RESUMO
Almost one century after the Sir Alexander Fleming's fortuitous discovery of penicillin and the identification of the fungal producer as Penicillium notatum, later Penicillium chrysogenum (currently reidentified as Penicillium rubens), the molecular mechanisms behind the massive production of penicillin titers by industrial strains could be considered almost fully characterized. However, this filamentous fungus is not only circumscribed to penicillin, and instead, it seems to be full of surprises, thereby producing important metabolites and providing expanded biotechnological applications. This review, in addition to summarizing the classical role of P. chrysogenum as penicillin producer, highlights its ability to generate an array of additional bioactive secondary metabolites and enzymes, together with the use of this microorganism in relevant biotechnological processes, such as bioremediation, biocontrol, production of bioactive nanoparticles and compounds with pharmaceutical interest, revalorization of agricultural and food-derived wastes or the enhancement of food industrial processes and the agricultural production.
Assuntos
Penicilinas , Penicillium chrysogenum , Penicillium chrysogenum/metabolismo , Penicillium chrysogenum/genética , Penicilinas/biossíntese , Penicilinas/metabolismo , Biotecnologia , Biodegradação Ambiental , Metabolismo Secundário , Microbiologia IndustrialRESUMO
Plant biomass is a promising substrate for biorefinery, as well as a source of bioactive compounds, platform chemicals, and precursors with multiple industrial applications. These applications depend on the hydrolysis of its recalcitrant structure. However, the effective biological degradation of plant cell walls requires several enzymatic groups acting synergistically, and novel enzymes are needed in order to achieve profitable industrial hydrolysis processes. In the present work, a feruloyl esterase (FAE) activity screening of Penicillium spp. strains revealed a promising candidate (Penicillium rubens Wisconsin 54-1255; previously Penicillium chrysogenum), where two FAE-ORFs were identified and subsequently overexpressed. Enzyme extracts were analyzed, confirming the presence of FAE activity in the respective gene products (PrFaeA and PrFaeB). PrFaeB-enriched enzyme extracts were used to determine the FAE activity optima (pH 5.0 and 50-55 °C) and perform proteome analysis by means of MALDI-TOF/TOF mass spectrometry. The studies were completed with the determination of other lignocellulolytic activities, an untargeted metabolite analysis, and upscaled FAE production in stirred tank reactors. The findings described in this work present P. rubens as a promising lignocellulolytic enzyme producer. KEY POINTS: ⢠Two Penicillium rubens ORFs were first confirmed to have feruloyl esterase activity. ⢠Overexpression of the ORFs produced a novel P. rubens strain with improved activity. ⢠The first in-depth proteomic study of a P. rubens lignocellulolytic extract is shown.
Assuntos
Penicillium chrysogenum , Penicillium , Penicillium chrysogenum/metabolismo , Proteômica/métodos , Penicillium/metabolismo , Extratos Vegetais/metabolismo , Proteínas Fúngicas/metabolismoRESUMO
In this work, we identified glucose and glycerol as tacrolimus repressing carbon sources in the important species Streptomyces tsukubaensis. A genome-wide analysis of the transcriptomic response to glucose and glycerol additions was performed using microarray technology. The transcriptional time series obtained allowed us to compare the transcriptomic profiling of S. tsukubaensis growing under tacrolimus producing and non-producing conditions. The analysis revealed important and different metabolic changes after the additions and a lack of transcriptional activation of the fkb cluster. In addition, we detected important differences in the transcriptional response to glucose between S. tsukubaensis and the model species Streptomyces coelicolor. A number of genes encoding key players of morphological and biochemical differentiation were strongly and permanently downregulated by the carbon sources. Finally, we identified several genes showing transcriptional profiles highly correlated to that of the tacrolimus biosynthetic pathway regulator FkbN that might be potential candidates for the improvement of tacrolimus production.
