RESUMEN
With the emergence of multidrug-resistant microorganisms, microbial agents have become a serious global threat, affecting human health and various plants. Therefore, new therapeutic alternatives, such as chitin-binding proteins, are necessary. Chitin is an essential component of the fungal cell wall, and chitin-binding proteins exhibit antifungal activity. In the present study, chitin-binding peptides isolated from Capsicum chinense seeds were characterized and evaluated for their in vitro antimicrobial effect against the growth of Candida and Fusarium fungi. Proteins were extracted from the seeds and subsequently the chitin-binding proteins were separated by chitin affinity chromatography. After chromatography, two fractions, Cc-F1 (not retained on the column) and Cc-F2 (retained on the column), were obtained. Electrophoresis revealed major protein bands between 6.5 and 26.6 kDa for Cc-F1 and only a ~ 6.5 kDa protein band for Cc-F2, which was subsequently subjected to mass spectrometry. The protein showed similarity with hevein-like and endochitinase and was then named Cc-Hev. Data are available via ProteomeXchange with identifier PXD054607. Next, we predicted the three-dimensional structure of the peptides and performed a peptide docking with (NAG)3. Subsequently, growth inhibition assays were performed to evaluate the ability of the peptides to inhibit microorganism growth. Cc-Hev inhibited the growth of C. albicans (up to 75% inhibition) and C. tropicalis (100% inhibition) and induced a 65% decrease in cell viability for C. albicans and 100% for C. tropicalis. Based on these results, new techniques to combat fungal diseases could be developed through biotechnological applications; therefore, further studies are needed.
Asunto(s)
Antifúngicos , Candida , Capsicum , Quitina , Quitinasas , Fusarium , Semillas , Semillas/química , Antifúngicos/farmacología , Antifúngicos/aislamiento & purificación , Antifúngicos/química , Antifúngicos/metabolismo , Quitina/metabolismo , Quitina/farmacología , Fusarium/efectos de los fármacos , Quitinasas/farmacología , Quitinasas/metabolismo , Quitinasas/química , Quitinasas/aislamiento & purificación , Candida/efectos de los fármacos , Candida/enzimología , Lectinas de Plantas/farmacología , Lectinas de Plantas/química , Lectinas de Plantas/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Péptidos/farmacología , Péptidos/química , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Simulación del Acoplamiento Molecular , Proteínas de Plantas/farmacología , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Péptidos Catiónicos AntimicrobianosRESUMEN
The current study provides a comprehensive look of the adsorption process of Candida rugosa lipase (CRL) on Ca2Fe2O5 iron oxide nanoparticles (NPs). Protein-support interactions were identified across a broad range of pH and ionic strengths (mM) through a response surface methodology, surface charge determination, and spectroscopic and in silico analyses. The maximum quantity of immobilized protein was achieved at an ionic strength of 50 mM and pH 4. However, this condition did not allow for the greatest hydrolytic activity to be obtained. Indeed, it was recorded at acidic pH, but at 150 mM, where evaluation of the recovered activity revealed hyperactivation of the enzyme. These findings were supported by adsorption isotherms performed under different conditions. Based on zeta potential measurements, electrostatic interactions contributed differently to protein-support binding under the conditions tested, showing a strong correlation with experimentally determined immobilization parameters. Raman spectra revealed an increase in hydrophobicity around tryptophan residues, whereas the enzyme immobilization significantly reduced the phenylalanine signal in CRL. This suggests that this residue was involved in the interaction with Ca2Fe2O2 and molecular docking analysis confirmed these findings. Fluorescence spectroscopy showed distinct behaviors in the CRL emission patterns with the addition of Ca2Fe2O5 at pH 4 and 7. The calculated thermodynamic parameters indicated that the contact would be mediated by hydrophobic interactions at both pHs, as well as by ionic ones at pH 4. In this approach, this work adds to our understanding of the design of biocatalysts immobilized in iron oxide NPs.
Asunto(s)
Candida , Candida/enzimología , Concentración de Iones de Hidrógeno , Lipasa/metabolismo , Concentración Osmolar , Enzimas Inmovilizadas/metabolismo , Simulación del Acoplamiento Molecular , Interacciones Hidrofóbicas e Hidrofílicas , Nanopartículas del Metal/químicaRESUMEN
Lipase immobilization using adsorption on magnetic nanoparticles, cross-linked enzyme aggregates (CLEA), and a combination of both techniques was investigated. Experimental designs were used for the optimization of the immobilization observing that the pH and ionic strength play a principal role during the lipase immobilization and its activity. For adsorption on magnetic nanoparticles and CLEA synthesis the optimal condition was pH and 100 mM. Besides, during the CLEA synthesis, glutaraldehyde concentration showed to be a significant effect on the enzyme activity. A comparison between a magnetic CLEA prepared with (Lip@mCLEA) and without (mCLEA) biological functionalized magnetic nanoparticles was made observing that the use of functionalized support showed the best performance activity. All biocatalytic systems developed gives to the enzyme thermal stability between 45 and 70 °C, being Lip@mCLEA the more stable biocatalyst. Similar behavior was observed at different pH, where both Lip@mCLEA and mCLEA showed stability at a range of pH 5 to 8. The immobilized biocatalysts showed the same affinity of the subtract that the free enzyme suggested that the enzyme structure not modified the active site. The combination of both types of immobilization show evidenced the importance of the biological functionalization of the support when magnetic CLEA is produced.
