RESUMO
The search for affordable enzymes with exceptional characteristics is fundamental to overcoming industrial and environmental constraints. In this study, a recombinant GH10 xylanase (Xyn10-HB) from the extremely alkaliphilic bacterium Halalkalibacterium halodurans C-125 cultivated at pH 10 was cloned and expressed in E. coli BL21(DE3). Removal of the signal peptide improved the expression, and an overall activity of 8 U/mL was obtained in the cell-free supernatant. The molecular weight of purified Xyn10-HB was estimated to be 42.6 kDa by SDS-PAGE. The enzyme was active across a wide pH range (5-10) with optimal activity recorded at pH 8.5 and 60 °C. It also presented good stability with a half-life of 3 h under these conditions. Substrate specificity studies showed that Xyn10-HB is a cellulase-free enzyme that conventionally hydrolyse birchwood and oat spelts xylans (Apparent Km of 0.46 mg/mL and 0.54 mg/mL, respectively). HPLC analysis showed that both xylans hydrolysis produced xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from 2 to 9. The conversion yield was 77% after 24 h with xylobiose and xylotriose as the main end-reaction products. When assayed on alkali-extracted wheat straw heteroxylan, the Xyn10-HB produced active XOS with antioxidant activity determined by the DPPH radical scavenging method (IC50 of 0.54 mg/mL after 4 h). Owing to its various characteristics, Xyn10-HB xylanase is a promising candidate for multiple biotechnological applications.
Assuntos
Endo-1,4-beta-Xilanases , Proteínas Recombinantes , Xilanos , Especificidade por Substrato , Hidrólise , Xilanos/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Clonagem Molecular , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Glucuronatos/metabolismo , Estabilidade Enzimática , Cinética , Peso Molecular , Oligossacarídeos/metabolismo , DissacarídeosRESUMO
The modification of the surgical polypropylene mesh and the polytetrafluoroethylene vascular prosthesis with cecropin A (small peptide) and puromycin (aminonucleoside) yielded very stable preparations of modified biomaterials. The main emphasis was placed on analyses of their antimicrobial activity and potential immunomodulatory and non-cytotoxic properties towards the CCD841 CoTr model cell line. Cecropin A did not significantly affect the viability or proliferation of the CCD 841 CoTr cells, regardless of its soluble or immobilized form. In contrast, puromycin did not induce a significant decrease in the cell viability or proliferation in the immobilized form but significantly decreased cell viability and proliferation when administered in the soluble form. The covalent immobilization of these two molecules on the surface of biomaterials resulted in stable preparations that were able to inhibit the multiplication of Staphylococcus aureus and S. epidermidis strains. It was also found that the preparations induced the production of cytokines involved in antibacterial protection mechanisms and stimulated the immune response. The key regulator of this activity may be related to TLR4, a receptor recognizing bacterial LPS. In the present study, these factors were produced not only in the conditions of LPS stimulation but also in the absence of LPS, which indicates that cecropin A- and puromycin-modified biomaterials may upregulate pathways leading to humoral antibacterial immune response.
Assuntos
Anti-Infecciosos , Materiais Biocompatíveis , Materiais Biocompatíveis/farmacologia , Lipopolissacarídeos , Anti-Infecciosos/farmacologia , Antibacterianos/farmacologia , Antibacterianos/química , Polímeros/farmacologia , Staphylococcus epidermidis , PuromicinaRESUMO
Biocatalysis processes based on oxidoreductases, such as fungal laccase, are important for discovering new organic compounds with broad structures and potential applications. They include bioactive compounds, which can be obtained through laccase-mediated oxidation of organic substrates having hydroxyl and/or amino groups especially, e.g., 5-aminosalicylic acid (5-ASA) is characterised for its potential for oxidation by a fungal laccase obtained from a Cerrena unicolor strain. The biotransformation process was optimised in terms of the buffer and co-solvent concentration, buffer pH value, and laccase activity. Selected crude dyes were analysed for their bioactive properties, toxicity, and suitability for the dyeing of wool fibres. The data obtained clearly indicated that a low concentration of the reaction buffer in the pH range from 5 to 6 and in the presence of 10% acetonitrile increased the rate of substrate oxidation and the amount of the product formed. The red-brown compound obtained via laccase-mediated oxidation of 5-aminosalicylic acid showed antioxidant properties and unique antimicrobial activity against Staphylococcus aureus and Staphylococcus epidermidis strains with the MIC value of 0.125 mg/mL detected for the purest dye. In addition, it was reported to have good wool fibre dyeing properties and no irritant effect after patch tests on a selected group with increased skin sensitivity.