Assuntos
Carbono/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Transcriptoma , Vias Biossintéticas/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Glucose/metabolismo , Glucose/farmacologia , Glicerol/metabolismo , Glicerol/farmacologia , Análise em Microsséries , Streptomyces/efeitos dos fármacos , Streptomyces/crescimento & desenvolvimento , Streptomyces coelicolor/genética , TacrolimoRESUMO
BACKGROUND: Some types of flavonoid intermediates seemed to be restricted to plants. Naringenin is a typical plant metabolite, that has never been reported to be produced in prokariotes. Naringenin is formed by the action of a chalcone synthase using as starter 4-coumaroyl-CoA, which in dicotyledonous plants derives from phenylalanine by the action of a phenylalanine ammonia lyase. RESULTS: A compound produced by Streptomyces clavuligerus has been identified by LC-MS and NMR as naringenin and coelutes in HPLC with a naringenin standard. Genome mining of S. clavuligerus revealed the presence of a gene for a chalcone synthase (ncs), side by side to a gene encoding a P450 cytochrome (ncyP) and separated from a gene encoding a Pal/Tal ammonia lyase (tal). Deletion of any of these genes results in naringenin non producer mutants. Complementation with the deleted gene restores naringenin production in the transformants. Furthermore, naringenin production increases in cultures supplemented with phenylalanine or tyrosine. CONCLUSION: This is the first time that naringenin is reported to be produced naturally in a prokariote. Interestingly three non-clustered genes are involved in naringenin production, which is unusual for secondary metabolites. A tentative pathway for naringenin biosynthesis has been proposed.
Assuntos
Flavanonas/biossíntese , Plantas/metabolismo , Streptomyces/metabolismo , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Aciltransferases/deficiência , Aciltransferases/genética , Sequência de Aminoácidos , Amônia-Liases/química , Amônia-Liases/deficiência , Amônia-Liases/genética , Amônia-Liases/metabolismo , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/deficiência , Sistema Enzimático do Citocromo P-450/genética , Flavanonas/análise , Flavanonas/química , Genoma Bacteriano , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Mutação , Fenilalanina/metabolismo , Plantas/química , Alinhamento de Sequência , Streptomyces/genética , Tirosina/metabolismoRESUMO
Coeliac disease (CD) is an immune-mediated enteropathy resulting from exposure to gluten in genetically predisposed individuals. Gluten proteins are partially digested by human proteases generating immunogenic peptides that cause inflammation in patients carrying HLA-DQ2 and DQ8 genes. Although intestinal dysbiosis has been associated with patients with CD, bacterial metabolism of gluten has not been studied in depth thus far. The aim of this study was to analyse the metabolic activity of intestinal bacteria associated with gluten intake in healthy individuals, CD patients and first-degree relatives of CD patients. Faecal samples belonging to twenty-two untreated CD patients, twenty treated CD patients, sixteen healthy volunteers on normal diet, eleven healthy volunteers on gluten-free diet (GFD), seventy-one relatives of CD patients on normal diet and sixty-nine relatives on GFD were tested for several proteolytic activities, cultivable bacteria involved in gluten metabolism, SCFA and the amount of gluten in faeces. We detected faecal peptidasic activity against the gluten-derived peptide 33-mer. CD patients showed differences in faecal glutenasic activity (FGA), faecal tryptic activity (FTA), SCFA and faecal gluten content with respect to healthy volunteers. Alterations in specific bacterial groups metabolising gluten such as Clostridium or Lactobacillus were reported in CD patients. Relatives showed similar parameters to CD patients (SCFA) and healthy volunteers (FTA and FGA). Our data support the fact that commensal microbial activity is an important factor in the metabolism of gluten proteins and that this activity is altered in CD patients.
