RESUMO
The objective of this study was to evaluate the activity of antioxidant enzymes, the functioning of the photosystem II and quality of C. xanthocarpa seedlings cultivated under intermittent water deficit and shading levels and the influence of shading on recovery potential after suspension of the stress conditions. The seedlings were subjected to three levels of shading (0, 30, and 70%), six periods of evaluation (start: 0 days; 1st and 2nd photosynthesis zero: 1st and 2nd P0; 1st and 2nd recovery: 1stand 2nd REC; and END), and two forms of irrigation (control: periodically irrigated to maintain 70% substrate water retention capacity, and intermittent irrigation: suspension of irrigation). The plants subjected to intermittent irrigation conditions at 0% shading showed a reduction in water potential (Ψw) and potential quantum efficiency of photosystem II (Fv/Fm) and maximum efficiency of the photochemical process (Fv/F0) and an increase in basal quantum production of the non-photochemical processes (F0/Fm). Superoxide dismutase (SOD) activity was higher in the leaves than in the roots. The C. xanthocarpa is a species sensitive to water deficit but presents strategies to adapt to an environment under temporary water restriction, which are more temporary are most efficient under shading. The seedlings with water deficit at all levels of shading exhibited higher protective antioxidant activity and lower quality at 0% shading. The shading minimizes prevents permanent damage to the photosystem II and after the re-irrigation, the evaluated characteristics showed recovery with respect to the control group, except POD and SOD activities in the leaves.
O objetivo deste estudo foi avaliar a atividade de enzimas antioxidantes, o funcionamento do fotossistema II e a qualidade de mudas de C. xanthocarpa cultivadas sob déficit hídrico intermitente e níveis de sombreamento e a influência do sombreamento sobre o potencial de recuperação após suspensão das condições de estresse. As mudas foram submetidas a três níveis de sombreamento (0, 30 e 70%), seis períodos de avaliação (início: 0 dias; 1ª e 2ª fotossíntese zero: 1ª e 2ª P0; 1ª e 2ª recuperação: 1ª e 2ª REC; e final), e duas formas de irrigação (controle: periodicamente irrigado para manter 70% da capacidade de retenção de água do substrato, e irrigação intermitente: suspensão da irrigação). As plantas submetidas às condições de irrigação intermitente a 0% de sombreamento apresentaram redução do potencial hídrico (Ψw) e eficiência quântica potencial do fotossistema II (Fv/Fm) e máxima eficiência do processo fotoquímico (Fv/F0) e aumento da produção quantica basal dos processos não fotoquímicos (F0/Fm). A atividade da superóxido dismutase (SOD) foi maior nas folhas do que nas raízes. C. xanthocarpa é uma espécie sensível ao déficit hídrico, mas apresenta estratégias para se adaptar a um ambiente com restrição hídrica temporária, sendo mais eficientes sob sombreamento. As mudas com déficit hídrico em todos os níveis de sombreamento exibiram maior atividade antioxidante protetora e menor qualidade no sombreamento 0%. O sombreamento minimiza danos permanentes ao fotossistema II e após a re-irrigação, as características avaliadas apresentaram recuperação em relação ao grupo controle, exceto atividades de POD e SOD nas folhas.