Assuntos
Lacase , Mesalamina , Animais , Lacase/metabolismo , Mesalamina/farmacologia , Oxirredução , Antioxidantes/química , Corantes/química , Concentração de Íons de HidrogênioRESUMO
Prosthetic infections are associated with high morbidity, mortality, and relapse rates, making them still a serious problem for implantology. Staphylococcus aureus is one of the most common bacterial pathogens causing prosthetic infections. In response to the increasing rate of bacterial resistance to commonly used antibiotics, this work proposes a method for combating pathogenic microorganisms by modifying the surfaces of synthetic polymeric biomaterials using proteolytic enzyme inhibitors (serine protease inhibitors-4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and puromycin). While using techniques based on the immobilization of biologically active molecules, it is important to monitor the changes occurring on the surface of the modified biomaterial, where spectroscopic techniques (e.g., FTIR) are ideal. ATR-FTIR measurements demonstrated that the immobilization of both inhibitors caused large structural changes on the surface of the tested vascular prostheses (polyester or polytetrafluoroethylene) and showed that they were covalently bonded to the surfaces of the biomaterials. Next, the bactericidal and antibiofilm activities of the tested serine protease inhibitors were determined using the CLSM microscopic technique with fluorescent staining. During LIVE/DEAD analyses, a significant decrease in the formation of Staphylococcus aureus biofilm after exposure to selected concentrations of native inhibitors (0.02-0.06 mg/mL for puromycin and 0.2-1 mg/mL for 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride) was demonstrated.
Assuntos
Anti-Infecciosos , Infecções Estafilocócicas , Sulfonas , Humanos , Prótese Vascular , Antibacterianos/farmacologia , Biofilmes , Inibidores de Serina Proteinase/farmacologia , Staphylococcus aureus , Materiais Biocompatíveis , Puromicina , Peptídeo HidrolasesRESUMO
In the present study, the potential of lead and cadmium removal by the extracellular polymeric substances (EPS) produced from Parachlorella kessleri and Chlorella vulgaris were investigated. Carbohydrates were the dominant components of EPS from both analyzed species. The contents of reducing sugars, uronic acids, and amino acids were higher in EPS synthesized by C. vulgaris than in EPS from P. kessleri. The analysis of the monosaccharide composition showed the presence of rhamnose, mannose and galactose in the EPS obtained from both species. The ICP-OES (inductively coupled plasma optical emission spectrometry) analyses demonstrated that C. vulgaris EPS showed higher sorption capacity in comparison to P. kessleri EPS. The sorption capacity of C. vulgaris EPS increased with the increase in the amount of metal ions. P. kessleri EPS had a maximum sorption capacity in the presence of 100 mg/L of metal ions. The FTIR analysis demonstrated that the carboxyl, hydroxyl, and carbonyl groups of EPS play a key role in the interactions with metal ions. The present study showed C. vulgaris EPS can be used as a biosorbent in bioremediation processes due to its biochemical composition, the presence of significant amounts of negatively charged uronic acids, and higher sorption capacity.
Assuntos
Chlorella vulgaris , Matriz Extracelular de Substâncias Poliméricas , Matriz Extracelular de Substâncias Poliméricas/química , Cádmio/química , Chlorella vulgaris/metabolismo , Metais/análise , Íons/análise , Ácidos Urônicos/metabolismoRESUMO
Oxalate oxidase (EC 1.2.3.4) is an oxalate-decomposing enzyme predominantly found in plants but also described in basidiomycete fungi. In this study, we investigated 23 fungi to determine their capability of oxalic acid degradation. After analyzing their secretomes for the products of the oxalic acid-degrading enzyme activity, three groups were distinguished among the fungi studied. The first group comprised nine fungi classified as oxalate oxidase producers, as their secretome pattern revealed an increase in the hydrogen peroxide concentration, no formic acid, and a reduction in the oxalic acid content. The second group of fungi comprised eight fungi described as oxalate decarboxylase producers characterized by an increase in the formic acid level associated with a decrease in the oxalate content in their secretomes. In the secretomes of the third group of six fungi, no increase in formic acid or hydrogen peroxide contents was observed but a decline in the oxalate level was found. The intracellular activity of OXO in the mycelia of Schizophyllum commune, Trametes hirsuta, Gloeophyllum trabeum, Abortiporus biennis, Cerrena unicolor, Ceriosporopsis mediosetigera, Trametes sanguinea, Ceriporiopsis subvermispora, and Laetiporus sulphureus was confirmed by a spectrophotometric assay.