Assuntos
Doença Celíaca/dietoterapia , Glutens/administração & dosagem , Glutens/metabolismo , Ácido Acético/metabolismo , Actinobacteria/isolamento & purificação , Actinobacteria/metabolismo , Adolescente , Adulto , Alelos , Ácido Butírico/metabolismo , Caproatos/metabolismo , Dieta Livre de Glúten , Fezes/química , Firmicutes/isolamento & purificação , Firmicutes/metabolismo , Antígenos HLA-DQ/metabolismo , Voluntários Saudáveis , Humanos , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Pessoa de Meia-Idade , Ácidos Pentanoicos/metabolismo , Propionatos/metabolismo , Proteobactérias/isolamento & purificação , Proteobactérias/metabolismo , Adulto JovemRESUMO
The PR-toxin is a potent mycotoxin produced by Penicillium roqueforti in moulded grains and grass silages and may contaminate blue-veined cheese. The PR-toxin derives from the 15 carbon atoms sesquiterpene aristolochene formed by the aristolochene synthase (encoded by ari1). We have cloned and sequenced a four gene cluster that includes the ari1 gene from P. roqueforti. Gene silencing of each of the four genes (named prx1 to prx4) resulted in a reduction of 65-75% in the production of PR-toxin indicating that the four genes encode enzymes involved in PR-toxin biosynthesis. Interestingly the four silenced mutants overproduce large amounts of mycophenolic acid, an antitumor compound formed by an unrelated pathway suggesting a cross-talk of PR-toxin and mycophenolic acid production. An eleven gene cluster that includes the above mentioned four prx genes and a 14-TMS drug/H(+) antiporter was found in the genome of Penicillium chrysogenum. This eleven gene cluster has been reported to be very poorly expressed in a transcriptomic study of P. chrysogenum genes under conditions of penicillin production (strongly aerated cultures). We found that this apparently silent gene cluster is able to produce PR-toxin in P. chrysogenum under static culture conditions on hydrated rice medium. Noteworthily, the production of PR-toxin was 2.6-fold higher in P. chrysogenum npe10, a strain deleted in the 56.8kb amplifiable region containing the pen gene cluster, than in the parental strain Wisconsin 54-1255 providing another example of cross-talk between secondary metabolite pathways in this fungus. A detailed PR-toxin biosynthesis pathway is proposed based on all available evidence.
Assuntos
Família Multigênica , Penicillium/genética , Vias Biossintéticas , Ácido Micofenólico/metabolismo , Naftóis/metabolismo , Penicillium/metabolismo , Penicillium chrysogenum/genética , Penicillium chrysogenum/metabolismoRESUMO
We described previously that an autoinducer molecule, identified as 1,3-diaminopropane (1,3-DAP), is secreted by Penicillium chrysogenum and Acremonium chrysogenum. Using pH-controlled fermentor cultures we have observed in this work that 1,3-DAP and spermidine clearly stimulate the biosynthesis of benzylpenicillin in P. chrysogenum, both in defined and in complex penicillin production media. Both 1,3-DAP and spermidine, but not putrescine (1,4-diaminobutane), produce a drastic increase in the transcript levels of the penicillin biosynthetic genes pcbAB, pcbC and penDE. These polyamines do not affect the expression of the global pH-stress regulator pacC gene, thus excluding that the effect of 1,3-DAP and spermidine is due to a modification of the pH control mechanism. Expression of the three penicillin biosynthetic genes is drastically reduced in a laeA-knock-down mutant of P. chrysogenum, which produces very low levels of benzylpenicillin. Interestingly, 1,3-DAP and spermidine revert the effect of the laeA knock-down mutation, completely restoring the levels of penicillin production. Furthermore, 1,3-DAP and spermidine enhanced the expression of laeA in the parental strain and restored the levels of laeA transcripts in the laeA knock-down mutant. Taken together these results indicate that the stimulatory effect of the inducer molecules 1,3-DAP and spermidine is exerted, at least in part, through the stimulation of the expression of laeA, a global regulator that acts epigenetically on the expression of secondary metabolite genes by heterochromatin reorganization.
Assuntos
Vias Biossintéticas/efeitos dos fármacos , Diaminas/metabolismo , Expressão Gênica/efeitos dos fármacos , Penicilina G/metabolismo , Penicillium chrysogenum/metabolismo , Espermidina/metabolismo , Transativadores/metabolismo , Vias Biossintéticas/genética , Meios de Cultura/química , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Penicillium chrysogenum/efeitos dos fármacos , Putrescina/metabolismoRESUMO
In this study, the use of spray-drying technology for encapsulating Flavourzyme® (protease-peptidase complex) was evaluated to overcome the limitations (low encapsulation efficiency and no large-scale production) of other encapsulation processes. To the best of our knowledge, spray drying has not been applied previously for the immobilization of this enzyme. Firstly, bovine serum albumin (BSA), as a model protein, was encapsulated by spray drying in chitosan and tripolyphoshate (TPP) cross-linked-chitosan shell matrices. The results showed that the chitosan-TPP microcapsules provided a high encapsulation efficiency and better protein stability compared to the non-crosslinked chitosan microcapsules. The effect of enzyme concentration and drying temperature were tested during the spray drying of Flavourzyme®. In this regard, an activity yield of 88.0% and encapsulation efficiency of 78.6% were obtained with a concentration of 0.1% (v/v) and an inlet temperature of 130 °C. Flavourzyme®-loaded chitosan microcapsules were also characterized in terms of their size and morphology using scanning electron microscopy and laser diffractometry.
