanism for hydrolysis.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Hidrolases/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Bactérias/genética , Calorimetria , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Hidrolases/genética , Cinética , Modelos Genéticos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/genética , Quinolonas/metabolismo , Percepção de Quorum/fisiologia , Proteínas Recombinantes/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismoRESUMO
Multiple sclerosis is a complex human neurodegenerative disease, characterized by the active destruction of the insulating myelin sheath around the axons in the central nervous system. The physical deterioration of myelin is mediated by hyperdeimination of myelin basic and other proteins, catalysed by the Ca2+ -dependent enzyme peptidylarginine deiminase 2 (PAD2). Thus, inhibition of PAD2 may be of value in treatment of this disease. Here, we have first characterized the in vitro kinetic properties of the human peptidylarginine deiminase isoform 2 (hPAD2). Phosphatidylserine and phosphatidylcholine reduced its Ca2+ dependence by almost twofold. Second, we have explored the putative inhibitory action of the methyl ester side chain of paclitaxel (TSME), which shares structural features with a synthetic PAD substrate, viz., the benzoyl-L-arginine ethyl ester (BAEE). Using the known crystallographic structure of the homologous enzyme hPAD4 and in silico molecular docking, we have shown that TSME interacted strongly with the catalytic site, albeit with a 100-fold lower affinity than BAEE. Despite paclitaxel having previously been shown to inhibit hPAD2 in vitro, the side chain of paclitaxel alone did not inhibit this enzyme's activity.
Assuntos
Antineoplásicos Fitogênicos , Cálcio/metabolismo , Hidrolases/metabolismo , Isoenzimas/metabolismo , Paclitaxel , Fosfolipídeos/metabolismo , Sequência de Aminoácidos , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/metabolismo , Domínio Catalítico , Humanos , Hidrolases/química , Hidrolases/genética , Isoenzimas/química , Isoenzimas/genética , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Esclerose Múltipla/enzimologia , Paclitaxel/química , Paclitaxel/metabolismo , Conformação Proteica , Proteína-Arginina Desiminase do Tipo 2 , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Alinhamento de SequênciaRESUMO
The integron/gene cassette systems identified in bacteria comprise a class of genetic elements that allow adaptation by acquisition of gene cassettes. Integron gene cassettes have been shown to facilitate the spread of drug resistance in human pathogens but their role outside a clinical setting has not been explored extensively. We sequenced 2145 integron gene cassettes from four marine sediment samples taken from the vicinity of Halifax Nova Scotia, Canada, increasing the number of gene cassettes obtained from environmental microbial communities by 10-fold. Sequence analyses reveals that the majority of these cassettes encode novel proteins and that this study is consistent with previous claims of high cassette diversity as we estimate a Chao1 diversity index of approximately 3000 cassettes from these samples. The functional distribution of environmental cassettes recovered in this study, when compared with that of cassettes from the only other source with significant sampling (Vibrio genomes) suggests that alternate selection regimes might be acting on these two gene pools. The majority of cassettes recovered in this study encode novel, unknown proteins. In instances where we obtained multiple alleles of a novel protein we demonstrate that non-synonymous versus synonymous substitution rates ratios suggest relaxed selection. Cassette-encoded proteins with known homologues represent a variety of functions and prevalent among these are isochorismatases; proteins involved in iron scavenging. Phylogenetic analysis of these isochorismatases as well as of cassette-encoded acetyltransferases reveals a patchy distribution, suggesting multiple sources for the origin of these cassettes. Finally, the two most environmentally similar sample sites considered in this study display the greatest overlap of cassette types, consistent with the hypothesis that cassette genes encode adaptive proteins.