RESUMO
The studies on natural compounds to diabetes mellitus treatment have been increasing in recent years. Research suggests that natural components can inhibit alpha-glucosidase activities, an important strategy in the management of blood glucose levels. In this work, for the first time in the literature, the compounds produced by Ganoderma lipsiense extracts were identified and evaluated on the inhibitory effect of these on alpha-glucosidase activity. Four phenolic compounds were identified by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) to crude extract from G. lipsiense grown in red rice medium (RCE) and synthetic medium (SCE), being syringic acid identified in both extracts. Gas chromatography-mass spectrometry (GC-MS) analysis showed fatty acids and their derivatives, terpene, steroid, niacin, and nitrogen compounds to SCE, while RCE was rich in fatty acids and their derivatives. Both extracts demonstrated alpha-glucosidase inhibition (RCE IC50 = 0.269 ± 8.25 mg mL-1; SCE IC50 = 0.218 ± 9.67 mg mL-1), and the purified hexane fraction of RCE (RHEX) demonstrated the highest inhibition of enzyme (81.1%). Studies on kinetic inhibition showed competitive inhibition mode to RCE, while SCE showed uncompetitive inhibition mode. Although the inhibitory effects of RCE and SCE were satisfactory, the present findings identified some unpublished compounds to G. lipsiense in the literature with important therapeutic properties.
Assuntos
Fermentação , Ganoderma/enzimologia , Micélio/enzimologia , alfa-Glucosidases/metabolismo , Glicemia , Cromatografia Líquida de Alta Pressão , Ácidos Graxos/química , Cromatografia Gasosa-Espectrometria de Massas , Inibidores de Glicosídeo Hidrolases/química , Humanos , Hipoglicemiantes/farmacologia , Concentração Inibidora 50 , Cinética , Fenóis/química , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
NADPH oxidases are enzymes that have been reported to generate reactive oxygen species (ROS) in animals, plants and many multicellular fungi in response to environmental stresses. Six genes of the NADPH oxidase complex components, including vvnoxa, vvnoxb, vvnoxr, vvbema, vvrac1 and vvcdc24, were identified based on the complete genomic sequence of the edible fungus Volvariella volvacea. The number of vvnoxa, vvrac1, vvbema and vvcdc24 transcripts fluctuated with ageing, and the gene expression patterns of vvnoxa, vvrac1 and vvbema were significantly positively correlated. However, the expression of vvnoxb and vvnoxr showed no significant difference during ageing. In hyphae subjected to mechanical injury stress, both O2- and H2O2 concentrations were increased. The expression of vvnoxa, vvrac1, vvbema and vvcdc24 was substantially upregulated, but vvnoxb and vvnoxr showed no response to mechanical injury stress at the transcriptional level. Additionally, the transcription of vvnoxa, vvrac1, vvbema and vvcdc24 could be repressed when the intracellular ROS were eliminated by diphenyleneiodonium (DPI) chloride and reduced glutathione (GSH) treatments. These results indicated a positive feedback loop involving NADPH oxidase and intracellular ROS, which might be the reason for the oxidative burst during injury stress.
Assuntos
Regulação Fúngica da Expressão Gênica , Micélio/genética , NADPH Oxidases/genética , Volvariella/enzimologia , Volvariella/genética , Proteínas Fúngicas/genética , Genoma Fúngico , Glutationa/farmacologia , Micélio/enzimologia , Oniocompostos/farmacologia , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória , Estresse FisiológicoRESUMO
Hydrolytic enzyme production (cellulases, laminarinases and xylanases) was studied in cultures of Lentinula edodes on sterilized coffee pulp. Samples of substrate colonized by mycelia were taken after 7, 14, 21, 28 and 35 days of incubation at 25°C (W1 to W5) and during the fruiting period at different stages: formation of primordia (PF), first harvest (H) and one week after the first harvest (PH). The enzymatic activity was lower during the early mycelial growth and showed higher levels during the formation and development of fruiting bodies. During the reproductive stage of the fungus, the samples were subjected to a soaking treatment; however, it was not possible to relate this soaking treatment to the increase in enzyme production. The levels of enzymatic activity suggest that secretion of the studied enzymes does not influence the adaptability of the strains to the substrate.