Assuntos
Enzimas/biossíntese , Estresse Fisiológico , Fotossíntese , Myrtaceae/crescimento & desenvolvimento , Myrtaceae/metabolismoRESUMO
The objective of this study was to evaluate the activity of antioxidant enzymes, the functioning of the photosystem II and quality of C. xanthocarpa seedlings cultivated under intermittent water deficit and shading levels and the influence of shading on recovery potential after suspension of the stress conditions. The seedlings were subjected to three levels of shading (0, 30, and 70%), six periods of evaluation (start: 0 days; 1st and 2nd photosynthesis zero: 1st and 2nd P0; 1st and 2nd recovery: 1stand 2nd REC; and END), and two forms of irrigation (control: periodically irrigated to maintain 70% substrate water retention capacity, and intermittent irrigation: suspension of irrigation). The plants subjected to intermittent irrigation conditions at 0% shading showed a reduction in water potential (Ψw) and potential quantum efficiency of photosystem II (Fv/Fm) and maximum efficiency of the photochemical process (Fv/F0) and an increase in basal quantum production of the non-photochemical processes (F0/Fm). Superoxide dismutase (SOD) activity was higher in the leaves than in the roots. The C. xanthocarpa is a species sensitive to water deficit but presents strategies to adapt to an environment under temporary water restriction, which are more temporary are most efficient under shading. The seedlings with water deficit at all levels of shading exhibited higher protective antioxidant activity and lower quality at 0% shading. The shading minimizes prevents permanent damage to the photosystem II and after the re-irrigation, the evaluated characteristics showed recovery with respect to the control group, except POD and SOD activities in the leaves.(AU)
O objetivo deste estudo foi avaliar a atividade de enzimas antioxidantes, o funcionamento do fotossistema II e a qualidade de mudas de C. xanthocarpa cultivadas sob déficit hídrico intermitente e níveis de sombreamento e a influência do sombreamento sobre o potencial de recuperação após suspensão das condições de estresse. As mudas foram submetidas a três níveis de sombreamento (0, 30 e 70%), seis períodos de avaliação (início: 0 dias; 1ª e 2ª fotossíntese zero: 1ª e 2ª P0; 1ª e 2ª recuperação: 1ª e 2ª REC; e final), e duas formas de irrigação (controle: periodicamente irrigado para manter 70% da capacidade de retenção de água do substrato, e irrigação intermitente: suspensão da irrigação). As plantas submetidas às condições de irrigação intermitente a 0% de sombreamento apresentaram redução do potencial hídrico (Ψw) e eficiência quântica potencial do fotossistema II (Fv/Fm) e máxima eficiência do processo fotoquímico (Fv/F0) e aumento da produção quantica basal dos processos não fotoquímicos (F0/Fm). A atividade da superóxido dismutase (SOD) foi maior nas folhas do que nas raízes. C. xanthocarpa é uma espécie sensível ao déficit hídrico, mas apresenta estratégias para se adaptar a um ambiente com restrição hídrica temporária, sendo mais eficientes sob sombreamento. As mudas com déficit hídrico em todos os níveis de sombreamento exibiram maior atividade antioxidante protetora e menor qualidade no sombreamento 0%. O sombreamento minimiza danos permanentes ao fotossistema II e após a re-irrigação, as características avaliadas apresentaram recuperação em relação ao grupo controle, exceto atividades de POD e SOD nas folhas.(AU)
Assuntos
Myrtaceae/crescimento & desenvolvimento , Myrtaceae/metabolismo , Enzimas/biossíntese , Fotossíntese , Estresse FisiológicoRESUMO
Yeast's beta-galactosidase is an intracellular enzyme, through which it is possible to determine in vivo its activity as a biocatalyst in the lactose hydrolysis. Permeabilization process was used for transforming the microorganisms cells into biocatalysts with an enhanced enzyme activity. The potential application of this enzyme technology in industrial process depends mainly on the enzyme activity. Beta-galactosidase enzyme that hydrolyzes lactose, for instance, is largely dependent on the reaction time and its stability under different physical conditions, such as pH, temperature and enzyme concentration. The objective of this study was to optimize the cellular permeabilization process of Kluyveromyces marxianusCCT 3172 and Saccharomyces fragilisCCT 7586 cultured in cheese whey for lactose hydrolysis. Box-Behnken design was carried out for cell permeabilization with three independent variables, ethanol concentration, permeabilization time and temperature. The best permeability conditions for K. marxianusCCT 3172 were 27% (v v-1) ethanol, 3 min at 20ºC, with specific enzymatic activity of 0.98 U mg-1.For S. fragilisCCT 7586, a specific enzymatic activity of 1.31 U mg-1was achieved using 45% (v v-1) of ethanol, 17 min. of reaction under 17ºC. Thus, it was concluded that cellular permeabilization with ethanolis an efficient process to determine beta-galactosidase activity.(AU)