RESUMO
Filamentous fungi produce an impressive variety of secondary metabolites; many of them have important biological activities. The biosynthesis of these secondary metabolites is frequently induced by plant-derived external elicitors and appears to also be regulated by internal inducers, which may work in a way similar to that of bacterial autoinducers. The biosynthesis of penicillin in Penicillium chrysogenum is an excellent model for studying the molecular mechanisms of control of gene expression due to a good knowledge of the biochemistry and molecular genetics of ß-lactam antibiotics and to the availability of its genome sequence and proteome. In this work, we first developed a plate bioassay that allows direct testing of inducers of penicillin biosynthesis using single colonies of P. chrysogenum. Using this bioassay, we have found an inducer substance in the conditioned culture broths of P. chrysogenum and Acremonium chrysogenum. No inducing effect was exerted by γ-butyrolactones, jasmonic acid, or the penicillin precursor δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine. The conditioned broth induced penicillin biosynthesis and transcription of the pcbAB, pcbC, and penDE genes when added at inoculation time, but its effect was smaller if added at 12 h and it had no effect when added at 24 h, as shown by Northern analysis and lacZ reporter studies. The inducer molecule was purified and identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR) as 1,3-diaminopropane. Addition of pure 1,3-diaminopropane stimulated the production of penicillin by about 100% compared to results for the control cultures. Genes for the biosynthesis of 1,3-diaminopropane have been identified in the P. chrysogenum genome.
Assuntos
Diaminas/metabolismo , Penicilinas/biossíntese , Penicillium chrysogenum/metabolismo , Acremonium/metabolismo , Bioensaio/métodos , Candida/química , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Meios de Cultivo Condicionados/química , Diaminas/isolamento & purificação , Diaminas/farmacologia , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Micélio/genética , Penicillium chrysogenum/efeitos dos fármacos , Penicillium chrysogenum/genética , Transcrição GênicaRESUMO
[This corrects the article DOI: 10.5334/tohm.600.].
RESUMO
Background: Studies have revealed controversial results regarding the diagnostic accuracy of plasma α-synuclein levels in patients with Parkinson's disease (PD). This study was aimed to analyze the diagnostic accuracy of plasma α-synuclein in PD versus healthy controls and patients with essential tremor (ET). Methods: In this cross-sectional study, we included de novo (n = 19) and advanced PD patients [OFF (n = 33), and On (n = 35) states], patients with ET (n = 19), and controls (n = 35). The total plasma α-synuclein levels were determined using an ELISA sandwich method. We performed adjusted multivariate regression analysis to estimate the association of α-synuclein levels with group conditions [controls, ET, and de novo, OFF and ON-PD]. We studied the diagnostic accuracy of plasma α-synuclein using the area under the curve (AUC). Results: The plasma α-synuclein levels were higher in controls compared to PD and ET (p < 0.0001), discriminating de novo PD from controls (AUC = 0.74, 95% CI 0.60-0.89), with a trend towards in advanced PD (OFF state) from ET (AUC = 0.69, 95% CI 0.53-0.84). Conclusions: This is the first study examining and comparing plasma α-synuclein levels in ET vs. PD and controls. Preliminary findings suggest that plasma α-synuclein levels might help to discriminate de novo and advanced PD from controls and ET.