Assuntos
Proteínas Fúngicas/metabolismo , Hidrolases/metabolismo , Cogumelos Shiitake/enzimologia , Adaptação Fisiológica , Coffea , Meios de Cultura , Hidrólise , Resíduos Industriais , Micélio/enzimologia , Reprodução , Cogumelos Shiitake/crescimento & desenvolvimento , Especificidade da EspécieRESUMO
Abstract Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669 bp) and pksT-2 (7901 bp) suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthase–acyltransferase domains through Agrobacterium -mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88.
Assuntos
Trichoderma/enzimologia , Proteínas Fúngicas/metabolismo , Policetídeo Sintases/metabolismo , Doenças das Plantas/microbiologia , Trichoderma/classificação , Trichoderma/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Dados de Sequência Molecular , Regulação Fúngica da Expressão Gênica , Alinhamento de Sequência , Sequência de Aminoácidos , Micélio/enzimologia , Micélio/genética , Policetídeo Sintases/genética , Policetídeo Sintases/químicaRESUMO
Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669bp) and pksT-2 (7901bp) suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthase-acyltransferase domains through Agrobacterium-mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88.
Assuntos
Proteínas Fúngicas/metabolismo , Policetídeo Sintases/metabolismo , Trichoderma/enzimologia , Sequência de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Micélio/enzimologia , Micélio/genética , Doenças das Plantas/microbiologia , Policetídeo Sintases/química , Policetídeo Sintases/genética , Alinhamento de Sequência , Trichoderma/classificação , Trichoderma/genéticaRESUMO
This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, ß-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. ß-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract.
Assuntos
Proteínas Fúngicas/metabolismo , Trichoderma/enzimologia , Quitinases/análise , Quitinases/metabolismo , Endo-1,4-beta-Xilanases/análise , Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/análise , Glicosídeo Hidrolases/análise , Glicosídeo Hidrolases/metabolismo , Micélio/química , Micélio/enzimologia , Micélio/crescimento & desenvolvimento , Paquistão , Trichoderma/química , Trichoderma/crescimento & desenvolvimentoRESUMO
Abstract This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract.
Assuntos
Quitinases/análise , Quitinases/química , Quitinases/enzimologia , Quitinases/crescimento & desenvolvimento , Quitinases/metabolismo , /análise , /química , /enzimologia , /crescimento & desenvolvimento , /metabolismo , Proteínas Fúngicas/análise , Proteínas Fúngicas/química , Proteínas Fúngicas/enzimologia , Proteínas Fúngicas/crescimento & desenvolvimento , Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/análise , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/enzimologia , Glicosídeo Hidrolases/crescimento & desenvolvimento , Glicosídeo Hidrolases/metabolismo , Micélio/análise , Micélio/química , Micélio/enzimologia , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Paquistão/análise , Paquistão/química , Paquistão/enzimologia , Paquistão/crescimento & desenvolvimento , Paquistão/metabolismo , Trichoderma/análise , Trichoderma/química , Trichoderma/enzimologia , Trichoderma/crescimento & desenvolvimento , Trichoderma/metabolismoRESUMO
The kingdom Fungi is represented by a large number of organisms, including pathogens that deteriorate the main structural components of wood, such as cellulose, hemicellulose and lignin. The aim of our work was to characterize the antifungal activity in Arthrobacter agilis UMCV2 and diverse amines against wood-decaying fungi. Four fungal organisms (designated as UMTM) were isolated from decaying wood samples obtained from a forest in Cuanajo-Michoacán, México. Two of them showed a clear enzymatic activity of cellulases, xylanases and oxido-reducing enzymes and were identified as Hypocrea (UMTM3 isolate) and Fusarium (UMTM13 isolate). In vitro, the amines showed inhibitory effect against UMTM growth and one of the amines, dimethylhexadecylamine (DMA16), exhibited strong potential as wood preventive treatment, against the attack of decaying fungi.