Assuntos
Permeabilidade , Kluyveromyces , beta-Galactosidase , Soro do Leite , Lactose , Leveduras , Queijo , Enzimas/biossínteseRESUMO
The aim of this study was to produce high yield of a local bacterial alkaline protease in the yeast system because the scientific involvement of microorganisms in enzyme production is still not given enough attention in Saudi Arabia. Soil samples were collected from the rhizosphere of some desert plants in Saudi Arabia. Ninety-three alkaline protease producing bacterial isolates were recovered on skimmed-milk agar at pH 9.4 and 45°C for 48 hr. Isolate D9 obtained from the rhizosphere of Heliotropium digynum at Dhahran City was the most potent isolate in respect to enzyme productivity (184.6 U/ml). The full gene of alkaline protease was amplified and showed the expected size (1300 bp). Restriction enzymes analysis also verified the integrity of the PCR product. The sequence of the protease gene revealed an open reading frame of 1329 nt correspond to the full length of the protease gene of isolate D9 encoding a 443 aa protein. After ligation of the amplified gene by the TA cloning method, digestion with appropriate restriction enzymes confirmed the integrity of the cloned gene. The insert was prepared by two PCRs that were conducted with a pair of primers specifically designed for this purpose. The digested and purified cloning vector pRS426/GAL1p-207-Glu-MS was ligated with the insert then transformed into various strains of Saccharomyces cerevisiae via the electroporation method. Maximum protease expression was done by recombinant OS303 in galactose containing media (145.5 U/ml) with an approximately 2-fold increase when compared with the wild OS303 strain., this may be due to ability to activate gal operon.
O objetivo deste estudo foi produzir alto rendimento de uma protease alcalina bacteriana local no sistema de leveduras, já que o envolvimento científico de microrganismos na produção de enzimas ainda não recebe atenção suficiente na Arábia Saudita. Amostras de solo foram coletadas da rizosfera de algumas plantas do deserto na Arábia Saudita. Noventa e três isolados bacterianos produtores de protease alcalina foram recuperados em ágar de leite desnatado a pH 9,4 e 45°C por 48 horas. O isolado D9 obtido da rizosfera de Heliotropium digynum na cidade de Dhahran foi o mais potente em relação à produtividade da enzima (184,6 U/ml). O gene completo da protease alcalina foi amplificado e apresentou o tamanho esperado (1300 pb). A análise de enzimas de restrição também verificou a integridade do produto de PCR. A sequência do gene da protease revelou uma fase de leitura aberta de 1329 nt, correspondendo ao comprimento total do gene da protease do isolado D9 que codifica uma proteína 443 aa. Após a ligação do gene amplificado pelo método de clonagem TA, a digestão com enzimas de restrição apropriadas confirmou a integridade do gene clonado. A inserção foi preparada por dois PCRs que foram conduzidos com um par de primers projetados especificamente para esta finalidade. O vetor de clonagem digerido e purificado pRS426/GAL1p-207-Glu-MS foi ligado com a inserção e então transformado em várias cepas de Saccharomyces cerevisiae por meio do método de eletroporação. A expressão máxima da protease foi feita por OS303 recombinante em meio contendo galactose (145,5 U/ml) com um aumento de aproximadamente duas vezes quando comparado com a cepa OS303 selvagem, e isso pode ser por causa da capacidade de ativar o operon gal.
Assuntos
Animais , Peptídeo Hidrolases , Saccharomyces cerevisiae , Leveduras , Ativadores de Enzimas , Enzimas/biossíntese , RizosferaRESUMO
Since ERT for several LSDs treatment has emerged at the beginning of the 1980s with Orphan Drug approval, patients' expectancy and life quality have been improved. Most LSDs treatment are based on the replaced of mutated or deficient protein with the natural or recombinant protein.One of the main ERT drawback is the high drug prices. Therefore, different strategies trying to optimize the global ERT biotherapeutic production have been proposed. LVs, a gene delivery tool, can be proposed as an alternative method to generate stable cell lines in manufacturing of recombinant proteins. Since LVs have been used in human gene therapy, clinical trials, safety testing assays and procedures have been developed. Moreover, one of the main advantages of LVs strategy to obtain manufacturing cell line is the short period required as well as the high protein levels achieved.In this chapter, we will focus on LVs as a recombinant protein production platform and we will present a case study that employs LVs to express in a manufacturing cell line, alpha-Galactosidase A (rhαGAL), which is used as ERT for Fabry disease treatment.