Asunto(s)
Enzimas Inmovilizadas/química , Proteínas Fúngicas/química , Lipasa/química , Nanopartículas de Magnetita/química , Candida/enzimología , Reactivos de Enlaces Cruzados/química , Estabilidad de Enzimas , Enzimas Inmovilizadas/metabolismo , Compuestos Férricos/química , Proteínas Fúngicas/metabolismo , Lipasa/metabolismoRESUMEN
Candida genus comprises several species that can be found in the oral cavity and the gastrointestinal and genitourinary tracts of healthy individuals. Under certain conditions, however, they behave as opportunistic pathogens that colonize these tissues, most frequently when the immune system is compromised by a disease or under certain medical treatments. To colonize the human host, these organisms require to express cell wall proteins (CWP) that allowed them to adhere and adapt to the reactive oxygen (ROS) and nitrogen (RNS) species produced in the macrophage during the respiratory burst. The aim of this study was to determine how four Candida species respond to the oxidative stress imposed by cumene hydroperoxide (CHP). To this purpose, C. albicans, C. glabrata, C. krusei and C. parapsilosis were exposed to this oxidant which is known to generate ROS in the membrane phospholipids. Accordingly, both mock and CHP-exposed cells were used to extract and analyze CWP and also to measure catalase activity and the levels of protein carbonylation. Results indicated that all four species express different CWP to neutralize ROS. Most relevant among these proteins were the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase, known as moonlight proteins because in addition to participate in glycolysis they play an important role in the cell response to ROS. In addition, a thiol-specific antioxidant enzyme (Tsa) was also found to counteract ROS.
Asunto(s)
Derivados del Benceno/farmacología , Candida/clasificación , Candida/metabolismo , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Antioxidantes/metabolismo , Candida/enzimología , Pared Celular/metabolismo , Tracto Gastrointestinal/microbiología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Macrófagos/inmunología , Boca/microbiología , Fosfopiruvato Hidratasa/metabolismo , Proteómica , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sistema Urogenital/microbiologíaRESUMEN
Candida dubliniensis (Cd) and Candida albicans (Ca) are the most frequently isolated yeasts in HIV+ patients. Some of the enzymes produced by these yeasts are considered virulence factors since they contribute to pathogenicity of Candida spp. The aim of the present study was to compare production of enzymes such as phospholipase (Ph), proteinase (P), and hemolysin (H) by Cd and Ca strains isolated from periodontal HIV-positive patients receiving and not receiving highly active antiretroviral therapy (HAART). Subgingival biofilm samples were obtained using paper points, and a sample of oral mucosa was taken using a swab. Phenotypic and molecular methods were used to isolate 39 strains of Candida, including 25 strains of Cd and 14 strains of Ca, obtained from 33 periodontal pocket samples and 6 oral mucosa samples collected from 15 HIV+ patients (8 receiving and 7 not receiving HAART). Malt egg-yolk agar, albumin agar and blood agar were used to evaluate pH, P and H production respectively. The strains were inoculated in duplicate and incubated at 37 ºC. Colony and halo diameters were measured. A greater proportion of Ca was observed in patients not receiving HAART, and a higher proportion of Cd was observed in those under HAART, Chi2 p< 0.001. Phospholipase production was observed in 92.9% percent of isolated Ca strains but in none of the isolated Cd strains. Proteinase production was high in Ca and Cd strains isolated from patients not receiving HAART. Hemolysin production was observed in all the studied strains, though it was significantly higher (p=0.04) in Ca and Cd strains isolated from patients not receiving HAART. To sum up, the proportion of Candida dubliniensis strains was highest in the subgingival biofilm of patients receiving HAART, and Cd strains were found to express fewer virulence factors than Ca strains.