Assuntos
Peróxido de Hidrogênio , Madeira , Madeira/microbiologia , Trametes/metabolismo , Ácido Oxálico/metabolismoRESUMO
Fungal laccase obtained from a Cerrena unicolor strain was used as an effective biocatalyst for the transformation of 8-anilino-1-naphthalenesulfonic acid into a green-coloured antibacterial compound, which can be considered as both an antimicrobial agent and a textile dye, simultaneously. The process of biosynthesis was performed in buffered solutions containing methanol as a co-solvent, allowing better solubilisation of substrate. The transformation process was optimised in terms of the buffer pH value, laccase activity, and concentrations of the substrate and co-solvent. The crude product obtained exhibited low cytotoxicity, antibacterial properties against Staphylococcus aureus and Staphylococcus epidermidis, and antioxidant properties. Moreover, the synthesised green-coloured compound proved non-allergenic and demonstrated a high efficiency of dyeing wool fibres.
Assuntos
Naftalenossulfonato de Anilina/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Corantes/química , Corantes/farmacologia , Lacase/metabolismo , Adulto , Idoso , Aliivibrio fischeri/efeitos dos fármacos , Naftalenossulfonato de Anilina/química , Antibacterianos/biossíntese , Antibacterianos/toxicidade , Antioxidantes/química , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Antioxidantes/toxicidade , Biocatálise , Linhagem Celular , Colo/efeitos dos fármacos , Corantes/metabolismo , Corantes/toxicidade , Células Epiteliais/efeitos dos fármacos , Feminino , Fibroblastos/efeitos dos fármacos , Fungos/enzimologia , Voluntários Saudáveis , Humanos , Hipersensibilidade , Lacase/química , Masculino , Pessoa de Meia-Idade , Oxirredução , Pele/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacosRESUMO
The water-soluble fraction of proteoglycan RS-89 isolated from the Rhodococcus opacus FCL89 and composed of 64.6% polysaccharide and 9.44% protein has been studied as regards its flocculating activity. The RS-89 polysaccharide component includes mannose, galactose and glucose at the molar ratio of 2.7: 1.3: 1. The basic factors affecting flocculating activity of the RS-89 have been established. Additionally, the kinetics of kaolin sedimentation without and with the bioflocculant was investigated. The presence of divalent metal ions had a positive effect on the flocculating activity of the RS-89. The addition of Ca2+ increased the RS-89 flocculating activity in comparison to the other studied metals. It was proved that the proteoglycan RS-89 achieved the highest flocculating activity at the concentration equal to 2 mg/L and in the presence of 10 mmol/L of Ca2+. The zeta potential values are less negative when there is an interaction between the kaolin particles and metal ions without the RS-89 in the tested systems. Therefore, the proposed mechanism to describe the proteoglycan interaction with kaolin particles in the presence of divalent ions includes charge neutralization and a bridging mechanism.