Assuntos
Enzimas/biossíntese , Técnicas de Transferência de Genes , Lentivirus , Enzimas/farmacologia , Doença de Fabry/terapia , Vetores Genéticos , Humanos , alfa-Galactosidase/biossíntese , alfa-Galactosidase/farmacologiaRESUMO
Estudos utilizando fungos endofíticos como produtores de metabólitos secundários de interesse biotecnológico vem sendo explorados. Assim, o presente trabalho objetivou avaliar a produção de enzimas por fungos filamentosos endofíticos, sendo escolhida a cepa 50 (C50), proveniente da coleção de cultura do Laboratório de Produtos Naturais e Biotecnologia (LPNBio) da UESB. A produção das enzimas amilase, celulase, invertase, lipase e poligalacturonase foi avaliada pelo o índice enzimático, atividade enzimática e verificado o tempo de fermentação de maior produtividade. Com exceção da invertase, a C50 apresentou atividade para as demais enzimas, destaque para lipase e poligalacturonase no tempo de 96 horas de fermentação. Estes resultados mostraram que a C50 tem potencial para ser explorada como produtora de enzimas.(AU)
Assuntos
Fungos , Ativação Enzimática , Fermentação , Enzimas/biossíntese , Enzimas/química , EndófitosRESUMO
Estudos utilizando fungos endofíticos como produtores de metabólitos secundários de interesse biotecnológico vem sendo explorados. Assim, o presente trabalho objetivou avaliar a produção de enzimas por fungos filamentosos endofíticos, sendo escolhida a cepa 50 (C50), proveniente da coleção de cultura do Laboratório de Produtos Naturais e Biotecnologia (LPNBio) da UESB. A produção das enzimas amilase, celulase, invertase, lipase e poligalacturonase foi avaliada pelo o índice enzimático, atividade enzimática e verificado o tempo de fermentação de maior produtividade. Com exceção da invertase, a C50 apresentou atividade para as demais enzimas, destaque para lipase e poligalacturonase no tempo de 96 horas de fermentação. Estes resultados mostraram que a C50 tem potencial para ser explorada como produtora de enzimas.
Assuntos
Ativação Enzimática , Enzimas/biossíntese , Enzimas/química , Fermentação , Fungos , EndófitosRESUMO
The establishment of an efficient and feasible biorefinery model depends on, among other factors, particularly the selection of the most appropriate microorganism. Mucor circinelloides is a dimorphic fungus species able to produce a wide variety of hydrolytic enzymes, lipids prone to biodiesel production, carotenoids, ethanol, and biomass with significant nutritional value. M. circinelloides also has been selected as a model species for genetic modification by being the first filamentous oleaginous species to have its genome fully characterized, as well as being a species characterized as a potential bioremediation agent. Considering the potential of replacing several nonrenewable feedstocks is widely dependent on fossil fuels, the exploitation of microbial processes and products is a desirable solution for promoting a green and sustainable future. Here, we introduce and thoroughly describe the recent and critical applications of this remarkable fungus within the context of developing a fungal-based biorefinery.
Assuntos
Carotenoides/biossíntese , Enzimas/biossíntese , Lipídeos/biossíntese , Mucor/química , Biocombustíveis , Biomassa , Carotenoides/química , Enzimas/química , Humanos , Metabolismo dos Lipídeos , Lipídeos/químicaRESUMO
Filamentous fungi are robust cell factories and have been used for the production of large quantities of industrially relevant enzymes. However, the production levels of heterologous proteins still need to be improved. Therefore, this article aimed to investigate the global proteome profiling of Aspergillus nidulans recombinant strains in order to understand the bottlenecks of heterologous enzymes production. About 250, 441 and 424 intracellular proteins were identified in the control strain Anid_pEXPYR and in the recombinant strains Anid_AbfA and Anid_Cbhl respectively. In this context, the most enriched processes in recombinant strains were energy pathway, amino acid metabolism, ribosome biogenesis, translation, endoplasmic reticulum and oxidative stress, and repression under secretion stress (RESS). The global protein profile of the recombinant strains Anid_AbfA and Anid_Cbhl was similar, although the latter strain secreted more recombinant enzyme than the former. These findings provide insights into the bottlenecks involved in the secretion of recombinant proteins in A. nidulans, as well as in regard to the rational manipulation of target genes for engineering fungal strains as microbial cell factories.