Assuntos
Aminas/farmacologia , Antibiose , Arthrobacter/fisiologia , Fusarium/crescimento & desenvolvimento , Hypocrea/crescimento & desenvolvimento , Madeira/microbiologia , Proteínas Fúngicas/metabolismo , Fungos/classificação , Fungos/isolamento & purificação , Fusarium/efeitos dos fármacos , Fusarium/enzimologia , Fusarium/isolamento & purificação , Hypocrea/efeitos dos fármacos , Hypocrea/enzimologia , Hypocrea/isolamento & purificação , México , Micélio/enzimologia , Técnicas de Tipagem Micológica , Pinus/microbiologiaRESUMO
A total of 95 yeast strains were isolated from the microbiota of different grapes collected at vineyards in southern Brazil. The yeasts were screened for ß-(1 â 3)-glucanases using a newly developed zymogram method that relies upon the appearance of clearance zones around growing colonies cultured on agarbotryosphaeran medium and also by submerged fermentation on nutrient medium containing botryosphaeran, a (1 â 3),(1 â 6)-ß-d-glucan. Among 14 ß-(1 â 3)-glucanase-positive yeasts identified, four strains produced the highest ß-glucanolytic activities and were evaluated for enzyme production on cellobiose, botryosphaeran, and mycelial biomass from Botryosphaeria rhodina (MAMB-05). Yeast strain 1WA1 produced the highest ß-(1 â 3)-glucanase and ß-glucosidase activities and was identified by molecular characterization as Aureobasidium pullulans. The physicochemical properties of the crude ß-glucanolytic enzyme preparation were characterized, and the preparation was used to hydrolyze several ß-d-glucans (laminarin, botryosphaeran, lasiodiplodan, pustulan, and curdlan). The production and physicochemical properties of the ß-glucanolytic preparation enable its potential applications in wine enology and production of prebiotics through hydrolysis of ß-d-glucans.
Assuntos
Ascomicetos/enzimologia , Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/metabolismo , Microbiota , Vitis/microbiologia , Ascomicetos/classificação , Ascomicetos/genética , Ascomicetos/isolamento & purificação , Brasil , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glucanos/química , Glucanos/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Hidrólise , Cinética , Micélio/química , Micélio/enzimologia , Micélio/genética , Especificidade por Substrato , Leveduras/classificação , Leveduras/genética , Leveduras/isolamento & purificação , beta-Glucanas/metabolismoRESUMO
Fungos basidiomicetos têm a capacidade de bioacumular metais pesados, no entanto existem poucos trabalhos sobre bioacumulação de zinco em micélio de Agaricussubrufescens. O objetivo deste trabalho foi avaliar a bioacumulação de zinco em micélio vegetativo de A. subrufescens cultivado em meio sólido e líquido. O fungo foi crescido em meio sólido ou em meio líquido a base de extrato de malte adicionado de ZnSO4 a fim de obter zero; 2,5; 5; 7,5; 10; 15 ou 20 ppm de zinco. Os meios foram inoculados e após 14 dias foi determinada a biomassa e a bioacumulação de zinco. A adição de zinco no meio de cultivo inibiu o crescimento micelial e induziu a bioacumulação na biomassa tanto no cultivo sólido como no líquido. Adições acima de 7,5 ppm de zinco inibiram totalmente o crescimento micelial. O fungo crescido em meio de cultivo líquido sofre maior inibição do crescimento com a adição de zinco e maior bioacumulação que no meio sólido.(AU)
Fungi basidiomycetes have the ability to bioaccumulate heavy metals, but there are few studies on zinc bioaccumulation in the mycelium of Agaricus subrufescens. The objective of this study is to evaluate the zinc bioaccumulation in the mycelium of A. subrufescens cultivated in solid and liquid culture media. Mycelium was grown on solid or liquid medium in malt extract base added with ZnSO4 to obtain zero, 2.5, 5, 7.5, 10, 15 or 20-ppm zinc. Mycelial biomass and zinc bioaccumulation were determined 14 days after inoculation in the culture media. Addition of zinc in culture medium inhibited mycelial growth and induced biomass bioaccumulation both in solid and in liquid culture. Additions higher than 7.5-ppm zinc completely inhibited mycelial growth in culture medium. Mycelial growth in liquid culture presented greater increase of growth inhibition with the addition of zinc and greater bioaccumulation than in solid medium.(AU)