Assuntos
Tremor Essencial , Doença de Parkinson , Biomarcadores , Estudos Transversais , Tremor Essencial/diagnóstico , Humanos , Doença de Parkinson/diagnóstico , alfa-SinucleínaRESUMO
BACKGROUND AND PURPOSE: Given the overlapping clinical manifestations and pathology, the differentiation between essential tremor (ET) and Parkinson's disease (PD) is difficult. Our aims were to examine the plasma metabolomics profiling and their association with motor and non-motor symptoms (NMS) in patients with PD, and to determine differences between de novo PD compared to moderate-advanced PD vs. controls and patients with ET. METHODS: Plasma samples were collected from 137 subjects including 35 age matched controls, 29 NOVO-PD, 35 PD and 38â¯ET patients. PD severity, motor and NMS including cognitive function were assessed using the UPDRS, NMS and PD cognitive rating scales, respectively. Metabolomics analysis was performed by UPLC-ESI-QToF-MS followed by unsupervised multivariate statistics. The area under the curve of the biomarkers according to distribution of their concentrations and the diagnosis of PD (NOVO-PD, advanced PD) vs ET and healthy controls was used as a measurement of diagnostic ability. RESULTS: Several acyl-carnitines, bilirubin, tyramine and tetrahydro-21-deoxycortisol (THS) presented good predictive accuracy (AUC higher than 0.8) for differentiating de novo PD and advanced PD from controls and ET, suggesting an alteration in the lipid oxidation pathway. In multivariate regression analysis, metabolite levels were not significantly associated with motor and NMS severity in PD. CONCLUSIONS: Diverse acyl-carnitines, bilirubin, tyramine and some adrenal gland derived metabolites are suggested as potential biomarkers able to distinguish between PD from controls and ET.
Assuntos
Bilirrubina/sangue , Carnitina/análogos & derivados , Cortodoxona/sangue , Tremor Essencial/diagnóstico , Doença de Parkinson/diagnóstico , Tiramina/sangue , Idoso , Biomarcadores/sangue , Carnitina/sangue , Estudos de Casos e Controles , Cognição , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The effect of the ripening time on the proteolytic process in cheeses made from ewe's milk during a 139-day ripening period was monitored by the use of capillary electrophoresis of pH 4.6 insoluble fraction. Totals of 18 and 21 peaks were recognized and matched in the electropherograms obtained with a fused-silica capillary and a neutral capillary (hydrophilically coated), respectively. These peaks correspond to intact ovine caseins and their hydrolysis products (alpha(s1)-casein I, alpha(s1)-casein II, alpha(s1)-casein III, alpha(s2)-casein, beta(1)-casein, beta(2)-casein, p-kappa-casein, alpha(s1)-I-casein, gamma(1)-casein, gamma(2)-casein, and gamma(3)-casein). The alpha(s)-caseins (alpha(s1)- and alpha(s2)-casein) displayed similar degradation pattern to one another, but different from those of beta-caseins (beta(1)- and beta(2)-casein). beta-Caseins were very much undergoing lesser degradation during the ripening time than alpha(s)-casein. Finally, partial least-squares regression and principal components regression were used to predict the ripening time in cheeses. The models obtained yielded good results since the root-mean-square error in prediction by cross validation was <8.6 days in all cases.
Assuntos
Caseínas/análise , Queijo/análise , Eletroforese Capilar , Manipulação de Alimentos/métodos , Ovinos , Análise de Variância , Animais , Caseínas/metabolismo , Feminino , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/metabolismo , Análise de Regressão , Fatores de TempoRESUMO
The traceability of olive oil is an unresolved issue that remains a challenge. In this field, DNA-based techniques are very powerful tools for discrimination that are less negatively influenced by environmental conditions than other techniques. More specifically, quantitative real time PCR (qPCR) achieves a high degree of sensitivity, although the DNA that it can directly isolate from these oils presents drawbacks. Our study reports the analysis of eight systems, in order to determine their suitability for olive detection in oil and oil-derived foodstuffs. The eight systems were analyzed on the basis of their sensitivity and specificity in the qPCR assay, their relative sensitivity to olive DNA detection and DNA mixtures, their sensitivity and specificity to olive in vegetable oils and the detection of olive in commercial products. The results show that the PetN-PsbM system, designed in this study, is a suitable and reliable technique in relation to olive oil and olive ingredients in both food authentication and food safety processes.