Assuntos
Aspergillus nidulans/química , Enzimas/biossíntese , Proteoma/análise , Proteínas Recombinantes/biossíntese , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Enzimas/genética , Proteínas Recombinantes/genéticaRESUMO
Background: Poly(DL-lactic acid), or PDLLA, is a biodegradable polymer that can be hydrolyzed by various types of enzymes. The protease produced by Actinomadura keratinilytica strain T16-1 was previously reported to have PDLLA depolymerase activity. However, few studies have reported on PDLLA-degrading enzyme production by bacteria. Therefore, the aims of this study were to determine a suitable immobilization material for PDLLA-degrading enzyme production and optimize PDLLA-degrading enzyme production by using immobilized A. keratinilytica strain T16-1 under various fermentation process conditions in a stirrer fermenter. Results: Among the tested immobilization materials, a scrub pad was the best immobilizer, giving an enzyme activity of 30.03 U/mL in a shake-flask scale. The maximum enzyme activity was obtained at aeration 0.25 vvm, agitation 170 rpm, 45°C, and 48 h of cultivation time. Under these conditions, a PDLLA-degrading enzyme production of 766.33 U/mL with 15.97 U/mL·h productivity was observed using batch fermentation in a 5-L stirrer fermenter. Increased enzyme activity and productivity were observed in repeated-batch (942.67 U/mL and 19.64 U/mL·h) and continuous fermentation (796.43 U/mL and 16.58 U/mL·h) at a dilution rate of 0.013/h. Scaled-up production of the enzyme in a 10-L stirrer bioreactor using the optimized conditions showed a maximum enzyme activity of 578.67 U/mL and a productivity of 12.06 U/mL·h. Conclusions: This research successfully scaled-up the enzyme production to 5 and 10 L in a stirrer fermenter and is helpful for many applications of poly(lactic acid).
Assuntos
Poliésteres/metabolismo , Actinomycetales/enzimologia , Enzimas/biossíntese , Biodegradação Ambiental , Reatores Biológicos , Enzimas/metabolismo , Enzimas Imobilizadas , FermentaçãoRESUMO
Abstract This study aimed to evaluate the biocontrol potential of bacteria isolated from different plant species and soils. The production of compounds related to phytopathogen biocontrol and/or promotion of plant growth in bacterial isolates was evaluated by measuring the production of antimicrobial compounds (ammonia and antibiosis) and hydrolytic enzymes (amylases, lipases, proteases, and chitinases) and phosphate solubilization. Of the 1219 bacterial isolates, 92% produced one or more of the eight compounds evaluated, but only 1% of the isolates produced all the compounds. Proteolytic activity was most frequently observed among the bacterial isolates. Among the compounds which often determine the success of biocontrol, 43% produced compounds which inhibit mycelial growth of Monilinia fructicola, but only 11% hydrolyzed chitin. Bacteria from different plant species (rhizosphere or phylloplane) exhibited differences in the ability to produce the compounds evaluated. Most bacterial isolates with biocontrol potential were isolated from rhizospheric soil. The most efficient bacteria (producing at least five compounds related to phytopathogen biocontrol and/or plant growth), 86 in total, were evaluated for their biocontrol potential by observing their ability to kill juvenile Mesocriconema xenoplax. Thus, we clearly observed that bacteria that produced more compounds related to phytopathogen biocontrol and/or plant growth had a higher efficacy for nematode biocontrol, which validated the selection strategy used.