Hongos basidiomicetos tienen la capacidad de bioacumular metales pesados, sin embargo hay pocos estudios sobre la bioacumulación de zinc en el micelio de Agaricus subrufescens. El objetivo de este estudio ha sido evaluar la bioacumulación de zinc en el micelio de A. subrufescens cultivado en medio sólido y líquido. El hongo ha crecido en medio sólido o líquido a base de extracto de malta agregado de ZnSO4 para obtener cero; 2.5, 5, 7.5, 10, 15 o 20 ppm de zinc. Los medios fueron inoculados y después de 14 días se determinó la biomasa y la bioacumulación de zinc. La adición de zinc en el medio del cultivo inhibió el crecimiento micelial y indujo la bioacumulación de la biomasa tanto en el cultivo sólido como en el líquido. Adiciones superiores a 7.5 ppm de zinc inhibieron completamente el crecimiento del micelio. El hongo crecido en medio de cultivo líquido sufre mayor inhibición del crecimiento con la adición de zinc y mayor bioacumulación que en el medio sólido.(AU)
Assuntos
Animais , Bioacumulação/análise , Bioacumulação/classificação , Micélio/química , Micélio/enzimologia , Zinco/análise , Zinco/químicaRESUMO
Fungos basidiomicetos têm a capacidade de bioacumular metais pesados, no entanto existem poucos trabalhos sobre bioacumulação de zinco em micélio de Agaricussubrufescens. O objetivo deste trabalho foi avaliar a bioacumulação de zinco em micélio vegetativo de A. subrufescens cultivado em meio sólido e líquido. O fungo foi crescido em meio sólido ou em meio líquido a base de extrato de malte adicionado de ZnSO4 a fim de obter zero; 2,5; 5; 7,5; 10; 15 ou 20 ppm de zinco. Os meios foram inoculados e após 14 dias foi determinada a biomassa e a bioacumulação de zinco. A adição de zinco no meio de cultivo inibiu o crescimento micelial e induziu a bioacumulação na biomassa tanto no cultivo sólido como no líquido. Adições acima de 7,5 ppm de zinco inibiram totalmente o crescimento micelial. O fungo crescido em meio de cultivo líquido sofre maior inibição do crescimento com a adição de zinco e maior bioacumulação que no meio sólido...
Fungi basidiomycetes have the ability to bioaccumulate heavy metals, but there are few studies on zinc bioaccumulation in the mycelium of Agaricus subrufescens. The objective of this study is to evaluate the zinc bioaccumulation in the mycelium of A. subrufescens cultivated in solid and liquid culture media. Mycelium was grown on solid or liquid medium in malt extract base added with ZnSO4 to obtain zero, 2.5, 5, 7.5, 10, 15 or 20-ppm zinc. Mycelial biomass and zinc bioaccumulation were determined 14 days after inoculation in the culture media. Addition of zinc in culture medium inhibited mycelial growth and induced biomass bioaccumulation both in solid and in liquid culture. Additions higher than 7.5-ppm zinc completely inhibited mycelial growth in culture medium. Mycelial growth in liquid culture presented greater increase of growth inhibition with the addition of zinc and greater bioaccumulation than in solid medium...
Hongos basidiomicetos tienen la capacidad de bioacumular metales pesados, sin embargo hay pocos estudios sobre la bioacumulación de zinc en el micelio de Agaricus subrufescens. El objetivo de este estudio ha sido evaluar la bioacumulación de zinc en el micelio de A. subrufescens cultivado en medio sólido y líquido. El hongo ha crecido en medio sólido o líquido a base de extracto de malta agregado de ZnSO4 para obtener cero; 2.5, 5, 7.5, 10, 15 o 20 ppm de zinc. Los medios fueron inoculados y después de 14 días se determinó la biomasa y la bioacumulación de zinc. La adición de zinc en el medio del cultivo inhibió el crecimiento micelial y indujo la bioacumulación de la biomasa tanto en el cultivo sólido como en el líquido. Adiciones superiores a 7.5 ppm de zinc inhibieron completamente el crecimiento del micelio. El hongo crecido en medio de cultivo líquido sufre mayor inhibición del crecimiento con la adición de zinc y mayor bioacumulación que en el medio sólido...