Las levaduras más aisladas en pacientes VIH+ son Candida dubliniensis (Cd) y Candida albicans (Ca). Algunas de sus enzimas constituyen factores de virulencia ya que favorecen la diseminación tisular. El objetivo fue comparar la producción de enzimas como fosfolipasa (F), proteinasa (P) y hemolisina (H) en cepas de Cd y Ca aisladas de pacientes VIH+ tratados y no tratados con antirretrovirales (TARGA). Se realizó la toma del biofilm de placa subgingival con conos de papel y la muestra de la mucosa bucal con hisopo. Se aislaron y tipificaron por métodos fenotípicos y moleculares 39 cepas: 25 de Cd y 14 Ca, obtenidas 33 de bolsas periodontales y 6 de mucosa bucal de 15 pacientes VIH+ (8 con y 7 sin tratamiento). Se utilizó agar malta con yema de huevo, agar albúmina y agar sangre para demostrar la producción de F, P y H, respectivamente. Se inocularon por duplicado e incubaron a 37°C. Se midieron los diámetros de las colonias y los de hidrólisis alrededor de las mismas. Se observó mayor proporción de Ca en los pacientes sin tratamiento y mayor proporción de Cd en los con tratamiento; Chi2 p< 0.001. El 92,9% de las Ca estudiadas, fueron productoras de fosfolipasa. En tanto que ninguna Cd produjo la enzima. En cuanto a la producción de proteinasa se observa una alta producción tanto en las cepas de Ca, como en las Cd aisladas en los pacientes no tratados. Todas las cepas estudiadas produjeron hemolisina, observándose una diferencia estadísticamente significativa (p=0,04) en ambas especies a favor de la alta producción de la enzima en las cepas obtenidas de pacientes no tratados. Podemos concluir que en el biofilm subgingival, en los pacientes bajo TARGA, se aíslan mayor proporción de Candida dubliniensis las cuales expresan menos factores de virulencia.
Asunto(s)
Terapia Antirretroviral Altamente Activa/métodos , Biopelículas/crecimiento & desarrollo , Candida albicans/enzimología , Candida albicans/aislamiento & purificación , Candida/enzimología , Candida/aislamiento & purificación , Candidiasis Bucal/microbiología , Encía/microbiología , Infecciones por VIH/complicaciones , Candida/clasificación , Candida/genética , Candida albicans/genética , Candidiasis Bucal/complicaciones , Genotipo , Infecciones por VIH/microbiología , Humanos , Mucosa Bucal/microbiología , Fenotipo , Reacción en Cadena de la Polimerasa , Factores de Virulencia/genéticaRESUMEN
ABSTRACT Candida dubliniensis (Cd) and Candida albicans (Ca) are the most frequently isolated yeasts in HIV+ patients. Some of the enzymes produced by these yeasts are considered virulence factors since they contribute to pathogenicity of Candida spp. The aim of the present study was to compare production of enzymes such as phospholipase (Ph), proteinase (P), and hemolysin (H) by Cd and Ca strains isolated from periodontal HIV-positive patients receiving and not receiving highly active antiretroviral therapy (HAART). Subgingival biofilm samples were obtained using paper points, and a sample of oral mucosa was taken using a swab. Phenotypic and molecular methods were used to isolate 39 strains of Candida, including 25 strains of Cd and 14 strains of Ca, obtained from 33 periodontal pocket samples and 6 oral mucosa samples collected from 15 HIV+ patients (8 receiving and 7 not receiving HAART). Malt egg-yolk agar, albumin agar and blood agar were used to evaluate pH, P and H production respectively. The strains were inoculated in duplicate and incubated at 37 ºC. Colony and halo diameters were measured. A greater proportion of Ca was observed in patients not receiving HAART, and a higher proportion of Cd was observed in those under HAART, Chi2 p< 0.001. Phospholipase production was observed in 92.9% percent of isolated Ca strains but in none of the isolated Cd strains. Proteinase production was high in Ca and Cd strains isolated from patients not receiving HAART. Hemolysin production was observed in all the studied strains, though it was significantly higher (p=0.04) in Ca and Cd strains isolated from patients not receiving HAART. To sum up, the proportion of Candida dubliniensis strains was highest in the subgingival biofilm of patients receiving HAART, and Cd strains were found to express fewer virulence factors than Ca strains.