Assuntos
Doenças das Plantas/microbiologia , Microbiologia do Solo , Bactérias/classificação , Fenômenos Fisiológicos Bacterianos , Bactérias/isolamento & purificação , Bactérias/genética , RNA Ribossômico 16S , Enzimas/biossíntese , Rizosfera , Amônia/metabolismo , Hidrólise , AntibioseRESUMO
This study aimed to evaluate the biocontrol potential of bacteria isolated from different plant species and soils. The production of compounds related to phytopathogen biocontrol and/or promotion of plant growth in bacterial isolates was evaluated by measuring the production of antimicrobial compounds (ammonia and antibiosis) and hydrolytic enzymes (amylases, lipases, proteases, and chitinases) and phosphate solubilization. Of the 1219 bacterial isolates, 92% produced one or more of the eight compounds evaluated, but only 1% of the isolates produced all the compounds. Proteolytic activity was most frequently observed among the bacterial isolates. Among the compounds which often determine the success of biocontrol, 43% produced compounds which inhibit mycelial growth of Monilinia fructicola, but only 11% hydrolyzed chitin. Bacteria from different plant species (rhizosphere or phylloplane) exhibited differences in the ability to produce the compounds evaluated. Most bacterial isolates with biocontrol potential were isolated from rhizospheric soil. The most efficient bacteria (producing at least five compounds related to phytopathogen biocontrol and/or plant growth), 86 in total, were evaluated for their biocontrol potential by observing their ability to kill juvenile Mesocriconema xenoplax. Thus, we clearly observed that bacteria that produced more compounds related to phytopathogen biocontrol and/or plant growth had a higher efficacy for nematode biocontrol, which validated the selection strategy used.
Assuntos
Bactérias/classificação , Fenômenos Fisiológicos Bacterianos , Doenças das Plantas/microbiologia , Microbiologia do Solo , Amônia/metabolismo , Antibiose , Bactérias/genética , Bactérias/isolamento & purificação , Enzimas/biossíntese , Hidrólise , RNA Ribossômico 16S , RizosferaRESUMO
Analysis of fungal secretomes is a prospection tool for the discovery of new catalysts with biotechnological applications. Since enzyme secretion is strongly modulated by environmental factors, evaluation of growth conditions is of utmost importance to achieve optimal enzyme production. In this work, a nonsequenced wood-rotting fungus, Lentinus crinitus, was used for secretome analysis by enzymatic assays and a proteomics approach. Enzyme production was assessed after the fungus was cultured in seven different carbon sources and three nitrogen-containing compounds. The biomass yields and secreted protein arrays differed drastically among growing conditions. A mixture of secreted extracts derived from solid and liquid cultures was inspected by shotgun mass spectrometry and two-dimensional gel electrophoresis (2-DE) prior to analysis via LC-MS/MS. Proteins were identified using mass spectrometry (MS)-driven BLAST. The spectrum of secreted proteins comprised CAZymes, oxidase/reductases, proteases, and lipase/esterases. Although preseparation by 2-DE improved the number of identifications (162) compared with the shotgun approach (98 identifications), the two strategies revealed similar protein patterns. Culture media with reduced water content stimulated the expression of oxidases/reductases, while hydrolases were induced during submerged fermentation. The diversity of proteins observed within both the CAZyme and oxidoreductase groups revealed in this fungus a powerful arsenal of enzymes dedicated to the breakdown and consumption of lignocellulose.
Assuntos
Proteínas Fúngicas/isolamento & purificação , Lentinula/química , Proteômica/métodos , Biomassa , Biotecnologia , Enzimas/análise , Enzimas/biossíntese , Enzimas/metabolismo , Proteínas Fúngicas/metabolismo , Lignina/metabolismoRESUMO
The development of new cost-effective bioprocesses for the production of cellulolytic enzymes is needed in order to ensure that the conversion of biomass becomes economically viable. The aim of this study was to determine whether a novel sequential solid-state and submerged fermentation method (SF) could be validated for different strains of the Trichoderma genus. Cultivation of the Trichoderma reesei Rut-C30 reference strain under SF using sugarcane bagasse as substrate was shown to be favorable for endoglucanase (EGase) production, resulting in up to 4.2-fold improvement compared with conventional submerged fermentation. Characterization of the enzymes in terms of the optimum pH and temperature for EGase activity and comparison of the hydrolysis profiles obtained using a synthetic substrate did not reveal any qualitative differences among the different cultivation conditions investigated. However, the thermostability of the EGase was influenced by the type of carbon source and cultivation system. All three strains of Trichoderma tested (T. reesei Rut-C30, Trichoderma harzianum, and Trichoderma sp INPA 666) achieved higher enzymatic productivity when cultivated under SF, hence validating the proposed SF method for use with different Trichoderma strains. The results suggest that this bioprocess configuration is a very promising development for the cellulosic biofuels industry.