Assuntos
Rhodococcus , Floculação , Concentração de Íons de Hidrogênio , ProteoglicanasRESUMO
Novel sustainable processes involving oxidative enzymatic catalysts are considered as an alternative for classical organic chemistry. The unique physicochemical and bioactive properties of novel bio-products can be obtained using fungal laccase as catalyst. Among them are textile biodyes synthesised during oxidation of substrates belonging to the amine and methoxy organic derivatives. The process of synthesis occurs in mild conditions of pH, temperature, and pressure, and without using harmful oxidants. The effect of fungal laccase activity on the substrates mixture transformation efficiency was analysed in terms of antimicrobial dye synthesis on a large scale. Three new phenazine dyes, obtained in the presence of laccase from Cerrena unicolor, were studied for their structure and properties. The phenazine core structure of the products was a result of tri-molecular transformation of aminomethoxybenzoic acid and aminonaphthalene sulfonic acid isomers. One of the compounds from the synthesised dye, namely 10-((2-carboxy-6-methoxyphenyl)amino)-11-methoxybenzo[a]phenazine-8-carboxylic acid, was able to inhibit the growth of Staphylococcus aureus. The high concentration of substrates (5 g/L) was efficiently transformed during 72 h in the mild conditions of pH 4 with the use of laccase with an activity of 200 U per g of the substrates mixture. The new bioactive dye exhibited excellent dyeing properties with concomitant antibacterial and antioxidative activity. The proposed enzyme-mediated synthesis represents an alternative eco-friendly route for the synthesis of novel antimicrobial compounds with high importance for the medical textile industry.
Assuntos
Corantes/química , Corantes/farmacologia , Fungos/enzimologia , Lacase/metabolismo , Têxteis , Antioxidantes/química , Antioxidantes/farmacologia , Biotransformação , Cromatografia Líquida de Alta Pressão , Eletroquímica , Concentração de Íons de Hidrogênio , Cinética , Estrutura Molecular , Oxirredução , Relação Estrutura-AtividadeRESUMO
Discovered in 1883, laccase is one of the first enzymes ever described. Now, after almost 140 years of research, it seems that this copper-containing protein with a number of unique catalytic properties is widely distributed across all kingdoms of life. Laccase belongs to the superfamily of multicopper oxidases (MCOs)-a group of enzymes comprising many proteins with different substrate specificities and diverse biological functions. The presence of cupredoxin-like domains allows all MCOs to reduce oxygen to water without producing harmful byproducts. This review describes structural characteristics and plausible evolution of laccase in different taxonomic groups. The remarkable catalytic abilities and broad substrate specificity of laccases are described in relation to other copper-containing MCOs. Through an exhaustive analysis of laccase roles in different taxa, we find that this enzyme evolved to serve an important, common, and protective function in living systems.
Assuntos
Lacase/química , Lacase/metabolismo , Bactérias/enzimologia , Bactérias/genética , Evolução Molecular , Fungos/enzimologia , Fungos/genética , Humanos , Lacase/genética , Filogenia , Domínios Proteicos , Especificidade por SubstratoRESUMO
Exopolymeric substances (EPS) can determine plant-microorganism interactions and have great potential as bioactive compounds. The different amounts of EPS obtained from cultures of three endophytic Fusarium culmorum strains with different aggressiveness-growth promoting (PGPF), deleterious (DRMO), and pathogenic towards cereal plants-depended on growth conditions. The EPS concentrations (under optimized culture conditions) were the lowest (0.2 g/L) in the PGPF, about three times higher in the DRMO, and five times higher in the pathogen culture. The EPS of these strains differed in the content of proteins, phenolic components, total sugars, glycosidic linkages, and sugar composition (glucose, mannose, galactose, and smaller quantities of arabinose, galactosamine, and glucosamine). The pathogen EPS exhibited the highest total sugar and mannose concentration. FTIR analysis confirmed the ß configuration of the sugars. The EPS differed in the number and weight of polysaccharidic subfractions. The EPS of PGPF and DRMO had two subfractions and the pathogen EPS exhibited a subfraction with the lowest weight (5 kDa). The three EPS preparations (ethanol-precipitated EP, crude C, and proteolysed P) had antioxidant activity (particularly high for the EP-EPS soluble in high concentrations). The EP-EPS of the PGPF strain had the highest antioxidant activity, most likely associated with the highest content of phenolic compounds in this EPS.