RESUMEN Las levaduras más aisladas en pacientes VIH+ son Candida dubliniensis (Cd) y Candida albicans (Ca). Algunas de sus enzimas constituyen factores de virulencia ya que favorecen la diseminación tisular. El objetivo fue comparar la producción de enzimas como fosfolipasa (F), proteinasa (P) y hemolisina (H) en cepas de Cd y Ca aisladas de pacientes VIH+ tratados y no tratados con antirretrovirales (TARGA). Se realizó la toma del biofilm de placa subgingival con conos de papel y la muestra de la mucosa bucal con hisopo. Se aislaron y tipificaron por métodos fenotípicos y moleculares 39 cepas: 25 de Cd y 14 Ca, obtenidas 33 de bolsas periodontales y 6 de mucosa bucal de 15 pacientes VIH+ (8 con y 7 sin tratamiento). Se utilizó agar malta con yema de huevo, agar albúmina y agar sangre para demostrar la producción de F, P y H, respectivamente. Se inocularon por duplicado e incubaron a 37°C. Se midieron los diámetros de las colonias y los de hidrólisis alrededor de las mismas. Se observó mayor proporción de Ca en los pacientes sin tratamiento y mayor proporción de Cd en los con tratamiento; Chi2 p< 0.001. El 92,9% de las Ca estudiadas, fueron productoras de fosfolipasa. En tanto que ninguna Cd produjo la enzima. En cuanto a la producción de proteinasa se observa una alta producción tanto en las cepas de Ca, como en las Cd aisladas en los pacientes no tratados. Todas las cepas estudiadas produjeron hemolisina, observándose una diferencia estadísticamente significativa (p=0,04) en ambas especies a favor de la alta producción de la enzima en las cepas obtenidas de pacientes no tratados. Podemos concluir que en el biofilm subgingival, en los pacientes bajo TARGA, se aíslan mayor proporción de Candida dubliniensis las cuales expresan menos factores de virulencia.
Asunto(s)
Humanos , Candida/aislamiento & purificación , Candida/enzimología , Candida albicans/aislamiento & purificación , Candida albicans/enzimología , Candidiasis Bucal/microbiología , Infecciones por VIH/complicaciones , Biopelículas/crecimiento & desarrollo , Terapia Antirretroviral Altamente Activa/métodos , Encía/microbiología , Fenotipo , Candida/clasificación , Candida/genética , Candida albicans/genética , Candidiasis Bucal/complicaciones , Infecciones por VIH/microbiología , Reacción en Cadena de la Polimerasa , Factores de Virulencia/genética , Genotipo , Mucosa Bucal/microbiologíaRESUMEN
In this study, the modulation of enzymatic biocatalysts were developed by the use of lipase B from Candida antarctica covalently immobilized on an eco-friendly support, cashew apple bagasse, activated with 10% glycidol-ethylenediamine-glutaraldehyde (GEG) under different immobilization strategies (5 mM or 100 mM ionic strength and in absence or presence of 0.5% (v/v) Triton X-100). The biocatalysts were characterized for thermal and organic solvents stabilities and compared with the soluble enzyme. The biocatalysts were then applied to the hydrolysis of the rac-indanyl acetate (2:1 ratio enzyme/substrate) at pH 7.0 and 30 °C for 24 h. For all the strategies evaluated, GEG promoted kinetic resolution of rac-indanyl acetate with maximum conversion (50%) and led to (R)-indanol with excellent enantiomeric excess (97%), maintaining the maximum conversion for five consecutive cycles of hydrolysis. Therefore, the use of cashew apple bagasse has proved to be a promising eco-friendly support for enzyme immobilization, since it resulted in stable biocatalysts for enzymatic kinetic resolution.
Asunto(s)
Acetatos/química , Basidiomycota/enzimología , Proteínas Fúngicas/química , Lipasa/química , Anacardium/metabolismo , Candida/enzimología , Estabilidad de Enzimas , Enzimas Inmovilizadas/química , Etilenodiaminas/química , Glutaral/química , Concentración de Iones de Hidrógeno , Cinética , Bases de Schiff , Solventes/química , Estereoisomerismo , Temperatura , Factores de TiempoRESUMEN
The opportunistic pathogens comprising the Candida haemulonii complex (C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera) are notable for their intrinsic resistance to different antifungal classes. Little is known about the virulence attributes in this emerging fungal complex. However, it is well-recognized that enzymes play important roles in virulence/pathogenesis of candidiasis. Herein, we aimed to identify aspartyl-type peptidases in 12 clinical isolates belonging to the C. haemulonii complex. All isolates were able to grow in a chemically defined medium containing albumin as the sole nitrogen source, and a considerable consumption of this protein occurred after 72-96 h. C. haemulonii var. vulnera isolates showed the lowest albumin degradation capability and the poorest growth rate. The measurement of secreted aspartyl peptidase (Sap) activity, using the cathepsin D fluorogenic substrate, varied from 91.6 to 413.3 arbitrary units and the classic aspartyl peptidase inhibitor, pepstatin A, significantly blocked the Sap released by C. haemulonii complex. No differences were observed in the Sap activity among the three fungal species. Flow cytometry, using a polyclonal antibody against Sap1-3 of C. albicans, detected homologous proteins at the surface of C. haemulonii complex (anti-Sap1-3-labeled cells ranged from 24.6 to 79.1%). Additionally, the immunoblotting assay, conducted with the same Sap1-3 antibody, recognized a protein of â¼50 kDa in all fungal isolates. A glimpse in the genome of these fungi revealed several potential proteins containing Sap1-3-like conserved domain. Altogether, our results demonstrated the potential of C. haemulonii species complex to produce Saps, an important virulence factor of Candida spp.