Assuntos
Animais , Bioacumulação/análise , Bioacumulação/classificação , Micélio/enzimologia , Micélio/química , Zinco/análise , Zinco/químicaRESUMO
The cultivation of Lentinus citrinus for mycelial biomass and protease production under different carbon and nitrogen sources was studied in submerged cultivation. The nutritional source concentration for protease production was evaluated using a full factorial design. For mycelial biomass maltose (4.94 mg/mL) and beef extract (5.45 mg/mL), carbon and nitrogen sources presented the best results, respectively. The maximum protease activity was 73.33 U/mL with fructose (30.0 g/L) and beef extract (10.0 g/L). Proteases showed maximum activity at 40°C and pH 7.0, which exhibited high stability at experimental conditions. The final part of this work was devoted to estimating the main thermodynamic parameters of the irreversible enzyme inactivation (ΔH* = 17.86 kJ/mol, ΔG* =102.09 kJ/mol, ΔS* = -260.76 J/mol×K) through residual activity tests carried out at 25-70°C, by making use of Arrhenius and Eyring plots.
Assuntos
Meios de Cultura/metabolismo , Proteínas Fúngicas/metabolismo , Lentinula/enzimologia , Micélio/crescimento & desenvolvimento , Peptídeo Hidrolases/metabolismo , Biomassa , Meios de Cultura/química , Estabilidade Enzimática , Proteínas Fúngicas/química , Cinética , Lentinula/química , Lentinula/crescimento & desenvolvimento , Lentinula/metabolismo , Micélio/química , Micélio/enzimologia , Micélio/metabolismo , Peptídeo Hidrolases/químicaRESUMO
Superoxide dismutase (SOD) activities of the oomycete Phytophthora cinnamomi were examined. Five polypeptides with manganese superoxide dismutase (MnSOD) activity were found in mycelium growing in liquid culture with relative molecular weights ranging from approximately 25 to 100 kDa. Comparison with characterized avocado SODs showed no evidence for the presence of either iron or copper/zinc SODs in P. cinnamomi. The level of activity of the MnSOD polypeptides decreased in the presence of avocado root or cell wall components. Growth of P. cinnamomi, measured as dry weight, increased when the mycelium was grown in the presence of superoxide anion (O(2) (-)), which was added exogenously. Our results suggest that the metabolism of O(2) (-) has an important role in the development of P. cinnamomi.
Assuntos
Proteínas Fúngicas/química , Micélio/enzimologia , Phytophthora/enzimologia , Superóxido Dismutase/química , Parede Celular/química , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/metabolismo , Peróxido de Hidrogênio/química , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Oxidantes/farmacologia , Persea/microbiologia , Phytophthora/efeitos dos fármacos , Phytophthora/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Cianeto de Potássio/química , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/metabolismo , Superóxidos/farmacologiaRESUMO
The aim of this work was the partial purification and subsequent evaluation of chitinase expression during the various growth phases of Paracoccidioides brasiliensis. Initially, PbCTS1r was expressed as a recombinant protein and displayed enzymatic activity against 4-MU-[N-acetylglucosamine (GlcNAc)]3 and 4-MU-(GlcNAc)2. Two proteins, 45 kDa and 39 kDa in size, were partially purified from P. brasiliensis yeast crude extract using cation-exchange chromatography coupled with HPLC and were characterised as PbCTS1 and PbCTS2, respectively. Anti-PbCTS1r antibody recognised two proteins in the crude extracts of yeast and the transitional stage between mycelial and yeast phases. In crude extracts of mycelium, only the 45 kDa protein was detected. However, quantitative real-time polymerase chain reaction led to the detection of small quantities of Pbcts2 transcript in the mycelial phase. In the yeast cell wall extract, only the 39 kDa protein was detected. Moreover, both proteins were secreted by the yeast parasitic phase, suggesting that these proteins participate in the modulation of the fungal environment. Phylogenetic analysis of the predicted PbCTS1 and PbCTS2 proteins indicated that they code for distinct chitinases in P. brasiliensis. During evolution, P. brasiliensis could have acquired the paralogues Pbcts1 and Pbcts2 for growth and survival in diverse environments in both saprophytic and parasitic phases.