Assuntos
Análise de Alimentos/métodos , Olea/genética , Azeite de Oliva/análise , Óleos de Plantas/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Óleo de Milho/análise , DNA de Plantas/análise , Eletroforese em Gel de Ágar , Ácidos Graxos Monoinsaturados/análise , Óleo de Brassica napus , Reprodutibilidade dos Testes , Óleo de Gergelim/análise , Óleo de Soja/análise , Óleo de GirassolRESUMO
The effect of the ripening time on the proteolytic process in cheeses manufactured from mixtures of cow's and ewe's milk during a 167-day ripening period was monitored by capillary electrophoresis of the pH 4.6-insoluble fraction. Totals of 21 and 16 peaks were recognized and matched in the electropherograms obtained with a fused-silica capillary and a neutral capillary (hydrophilically coated), respectively. These peaks corresponded to intact bovine and ovine caseins and their hydrolysis products (e.g., alpha(s1)-casein, gamma-caseins). In 167-day-old cheeses, bovine alpha(s0)-casein (alpha(s1)-casein 9P) had been completely degraded and 6% of the residual bovine alpha(s1)-casein remained intact. Breakdown of the beta-casein fraction was lower than that of the alpha(s)-casein fraction. Finally, partial least-squares regression and principal component regression were used to predict the ripening time in cheeses. The root-mean-square errors in prediction by cross-validation were <7.8 days in all cases.
Assuntos
Caseínas/análise , Queijo/análise , Eletroforese Capilar , Tecnologia de Alimentos/métodos , Animais , Caseínas/metabolismo , Bovinos , Fibrinolisina/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Leite/química , Análise de Regressão , Ovinos , Fatores de TempoRESUMO
Gluten, a common component in the human diet, is capable of triggering coeliac disease pathogenesis in genetically predisposed individuals. Although the function of human digestive proteases in gluten proteins is quite well known, the role of intestinal microbiota in the metabolism of proteins is frequently underestimated. The aim of this study was the isolation and characterisation of the human gut bacteria involved in the metabolism of gluten proteins. Twenty-two human faecal samples were cultured with gluten as the principal nitrogen source, and 144 strains belonging to 35 bacterial species that may be involved in gluten metabolism in the human gut were isolated. Interestingly, 94 strains were able to metabolise gluten, 61 strains showed an extracellular proteolytic activity against gluten proteins, and several strains showed a peptidasic activity towards the 33-mer peptide, an immunogenic peptide in patients with coeliac disease. Most of the strains were classified within the phyla Firmicutes and Actinobacteria, mainly from the genera Lactobacillus, Streptococcus, Staphylococcus, Clostridium and Bifidobacterium. In conclusion, the human intestine exhibits a large variety of bacteria capable of utilising gluten proteins and peptides as nutrients. These bacteria could have an important role in gluten metabolism and could offer promising new treatment modalities for coeliac disease.
Assuntos
Bactérias/classificação , Bactérias/metabolismo , Glutens/metabolismo , Intestinos/microbiologia , Microbiota , Actinobacteria/metabolismo , Adulto , Bactérias/enzimologia , Bactérias/isolamento & purificação , Biodiversidade , Doença Celíaca/microbiologia , Meios de Cultura , Fezes/microbiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeo Hidrolases/metabolismo , Adulto JovemRESUMO
A vicilin-like globulin seed storage protein, termed convicilin, was isolated for the first time from Korean pine (Pinus koraiensis). SDS-PAGE analysis revealed that Korean pine convicilin was post-translationally processed. The N-terminal peptide sequences of its components were determined. These peptides could be mapped to a protein translated from an embryo abundant transcript isolated in this study. Similar to vicilin, native convicilin appeared to be homotrimeric. Differential scanning calorimetry (DSC) analyses revealed that this protein is less resistant to thermal treatment than Korean pine vicilin. Its transition temperature was 75.57 °C compared with 84.13 °C for vicilin. The urea induced folding-unfolding equilibrium of pine convicilin monitored by intrinsic fluorescence could be interpreted in terms of a two-state model, with a Cm of 4.41 ± 0.15 M.