Asunto(s)
Antifúngicos/farmacología , Candida/efectos de los fármacos , Candida/enzimología , Candidiasis/microbiología , Dipeptidasas/metabolismo , Candida/clasificación , Candida albicans/efectos de los fármacos , Candida albicans/enzimología , Resistencia a Múltiples Medicamentos , Humanos , Pepstatinas/farmacología , Inhibidores de Proteasas/farmacología , Análisis de Secuencia de ProteínaRESUMEN
We isolated two Candida pseudointermedia strains from the Atlantic rain forest in Brazil, and analyzed cellobiose metabolization in their cells. After growth in cellobiose medium, both strains had high intracellular ß-glucosidase activity [~ 200 U (g cells)-1 for 200 mM cellobiose and ~ 100 U (g cells)-1 for 2 mM pNPßG] and negligible periplasmic cellobiase activity. During batch fermentation, the strain with the best performance consumed all the available cellobiose in the first 18 h of the assay, producing 2.7 g L-1 of ethanol. Kinetics of its cellobiase activity demonstrated a high-affinity hydrolytic system inside cells, with Km of 12.4 mM. Our data suggest that, unlike other fungal species that hydrolyze cellobiose extracellularly, both analyzed strains transport it to the cytoplasm, where it is then hydrolyzed by high-affinity intracellular ß-glucosidases. We believe this study increases the fund of knowledge regarding yeasts from Brazilian microbiomes.
Asunto(s)
Candida/enzimología , Celobiosa/metabolismo , Madera/metabolismo , Madera/microbiología , beta-Glucosidasa/metabolismo , Brasil , Candida/aislamiento & purificación , Candida/metabolismo , Metabolismo de los Hidratos de Carbono , Etanol/metabolismo , Fermentación , Hidrólisis , CinéticaRESUMEN
This work aims to study the immobilization of Candida rugosa lipase (CRL) onto corn straw residue. For this purpose, chemical, morphological, and textural characteristics of the corn straw; immobilization process by adsorption; and immobilized enzyme activity and storage stability were evaluated. The corn straw presented isoelectric point of 7.0, surface with hydroxyl bands being favorable to the immobilization process. An irregular surface was also observed with fibers and pores, which are mesoporous and macroporous, characteristics that demonstrate efficiency in mass transfer mechanisms. Upon immobilization, it was observed that adsorption velocity is proportional to the square of the available adsorption sites (pseudo-second-order), and that the immobilization occurs in monolayers (Langmuir isotherm). The adsorption process was favorable and considered as a chemical adsorption mechanism. After immobilization, the optimum temperature increased, the optimum pH reduced, and the affinity of the biocatalyst for the substrate decreased. Corn straw derivative demonstrated good thermal stability. Regarding storage stability, there was approximately 12% loss of activity after 60 days of storage at 4 °C. Considering that no treatment was applied to the corn straw, this result is satisfactory and shows good affinity between this support and CRL.
Asunto(s)
Enzimas Inmovilizadas/metabolismo , Lipasa/metabolismo , Zea mays/química , Adsorción , Candida/enzimología , Frío , Estabilidad de Enzimas , Punto IsoeléctricoRESUMEN
Sustainability in chemistry heavily relies on heterogeneous catalysis. Enzymes, the main catalyst for biochemical reactions in nature, are an elegant choice to catalyze reactions due to their high activity and selectivity, although they usually suffer from lack of robustness. To overcome this drawback, enzyme-decorated nanoporous heterogeneous catalysts were developed. Three different approaches for Candida antarctica lipaseâ B (CAL-B) immobilization on a covalent organic framework (PPF-2) were employed: physical adsorption on the surface, covalent attachment of the enzyme in functional groups on the surface and covalent attachment into a linker added post-synthesis. The influence of the immobilization strategy on the enzyme uptake, specific activity, thermal stability, and the possibility of its use through multiple cycles was explored. High specific activities were observed for PPF-2-supported CAL-B in the esterification of oleic acid with ethanol, ranging from 58 to 283â U mg-1 , which was 2.6 to 12.7â times greater than the observed for the commercial Novozyme 435.