Assuntos
Antioxidantes/química , Antioxidantes/farmacologia , Grão Comestível/efeitos dos fármacos , Matriz Extracelular de Substâncias Poliméricas/química , Matriz Extracelular de Substâncias Poliméricas/metabolismo , Fusarium/fisiologia , Antioxidantes/isolamento & purificação , Grão Comestível/microbiologia , Interações Hospedeiro-PatógenoRESUMO
Three serine protease inhibitors (AEBSF, soy inhibitor, α1-antitrypsin) were covalently immobilized on the surface of three polymer prostheses with the optimized method. The immobilization efficiency ranged from 11 to 51%, depending on the chosen inhibitor and biomaterial. The highest activity for all inhibitors was observed in the case of immobilization on the surface of the polyester Uni-Graft prosthesis, and the preparations obtained showed high stability in the environment with different pH and temperature values. Modification of the Uni-Graft prosthesis surface with the synthetic AEBSF inhibitor and human α1-antitrypsin inhibited the adhesion and multiplication of Staphylococcus aureus subs. aureus ATCC® 25923TM and Candida albicans from the collection of the Department of Genetics and Microbiology, UMCS. Optical profilometry analysis indicated that, after the immobilization process on the surface of AEBSF-modified Uni-Graft prostheses, there were more structures with a high number of protrusions, while the introduction of modifications with a protein inhibitor led to the smoothing of their surface.
Assuntos
Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/metabolismo , Inibidores de Serina Proteinase/farmacologia , Antibacterianos/farmacologia , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/farmacologia , Candida albicans/efeitos dos fármacos , Endopeptidases , Humanos , Polímeros , Staphylococcus aureus/efeitos dos fármacos , Sulfonas/química , Sulfonas/farmacologia , alfa 1-Antitripsina/química , alfa 1-Antitripsina/farmacologiaRESUMO
We have described for the first time the localisation of oxalate oxidase (OXO, EC 1.2.3.4) in Abortiporus biennis cells, using histochemical and immunochemical methods coupled with transmission electron microscopy. Rabbit anti-oxalate oxidase immunoglobulins with anti-rabbit secondary antibody conjugated with 10-nm gold particles were used. Moreover, the formation of electron dense precipitation of reaction of diaminobenzidine (DAB) with horseradish peroxidase (HRP) for histochemical localisation of the enzyme was found. OXO was localised close to the membranous structures of the cell membranes, in membranous vesicles located close to the outer cell membrane, and vacuolar membranes surrounding vacuoles. The positive immunoreaction to OXO was also intense in cell wall areas. Moreover, we proved that gene coding for OXO was expressed in the same cultures. Corresponding mRNA was isolated, full length cDNA was synthesized, cloned and sequenced. Two copies of cupin domains were found in the sequence of amino-acids conserved domain coding for the cupin enzyme. Comparison of the genomic DNA and cDNA sequences has revealed the presence of seventeen introns in the gene. The isoelectric point of the protein was estimated at pH 4.5 and several possible N-glycosylation sites were predicted.
Assuntos
Basidiomycota/enzimologia , Basidiomycota/genética , Oxirredutases/genética , Oxirredutases/metabolismo , Basidiomycota/ultraestrutura , DNA Complementar , Ativação Enzimática , Imuno-Histoquímica , Oxirredutases/química , Oxirredutases/isolamento & purificação , Transporte Proteico , Análise de Sequência de DNARESUMO
Transformation of 2-amino-3-methoxybenzoic acid into novel and eco-friendly orange dye (N15) was performed using native and immobilised laccase (LAC) from Pleurotus ostreatus strain. A several parameters affecting laccase-mediated transformation efficiency included the selection of type and pH value of buffer, reaction temperature, substrate and laccase concentration as well as the type of carrier and LAC storage conditions were evaluated. The optimal conditions for N15 dye synthesis were 40â¯mM sodium-tartrate buffer pH 5.5 containing 3â¯mM of the substrate, efficiently transformed by 2 U of free laccase per 1â¯mmol of the substrate. Laccase was immobilised on porous Purolite® carriers, which had never been tested as a support for oxidoreductases. Immobilised laccase, characterised by a high immobilisation yield, was obtained by adsorption of laccase on a porous acrylic carrier with octadecyl groups (C18) incubated in optimum conditions of 40â¯mM phosphate buffer pH 7.0 containing 1â¯mg of laccase per 1â¯g of the carrier (wet mass). The immobilised LAC showed the highest storage stability for 21 days and higher thermostability at 40â¯â and 60â¯â in comparison to its native form. The N15 dye showed good dyeing properties towards natural fibres, and the dyed fibre demonstrated resistance to different physicochemical factors during use, which was confirmed by commercial quality tests. The N15 dye is a phenazine, i.e. a heterogenic compound containing amino-, methoxy-, and three carboxyl functional groups with the molecular weight of approximately 449.37 U.