Assuntos
Biotecnologia/métodos , Enzimas/biossíntese , Trichoderma/enzimologia , Celulase/metabolismo , Celulose/metabolismo , Ativação Enzimática , Estabilidade Enzimática , Fermentação , Meia-Vida , Concentração de Íons de Hidrogênio , Hidrólise , Reprodutibilidade dos Testes , Saccharum/química , TemperaturaAssuntos
Enzimas/química , Glicoproteínas/química , Animais , Enzimas/biossíntese , Enzimas/síntese química , Enzimas/metabolismo , Enzimas/farmacologia , Glicoproteínas/biossíntese , Glicoproteínas/síntese química , Glicoproteínas/metabolismo , Glicoproteínas/farmacologia , Glicosilação , Humanos , Modelos MolecularesRESUMO
To explore the molecular mechanisms that prevail during the establishment of the arbuscular mycorrhiza symbiosis involving the genus Glomus, we transcriptionally analysed spores of Glomus intraradices BE3 during early hyphal growth. Among 458 transcripts initially identified as being expressed at presymbiotic stages, 20% of sequences had homology to previously characterized eukaryotic genes, 30% were homologous to fungal coding sequences, and 9% showed homology to previously characterized bacterial genes. Among them, GintPbr1a encodes a homolog to Phenazine Biosynthesis Regulator (Pbr) of Burkholderia cenocepacia, an pleiotropic regulatory protein that activates phenazine production through transcriptional activation of the protein D isochorismatase biosynthetic enzyme phzD (Ramos et al., 2010). Whereas GintPbr1a is expressed during the presymbiotic phase, the G. intraradices BE3 homolog of phzD (BGintphzD) is transcriptionally active at the time of the establishment of the arbuscular mycorrhizal symbiosis. DNA from isolated bacterial cultures found in spores of G. intraradices BE3 confirmed that both BGintPbr1a and BGintphzD are present in the genome of its potential endosymbionts. Taken together, our results indicate that spores of G. intraradices BE3 express bacterial phenazine biosynthetic genes at the onset of the fungal-plant symbiotic interaction.
Assuntos
Sequência de Bases , Enzimas/biossíntese , Fenazinas/análise , Hifas/crescimento & desenvolvimento , Técnicas In Vitro , Micorrizas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase/métodos , Simbiose/genética , Ativação Enzimática , Métodos , Prevalência , Esporos BacterianosRESUMO
Chlamydomonas reinhardtii has many advantages compared with traditional systems for the molecular farming of recombinant proteins. These include low production costs, rapid scalability at pilot level, absence of human pathogens and the ability to fold and assemble complex proteins accurately. Currently, the successful expression of several proteins with pharmaceutical relevance has been reported from the nuclear and the chloroplastic genome of this alga, demonstrating its usefulness for biotechnological applications. However, several factors affect the level of recombinant protein expression in Chlamydomonas such as enhancer elements, codon dependency, sensitivity to proteases and transformation-associated genotypic modification. The present review outlines a number of strategies to increase protein yields and summarizes recent achievements in algal protein production including biopharmaceuticals such as vaccines, antibodies, hormones and enzymes with implications on health-related approaches. The current status of bioreactor developments for algal culture and the challenges of scale-up and optimization processes are also discussed.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Indústria Farmacêutica/métodos , Microalgas/metabolismo , Proteínas Recombinantes/biossíntese , Reatores Biológicos , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Enzimas/biossíntese , Hormônios/biossíntese , Humanos , Microalgas/genética , Microalgas/crescimento & desenvolvimento , Agricultura Molecular , Planticorpos/metabolismo , Plantas Geneticamente Modificadas , VacinasRESUMO