Asunto(s)
Enzimas Inmovilizadas/química , Proteínas Fúngicas/química , Lipasa/química , Estructuras Metalorgánicas/química , Adsorción , Biocatálisis , Candida/enzimología , Esterificación , Modelos Moleculares , Nanoporos/ultraestructura , Ácido Oléico/químicaRESUMEN
Candida haemulonii complex (C. haemulonii, C. haemulonii var. vulnera and C. duobushaemulonii) consists of emergent multidrug-resistant pathogens that cause bloodstream and deep-seated infections. However, little is known about their virulence factors. Herein, we evaluated the presence of extracellular serine peptidases in this fungal complex. Serine peptidase activity was measured by spectrophotometry using chromogenic peptide substrates to the S1 family. Chymotrypsin-, trypsin- and elastase-like activities were detected in all fungal isolates. Since higher chymotrypsin- and trypsin-like activities were observed from the cleavage of N-succinyl-Ala-Ala-Pro-Phe-pNa and N-benzoyl-Phe-Val-Arg-pNa, respectively, these substrates were selected for further experiments. Overall, pHs 7.0 and 9.0 were those in which higher chymotrypsin- and trypsin-like activities were observed, respectively, displaying higher hydrolytic activities at 37-45°C. Additionally, the serine peptidases produced by C. haemulonii complex were inhibited by PMSF and AEBSF in a typically concentration-dependent manner. Although the Michaelis constant (Km) values obtained for chymotrypsin-like peptidases were similar, greater differences were observed for trypsin-like enzymes secreted by the different fungal isolates. This is the first time that peptidases belonging to the S1 family are described in the C. haemulonii species complex. Thus, these data open the doors for more detailed studies into potential roles of these peptidases in fungal virulence.
Asunto(s)
Candida/enzimología , Quimotripsina/metabolismo , Farmacorresistencia Fúngica Múltiple , Tripsina/metabolismo , Candida/clasificación , Medios de Cultivo , Espectrofotometría , TemperaturaRESUMEN
Lipase B from Candida antarctica (CalB) is the most widely used lipase, including in many industrial sectors, such as in biodiesel and pharmaceuticals production. CalB has been produced by heterologous expression using Pichia pastoris under PGK constitutive promoter (named LipB). Here, we have studied the structural features of commercial CalB and LipB enzymes using circular dichroism and fluorescence under different conditions. In the presence of denaturing agents CalB was more stable than LipB, in contrast, at increasing temperatures, LipB was more thermostable than CalB. Mass spectrometry data indicates that both enzymes have an insertion of amino acids related to α-factor yeast signal, however LipB enzyme showed the addition of nine residues at the N-terminal while CalB showed only four residues. Molecular modeling of LipB showed the formation of an amphipathic α-helix in N-terminal region that was not observed in CalB. This data suggests that this new α-helix possess could be involved in LipB thermostability. These results associated with new structural studies may provide information to the design of novel biocatalysts.
Asunto(s)
Candida/enzimología , Proteínas Fúngicas/química , Lipasa/química , Proteínas Recombinantes de Fusión , Secuencia de Aminoácidos , Candida/genética , Activación Enzimática , Estabilidad de Enzimas , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Hidrólisis , Lipasa/genética , Lipasa/aislamiento & purificación , Lipasa/metabolismo , Modelos Moleculares , Conformación Proteica , Relación Estructura-Actividad , Temperatura , TermodinámicaRESUMEN
In this communication, lipase A from Candida antarctica (CALA) was immobilized by covalent bonding on magnetic nanoparticles coated with chitosan and activated with glutaraldehyde, labelled CALA-MNP, (immobilization parameters: 84.1% ± 1.0 for immobilization yield and 208.0 ± 3.0 U/g ± 1.1 for derivative activity). CALA-MNP biocatalyst was characterized by X-ray Powder Diffraction (XRPD), Fourier Transform Infrared (FTIR) spectroscopy, Thermogravimetry (TG) and Scanning Electron Microscope (SEM), proving the incorporation of magnetite and the immobilization of CALA in the chitosan matrix. Besides, the immobilized biocatalyst showed a half-life 8-11 times higher than that of the soluble enzyme at pH 5-9. CALA showed the highest activity at pH 7, while CALA-MNP presented the highest activity at pH 10. The immobilized enzyme was more active than the free enzyme at all studied pH values, except pH 7.
Asunto(s)
Candida/enzimología , Quitosano/química , Lipasa/metabolismo , Nanopartículas de Magnetita/química , Estabilidad de Enzimas , Enzimas Inmovilizadas/metabolismo , Proteínas Fúngicas/metabolismoRESUMEN
In the last half century there was a significant increase in the incidence of fungal infections being likely to become a global health priority. The sophisticated degree of host-Candida interaction is the product of different virulence strategies used by the fungus to invade the tissues and the various defense mechanisms that it develops to control it. There is a significant amount of literature that indicates that this opportunistic commensal fungus has components that can be considered virulence factors related to the stage of the infectious process. Among the virulence factors of this fungus can be mentioned the adherence to cell surfaces, the formation of biofilms and the production of hydrolytic enzymes. The most studied hydrolases secreted by C. albicans are aspartyl proteinases, phospholipases and esterases, while lipases have been the least studied. These enzymes would have the function to facilitate active penetration into the cells, participating in the digestion and synthesis of lipid esters for their nutrition and contributing to the invasion of the tissue by hydrolyzing the lipid components of the host cell membranes. There is also bibliographic evidence that these enzymes are capable to damage cells and molecules of the immune system to avoid the antimicrobial activity.Taking into account the foregoing, this review provides an updated description of biochemical and molecular characteristics of the lipases secreted by Candida, its role as a virulence factor and its potential for the development of new antifungal drugs.