Assuntos
Pinus/metabolismo , Proteínas de Armazenamento de Sementes/isolamento & purificação , Proteínas de Armazenamento de Sementes/metabolismo , Eletroforese em Gel de Poliacrilamida , Dobramento de Proteína , Proteínas de Armazenamento de Sementes/química , TemperaturaRESUMO
A single gene cluster of Penicillium chrysogenum contains genes involved in the biosynthesis and secretion of the mycotoxins roquefortine C and meleagrin. Five of these genes have been silenced by RNAi. Pc21g15480 (rds) encodes a nonribosomal cyclodipeptide synthetase for the biosynthesis of both roquefortine C and meleagrin. Pc21g15430 (rpt) encodes a prenyltransferase also required for the biosynthesis of both mycotoxins. Silencing of Pc21g15460 or Pc21g15470 led to a decrease in roquefortine C and meleagrin, whereas silencing of the methyltransferase gene (Pc21g15440; gmt) resulted in accumulation of glandicolin B, indicating that this enzyme catalyzes the conversion of glandicolin B to meleagrin. All these genes are transcriptionally coregulated. Our results prove that roquefortine C and meleagrin derive from a single pathway.
Assuntos
Indóis/metabolismo , Família Multigênica , Ovomucina/biossíntese , Penicillium chrysogenum/genética , Sítios de Ligação , Biocatálise , Dimetilaliltranstransferase/antagonistas & inibidores , Dimetilaliltranstransferase/genética , Dimetilaliltranstransferase/metabolismo , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Indóis/química , Metiltransferases/antagonistas & inibidores , Metiltransferases/genética , Metiltransferases/metabolismo , Micotoxinas/biossíntese , Oxirredutases/genética , Oxirredutases/metabolismo , Penicillium chrysogenum/metabolismo , Piperazinas/química , Piperazinas/metabolismo , Prenilação de Proteína , Estrutura Terciária de Proteína , Interferência de RNARESUMO
Information relating to the resistance of food allergens to thermal and/or chemical denaturation is critical if a reduction in protein allergenicity is to be achieved through food-processing means. This study examined the changes in the secondary structure of an almond allergen, amandin, and its acidic and basic polypeptides as a result of thermal and chemical denaturation. Amandin ( approximately 370 kDa) was purified by cryoprecipitation followed by gel filtration chromatography and subjected to thermal (13-96 degrees C) and chemical (urea and dithiothreitol) treatments. Changes in the secondary structure of the protein were followed using circular dichroism spectroscopy. The secondary structure of the hexameric amandin did not undergo remarkable changes at temperatures up to 90 degrees C, although protein aggregation was observed. In the presence of a reducing agent, irreversible denaturation occurred with the following experimental values: T(m) = 72.53 degrees C (transition temperature), DeltaH = 87.40 kcal/mol (unfolding enthalpy), and C(p) = 2.48 kcal/(mol degrees C) (heat capacity). The concentration of urea needed to achieve 50% denaturation was 2.59 M, and the Gibbs free energy of chemical denaturation was calculated to be DeltaG = 3.82 kcal/mol. The basic and acidic polypeptides of amandin had lower thermal stabilities than the multimeric protein.
Assuntos
Antígenos de Plantas/química , Peptídeos/química , Prunus/química , Prunus/imunologia , TermodinâmicaRESUMO
Seed storage proteins are accumulated during seed development and act as a reserve of nutrition for seed germination and young sprout growth. Plant seeds play an important role in human nutrition by providing a relatively inexpensive source of protein. However, many plant foods contain allergenic proteins, and the number of people suffering from food allergies has increased rapidly in recent years. The 11S globulins are the most widespread seed storage proteins, present in monocotyledonous and dicotyledonous seeds as well as in gymnosperms (conifers) and other spermatophytes. This family of proteins accounts for a number of known major food allergens. They are of interest to both the public and industry due to food safety concerns. Because of the interests in the structural basis of the allergenicity of food allergens, we sought to determine the crystal structure of Pru1, the major component of the 11 S storage protein from almonds. The structure was refined to 2.4 A, and the R/Rfree for the final refined structure is 17.2/22.9. Pru1 is a hexamer made of two trimers. Most of the back-to-back trimer-trimer association was contributed by monomer-monomer interactions. An alpha helix (helix 6) at the C-terminal end of the acidic domain of one of the interacting monomers lies at the cleft of the two protomers. The residues in this helix correspond to a flexible region in the peanut allergen Ara h 3 that encompasses a previously defined linear IgE epitope.