Assuntos
Corantes/metabolismo , Enzimas Imobilizadas/metabolismo , Lacase/metabolismo , Pleurotus/enzimologia , Ácido Vanílico/análogos & derivados , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Têxteis , Ácido Vanílico/químicaRESUMO
Four bacterial EPSs extracted from Rhizobium leguminosarum bv. trifolii Rt24.2, Sinorhizobium meliloti Rm1021, Bradyrhizobium japonicum USDA110, and Bradyrhizobium elkanii USDA76 were determined towards their metal ion adsorption properties and possible modification of Cerrena unicolor laccase properties. The highest magnesium and iron ion-sorption capacity (~ 42 and ~ 14.5%, respectively) was observed for EPS isolated from B. japonicum USDA110. An evident influence of EPSs on the stability of laccase compared to the control values (without EPSs) was shown after 30-day incubation at 25 °C. The residual activity of laccases was obtained in the presence of Rh76EPS and Rh1021EPS, i.e., 49.5 and 41.5% of the initial catalytic activity, respectively. This result was confirmed by native PAGE electrophoresis. The EPS effect on laccase stability at different pH (from 3.8 to 7.0) was also estimated. The most significant changes at the optimum pH value (pH 5.8) was observed in samples of laccase stabilized by Rh76EPS and Rh1021EPS. Cyclic voltamperometry was used for analysis of electrochemical parameters of laccase stabilized by bacterial EPS and immobilized on single-walled carbon nanotubes (SWCNTs) with aryl residues. Laccases with Rh76EPS and Rh1021EPS had an evident shift of the value of the redox potential compared to the control without EPS addition. In conclusion, the results obtained in this work present a new potential use of bacterial EPSs as a metal-binding component and a modulator of laccase properties especially stability of enzyme activity, which can be a very effective tool in biotechnology and industrial applications.
Assuntos
Bactérias/química , Basidiomycota/enzimologia , Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Lacase/química , Metais/química , Polissacarídeos Bacterianos/química , Estabilidade Enzimática , Concentração de Íons de HidrogênioRESUMO
Extensive research efforts have been dedicated to describing degradation of wood, which is a complex process; hence, microorganisms have evolved different enzymatic and non-enzymatic strategies to utilize this plentiful plant material. This review describes a number of fungal and bacterial organisms which have developed both competitive and mutualistic strategies for the decomposition of wood and to thrive in different ecological niches. Through the analysis of the enzymatic machinery engaged in wood degradation, it was possible to elucidate different strategies of wood decomposition which often depend on ecological niches inhabited by given organism. Moreover, a detailed description of low molecular weight compounds is presented, which gives these organisms not only an advantage in wood degradation processes, but seems rather to be a new evolutionatory alternative to enzymatic combustion. Through analysis of genomics and secretomic data, it was possible to underline the probable importance of certain wood-degrading enzymes produced by different fungal organisms, potentially giving them advantage in their ecological niches. The paper highlights different fungal strategies of wood degradation, which possibly correlates to the number of genes coding for secretory enzymes. Furthermore, investigation of the evolution of wood-degrading organisms has been described.
Assuntos
Fungos/enzimologia , Genoma Fúngico/genética , Lignina/metabolismo , Madeira/microbiologia , Bactérias/classificação , Bactérias/enzimologia , Bactérias/genética , Evolução Biológica , Fungos/classificação , Fungos/genéticaRESUMO
In this study, the transcriptomic-based response of the white rot fungus Abortiporus biennis to oxalic acid induction was reported. The whole transcriptome of A. biennis was analysed using the RNA-based sequencing technology and Solid 5500 platform. De novo assembly of reads generated 37,719 contigs. A molecular function for 26,280 unique transcripts was assigned. The analysis of the A. biennis transcriptome predicted 635 hypothetical open reading frames encoding carbohydrate active enzymes distributed in 122 families. 82 genes were identified, whose expression level was significantly changed after oxalic acid addition. Among them, 18 genes were up-regulated and 64 genes were down-regulated. Genes coding for putative cellulose and hemicellulose degrading enzymes were predominantly up-regulated in the mycelium induced with oxalic acid; it was in the case of cellulases and xylanases (hemicellulases), in particular, ß-glucosidase and endo-1,4-ß-xylanases. On the contrary, several genes coding for lignolytic enzymes were down-regulated, with the significant repression level in the case of versatile peroxidase. Finally, we identified putative genes involved in oxalate metabolism. Among the transcripts detected in the A. biennis transcriptome, one was annotated as coding for putative oxalate decarboxylase (ODC) and nine transcripts were annotated as formate dehydrogenases (FDH). The addition of oxalic acid to the culture caused upregulation of the gene coding for ODC and three genes for FDH. Amongst the transcripts of putative FDH genes, one designated as NODE_36057, demonstrated the highest induction level recorded in this study after the oxalic acid addition.