En el último medio siglo se produjo un aumento significativo en la incidencia de infecciones fúngicas siendo probable que se conviertan en una prioridad de salud global. El sofisticado grado de interacción hospedador-Candida es producto de diferentes estrategias de virulencia que utiliza el hongo para invadir los tejidos y de los diversos mecanismos de defensa que este último desarrolla para controlarlo. Existe bibliografía que indica que este hongo comensal oportunista posee componentes que pueden ser considerados factores de virulencia asociados a la etapa del proceso infeccioso. Dentro de los factores de virulencia de este hongo pueden mencionarse la adherencia a las superficies celulares, la formación de biofilms y la producción de enzimas hidrolíticas. Las hidrolasas secretadas por C. albicans más estudiadas son las aspartil proteinasas, las fosfolipasas y las esterasas, mientras que las lipasas han sido las menos exploradas. Estas enzimas tendrían como función facilitar la penetración activa en las células, participar en la digestión y síntesis de ésteres de lípidos para su nutrición y contribuir a la invasión del tejido al hidrolizar los componentes lipídicos de las membranas celulares del hospedador. También hay evidencia bibliográfica que indica que estas enzimas son capaces de dañar células y moléculas del sistema inmune para evitar la actividad antimicrobiana. Teniendo en cuenta lo precedente, esta revisión, proporciona una actualizada descripción de las características bioquímicas y moleculares de las lipasas secretadas por el hongo Candida, su rol como factor de virulencia y su potencial para el desarrollo de nuevos fármacos antifúngicos.
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Candida/enzimología , Lipasa , Candida/patogenicidad , Humanos , Lipasa/química , Lipasa/clasificación , Lipasa/genética , Lipasa/fisiología , Factores de VirulenciaRESUMEN
BACKGROUND: Yeasts of the Candida genus are one of the most common causes of bloodstream infections associated with high rates of morbidity and mortality, mainly affecting immunocompromised patients. We aimed to identify yeasts obtained from blood cultures of patients interned at tertiary hospitals in Brazil. METHODS: We evaluated some of the major virulence factors of Candida spp., including the ability to adhere to human buccal epithelial cells, biofilm formation, hemolytic and phospholipase activity. RESULTS: We analyzed 70 isolates of Candida spp. obtained from March 2011 and March 2015. Candida spp. showed different peculiarities in terms of expression of virulence factors evaluated in vitro. C. albicans strains were more adherent to HBEC than all the other Candida species. C. tropicalis strains were considered strong biofilm producers. Strains belonging to the C. parapsilosis species complex were able to produce hemolysins, while C. glabrata was also able to lyse erythrocytes and to produce phospholipase. CONCLUSION: These results suggest that Non-Candida albicans Candida species are also able to express virulence factors which play an important role in bloodstream infectious caused by these yeasts.
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Candida/aislamiento & purificación , Candida/patogenicidad , Candidemia/epidemiología , Factores de Virulencia/metabolismo , Biopelículas/crecimiento & desarrollo , Cultivo de Sangre , Brasil/epidemiología , Candida/enzimología , Candida albicans/aislamiento & purificación , Candida albicans/patogenicidad , Candida glabrata/aislamiento & purificación , Candida glabrata/patogenicidad , Candidemia/microbiología , Células Epiteliales/microbiología , Proteínas Hemolisinas/metabolismo , Humanos , Boca , Fosfolipasas/metabolismo , Centros de Atención TerciariaRESUMEN
The effect of gene dosage on the production of Candida antarctica lipase B (CalB) in the methylotrophic yeast Komagataella phaffii, at high densities in a simple medium containing crude glycerin as the sole carbon source, is described. The use of crude glycerin, the main by-product of biodiesel production from vegetable oils, will reduce the production cost of the bioprocess. Two K. phaffii strains were constructed with one or three copies of LipB, an optimized version of the gene encoding CalB under the control of the constitutive PPGK1 promoter. These two constructs were tested and compared on batches using minimal-salts medium with crude glycerin. The strain with three copies achieved a higher enzyme yield (48,760 U/L, 2.3-fold higher than the one-copy strain), with 42 g/L biomass, with no effects on growth.