Assuntos
Quitinases/metabolismo , Micélio/enzimologia , Paracoccidioides/enzimologia , Cromatografia Líquida de Alta Pressão , Quitinases/genética , DNA Complementar/genética , DNA Fúngico/genética , Regulação Enzimológica da Expressão Gênica , Micélio/crescimento & desenvolvimento , Filogenia , Paracoccidioides/genética , Paracoccidioides/crescimento & desenvolvimento , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The aim of this work was the partial purification and subsequent evaluation of chitinase expression during the various growth phases of Paracoccidioides brasiliensis. Initially, PbCTS1r was expressed as a recombinant protein and displayed enzymatic activity against 4-MU-[N-acetylglucosamine (GlcNAc)]3 and 4-MU-(GlcNAc)2. Two proteins, 45 kDa and 39 kDa in size, were partially purified from P. brasiliensis yeast crude extract using cation-exchange chromatography coupled with HPLC and were characterised as PbCTS1 and PbCTS2, respectively. Anti-PbCTS1r antibody recognised two proteins in the crude extracts of yeast and the transitional stage between mycelial and yeast phases. In crude extracts of mycelium, only the 45 kDa protein was detected. However, quantitative real-time polymerase chain reaction led to the detection of small quantities of Pbcts2 transcript in the mycelial phase. In the yeast cell wall extract, only the 39 kDa protein was detected. Moreover, both proteins were secreted by the yeast parasitic phase, suggesting that these proteins participate in the modulation of the fungal environment. Phylogenetic analysis of the predicted PbCTS1 and PbCTS2 proteins indicated that they code for distinct chitinases in P. brasiliensis. During evolution, P. brasiliensis could have acquired the paralogues Pbcts1 and Pbcts2 for growth and survival in diverse environments in both saprophytic and parasitic phases.
Assuntos
Quitinases/metabolismo , Micélio/enzimologia , Paracoccidioides/enzimologia , Quitinases/genética , Cromatografia Líquida de Alta Pressão , DNA Complementar/genética , DNA Fúngico/genética , Regulação Enzimológica da Expressão Gênica , Micélio/crescimento & desenvolvimento , Paracoccidioides/genética , Paracoccidioides/crescimento & desenvolvimento , Filogenia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Lipases are widely used in the industry for different purposes. Although these enzymes are mainly produced by submerged fermentation, lipase production by solid-state fermentation (SSF) has been gaining interest due to the advantages of this type of culture. Major advantages are higher production titers and productivity, less catabolite repression, and use of the dried fermented material as biocatalyst. This chapter describes a traditional methodology to produce fungal (Rhizopus homothallicus) lipases by SSF using perlite as inert support. The use of different devices (glass columns or Erlenmeyer flasks) and type of inoculum (spores or growing mycelium) is considered so that lipase production by SSF could be easily performed in any laboratory.
Assuntos
Lipase/biossíntese , Micélio/enzimologia , Rhizopus/enzimologia , Esporos Fúngicos/enzimologia , Óxido de Alumínio/química , Reatores Biológicos , Biotecnologia , Contagem de Colônia Microbiana , Fermentação , Vidro/química , Concentração de Íons de Hidrogênio , Dióxido de Silício/químicaRESUMO
Xylanolytic enzymes produced by Lentinula edodes UFV70, cultivated in eucalyptus sawdust/rice bran medium, were stable at 50, 60 and 65ºC for 21 hours, losing only 15-25% activity. Fungus incubation at 50ºC for 12 hours and at 65ºC for 24 hours increased the amount of xylose produced.