Assuntos
Basidiomycota/efeitos dos fármacos , Basidiomycota/enzimologia , Basidiomycota/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica/fisiologia , Ácido Oxálico/farmacologia , Sequência de Bases , Basidiomycota/metabolismo , Celulases/genética , Regulação para Baixo , Endo-1,4-beta-Xilanases/genética , Formiato Desidrogenases/genética , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Genes Fúngicos , Glicosídeo Hidrolases/genética , Micélio/efeitos dos fármacos , Micélio/enzimologia , Oxirredutases/genética , RNA Fúngico/isolamento & purificação , Transcriptoma , Madeira/microbiologia , beta-Glucosidase/genéticaRESUMO
From the earliest times, medicine has focused on finding the most suitable and effective treatment for every patient. At present, a dynamic development of diagnostic methods and techniques for designing new drugs allows to create therapies for many diseases at the molecular level. Among the many drugs appearing on the medical market every year, special attention should be paid to those whose action is based on the inhibition of proteolytic enzyme activity. Protease inhibitors are a diverse group of biologically active molecules for which antiviral, antimicrobial, antifungal, antiparasitic or anticancer effects have been documented. Successes in the treatment of HIV infection, hepatitis C and influenza diseases certainly encourage researchers to look for new inhibitors that could be used in new therapies. This paper provides an overview of selected information on enzyme inhibitors, especially protease inhibitors, which are already registered medicines and substances that are promising candidates for medical use.
Assuntos
Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/enzimologia , Hepatite C/tratamento farmacológico , Hepatite C/enzimologia , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/enzimologiaRESUMO
Pharmaceuticals and other biologically active substances are produced in increasing numbers. Because of increased usage and improper storage, they pass into surface water, ground water and drinking water directly or through wastewaters. This is a threat to many living organisms, including humans, because of hormonal imbalances primarily related to reproductive processes or the problem of microbial drug resistance. Due to the scale of the emission and limited possibilities of decomposition of these pollutants by physico-chemical methods it is necessary to develop new efficient processes. One of the proposed solutions is the use of tools offered by biocatalysis. Thanks to the biocatalysis process, a wide range of biologically active compounds can be removed, by using of enzymes with low substrate specificity and operating in environmentally friendly conditions. Recent studies indicate the effectiveness of those methods used in the removal of pollutants of different chemical structure, with the formation of non-toxic metabolites.
Assuntos
Biocatálise , Enzimas/metabolismo , Poluentes Químicos da Água/isolamento & purificação , Poluentes Químicos da Água/metabolismo , Purificação da Água/métodos , Água/química , Especificidade por SubstratoRESUMO
Oxalate oxidase was identified in mycelial extracts of a basidiomycete Abortiporus biennis strain. Intracellular enzyme activity was detected only after prior lowering of the pH value of the fungal cultures by using oxalic or hydrochloric acids. This enzyme was purified using size exclusion chromatography (Sephadex G-25) and ion-exchange chromatography (DEAE-Sepharose). This enzyme exhibited optimum activity at pH 2 when incubated at 40°C, and the optimum temperature was established at 60°C. Among the tested organic acids, this enzyme exhibited specificity only towards oxalic acid. Molecular mass was calculated as 58 kDa. The values of Km for oxalate and Vmax for the enzyme reaction were 0.015 M and 30 mmol min(-1), respectively.