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Candida/enzimología , Candida/genética , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Lipasa/biosíntesis , Lipasa/genética , Pichia/genética , Saccharomycetales/metabolismo , Candida/metabolismo , Dosificación de Gen/genética , Glicerol/metabolismo , Regiones Promotoras Genéticas/genética , Saccharomycetales/genéticaRESUMEN
This work aimed to evaluate the inhibition of Candida rugosa lipase by five guanylhydrazone derivatives through biological, biophysical and theoretical studies simulating physiologic conditions. The compound LQM11 (IC50â¯=â¯14.70⯵M) presented the highest inhibition against the enzyme. Therefore, for a better understanding of the interaction process, spectroscopic and theoretical studies were performed. Fluorescence and UV-vis assays indicate a static quenching mechanism with non-fluorescent supramolecular complex formation and changing the native protein structure. The binding process was spontaneous (ΔGâ¯<â¯0) and electrostatic forces (ΔHâ¯<â¯0 and ΔSâ¯>â¯0) played a preferential role in stabilizing the complex ligand-lipase. The compounds were classified as non-competitive inhibitors using orlistat as a reference in competition studies. Based on the 1H NMR assays it was possible to propose the sites of ligand (epitope) that bind preferentially to the enzyme and the theoretical studies were consistent with the experimental results. Finally, LQM11 was efficient as a lipase inhibitor of the crude intestinal extract of larvae of Rhynchophorus palmarum, an important agricultural plague, showing potential for control of this pest. Within this context, the real potential of this biotechnological application deserves further studies.
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Candida/enzimología , Inhibidores Enzimáticos/farmacología , Hidrazonas/farmacología , Lipasa/antagonistas & inhibidores , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Animales , Biotecnología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Hidrazonas/química , Hidrazonas/aislamiento & purificación , Lipasa/metabolismo , Estructura Molecular , Termodinámica , Gorgojos/químicaRESUMEN
With the use of genetic engineering, modified and sometimes more efficient enzymes can be created for different purposes, including industrial applications. However, building modified enzymes depends on several in vitro experiments, which may result in the process being expensive and time-consuming. Therefore, computational approaches could reduce costs and accelerate the discovery of new technological products. In this study, we present a method, called structural signature variation (SSV), to propose mutations for improving enzymes' activity. SSV uses the structural signature variation between target enzymes and template enzymes (obtained from the literature) to determine if randomly suggested mutations may provide some benefit for an enzyme, such as improvement of catalytic activity, half-life, and thermostability, or resistance to inhibition. To evaluate SSV, we carried out a case study that suggested mutations in ß-glucosidases: Essential enzymes used in biofuel production that suffer inhibition by their product. We collected 27 mutations described in the literature, and manually classified them as beneficial or not. SSV was able to classify the mutations with values of 0.89 and 0.92 for precision and specificity, respectively. Then, we used SSV to propose mutations for Bgl1B, a low-performance ß-glucosidase. We detected 15 mutations that could be beneficial. Three of these mutations (H228C, H228T, and H228V) have been related in the literature to the mechanism of glucose tolerance and stimulation in GH1 ß-glucosidase. Hence, SSV was capable of detecting promising mutations, already validated by in vitro experiments, that improved the inhibition resistance of a ß-glucosidase and, consequently, its catalytic activity. SSV might be useful for the engineering of enzymes used in biofuel production or other industrial applications.
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Biología Computacional/métodos , Mutación/genética , beta-Glucosidasa/química , beta-Glucosidasa/genética , Candida/enzimología , Lipasa/genética , Modelos MolecularesRESUMEN
The Candida haemulonii complex (Candida haemulonii, Candida haemulonii var. vulnera, and Candida duobushaemulonii) comprises emerging opportunistic human fungal pathogens with recognized multidrug-resistance profiles. Little is known about the virulence markers produced by this fungal complex. However, it is recognized that Candida spp. express a large array of peptidases, which play multiple roles in different aspects of fungal-host interactions. In the present study, we have identified proteolytic enzymes in clinical isolates of the C. haemulonii complex using zymographic assays. Peptidases able to hydrolyze gelatin, casein, albumin, hemoglobin, and immunoglobulin G were detected in cell-free supernatants and cellular extracts taken from the three species forming the C. haemulonii complex. Overall, peptidases were preferentially evidenced at physiological pH and temperatures of 37-42 °C, with molar masses between 35 and 85 kDa. Peptidase profiles of C. haemulonii and C. haemulonii var. vulnera isolates were quite similar, contrasting to the peptidases produced by C. duobushaemulonii. Almost all peptidases were inhibited by phenylmethanesulfonyl fluoride (PMSF), thus classifying them as serine-type peptidases. Additionally, proteolytic cleavage of soluble azoalbumin was blocked by PMSF (65-95% inhibition depending on the fungal isolate). These unprecedented results have demonstrated the capability of the C. haemulonii complex to produce serine-type peptidases with an ability to cleave a broad spectrum of proteins, including key host components.