Assuntos
Biomassa , Cogumelos Shiitake/isolamento & purificação , Micélio/enzimologia , Xilanos/isolamento & purificação , Xilose/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Ensaios Enzimáticos Clínicos , Ativação Enzimática , MétodosRESUMO
Acid phosphatase (ACP) enzymes are involved in the mobilization of soil phosphorus (P) and polyphosphate accumulated in the fungal tissues of ectomycorrhizal roots, thereby influencing the amounts of P that are stored in the fungus and transferred to the host plant. This study evaluated the effects of ectomycorrhizal morphotype and soil fertility on ACP activity in the extraradical mycelium (ACP(myc)), the mantle (ACP(mantle)) and the Hartig net region (ACP(Hartig)) of ectomycorrhizal Nothofagus obliqua seedlings. ACP activity was quantified in vivo using enzyme-labelled fluorescence-97 (ELF-97) substrate, confocal laser microscopy and digital image processing routines. There was a significant effect of ectomycorrhizal morphotype on ACP(myc), ACP(mantle) and ACP(Hartig), while soil fertility had a significant effect on ACP(myc) and ACP(Hartig). The relative contribution of the mantle and the Hartig net region to the ACP activity on the ectomycorrhizal root was significantly affected by ectomycorrhizal morphotype and soil fertility. A positive correlation between ACP(Hartig) and the shoot P concentration was found, providing evidence that ACP activity at the fungus:root interface is involved in P transfer from the fungus to the host. It is concluded that the spatial distribution of ACP in ectomycorrhizas varies as a function of soil fertility and colonizing fungus.
Assuntos
Fosfatase Ácida/metabolismo , Ascomicetos/enzimologia , Basidiomycota/enzimologia , Magnoliopsida/enzimologia , Micorrizas/enzimologia , Fósforo/metabolismo , Ascomicetos/fisiologia , Basidiomycota/fisiologia , Transporte Biológico , Modelos Lineares , Magnoliopsida/microbiologia , Magnoliopsida/fisiologia , Micélio/enzimologia , Micélio/fisiologia , Micorrizas/fisiologia , Raízes de Plantas/enzimologia , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Brotos de Planta/metabolismo , Plântula/enzimologia , Plântula/microbiologia , Plântula/fisiologia , Solo/química , SimbioseRESUMO
Transformation reactions on 3ß,17ß-dihydroxyandrost-5-ene using free fungal cells were compared with those carried out by macerated mycelia, immobilized in calcium alginate beads. Six fungi were utilized in this study, namely Rhizopus oryzae ATCC 11145, Mucor plumbeus ATCC 4740, Cunninghamella echinulata var. elegans ATCC 8688a, Aspergillus niger ATCC 9142, Phanerochaete chrysosporium ATCC 24725 and Whetzelinia sclerotiorum ATCC 18687. The results show, for the first time, that encapsulated mycelial fragments essentially carry out the same bioconversions as those observed with growing cells. As the immobilized cells were "resting", the products formed were free of contamination by natural products, and this greatly aided the purification of the metabolites. Conditions for bead preparation were optimized. Furthermore, it was noted that the beads could be reused, once they had been subjected to a rejuvenation process.
Assuntos
Alginatos/química , Androstenos/química , Células Imobilizadas/enzimologia , Fungos/enzimologia , Micélio/enzimologia , Biocatálise , Biotransformação , Células Imobilizadas/química , Fermentação , Fungos/química , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Micélio/química , ReciclagemRESUMO
A protein species preferentially expressed in yeast cells with a molecular mass of 80 kDa and isoeletric point (pI) of 7.79 was isolated from the proteome of Paracoccidioides brasiliensis and characterized as an aconitase (ACO) (E.C. 4.2.1.3). ACO is an enzyme that catalyzes the isomerization of citrate to isocitrate in both the Krebs cycle and the glyoxylate cycle. We report the cloning and characterization of the cDNA encoding the ACO of P. brasiliensis (PbACO). The cDNA showed a 2361 bp open reading frame (ORF) and encoded a predicted protein with 787 amino acids. Polyclonal antibodies against the purified recombinant PbACO was obtained in order to analyze the subcellular localization of the molecule in P. brasiliensis. The protein is present in the extracellular fluid, cell wall enriched fraction, mitochondria, cytosol and peroxisomes of yeast cells as demonstrated by western blot and immunocytochemistry analysis. The expression analysis of the Pbaco gene was performed by quantitative real-time RT-PCR and results demonstrated an increased expression in yeast cells compared to mycelia. Real-time RT-PCR assays was also used to evaluate the Pbaco expression when the fungus grows on media with acetate and ethanol as sole carbon sources and in different iron levels. The results demonstrated that Pbaco transcript is over expressed in acetate and ethanol as sole carbon sources and in high-iron conditions.