The aim of this study is to describe the ultrastructure of the hepatopancreas of P. argentinus in intermoult. P. argentinus hepatopancreas was studied using standard TEM techniques. Each tubule consists of four cellular types: E (embryonic), F (fibrillar), R (resorptive) and B (blister like). E-cells have embryonic features and some of them were found in mitosis. F, R and B cells possess an apical brush border. F-cells have a central or basal nucleus, a conspicuous RER, and dilated Golgi cisternae. R cells show a polar organization of organelles in three areas: apical, with numerous mitochondria and sER tubules, a central area with the nucleus and RER, and a basal area containing a sER-like tubule system and mitochondria. B-cells were observed at different stages of their life cycle. In an early differentiation stage they comprise an apical endocytotic complex and Golgi vesicles. The fusion of endocytotic and Golgi vesicles originates subapical vacuoles. During maturation, a big central vacuole is formed by coalescence of subapical vacuoles. The central vacuole is eliminated by holocrine secretion. The ultrastructure suggests that F-cells synthesize proteins, R-cells storage nutrients and B-cells have a secretory or excretory function, and confirms the independent origin of F, B and R cells from the embryonic cells.
Assuntos
Células Epiteliais/ultraestrutura , Fígado/ultraestrutura , Palaemonidae/ultraestrutura , Pâncreas/ultraestrutura , Animais , Diferenciação Celular/fisiologia , Fenômenos Fisiológicos do Sistema Digestório , Enzimas/biossíntese , Enzimas/metabolismo , Células Epiteliais/metabolismo , Feminino , Fígado/embriologia , Fígado/fisiologia , Masculino , Microscopia Eletrônica de Transmissão , Organelas/fisiologia , Organelas/ultraestrutura , Palaemonidae/embriologia , Palaemonidae/fisiologia , Pâncreas/embriologia , Pâncreas/fisiologiaRESUMO
En la búsqueda de alternativas para mejorar la expresión de la enzima Iduronato Sulfatasa (IDSh) en la levaduraPichia pastoris, se realizó una Revisión Sistemática de la Literatura con el fin de recopilar información quepermitiera relacionar los niveles de expresión de proteínas humanas recombinantes con la señal de secreción y las características propias de la molécula a expresar. Se hallaron 349 publicaciones de las cuales sólo 7 (2)porciento reportaron la expresión de proteínas que cumplían con los criterios de inclusión manejados en el estudio. Con la información obtenida en los 7 artículos se realizó una prueba de hipótesis tomando como muestras los datos recopilados y un análisis cualitativo de la información, con los cuales se evidenció que al reemplazar la señal de secreción nativa por el a-Factor como péptido líder se incrementa el nivel de expresión de proteínas humanas recombinantes en P. pastoris (p=0.053). Se encontró que la eliminación de la secuencia que codifica para el péptido nativo heterólogo en el ADNc de la proteína, es imprescindible para que el a-Factor pueda favorecer la secreción de proteínas heterólogas y por consiguiente incrementar el nivel de expresión. En el caso de la IDShr se halló que en la construcción GS115/pPIC9-IDS, aparecen dos secuencias de péptido señal al mismo tiempo, la nativa de la IDSh y la putativa proveniente de Saccharomyces cerevisiae; sin embargo, se han obtenidos expresiones hasta de ~30mmol/h mg de proteína, lo que deja la incógnita de un posible conflicto en el reconocimiento erróneo de una u otra señal de secreción, teniendo en cuenta el grado de hidrofobicidad de ambas.
Assuntos
Animais , Enzimas/análise , Enzimas/biossíntese , Enzimas/classificação , Iduronato Sulfatase/análise , Iduronato Sulfatase/classificação , Iduronato Sulfatase/deficiência , Pichia/classificação , Peptídeo C/análise , Peptídeo C/classificação , Thlaspi bursa pastoris/análiseRESUMO
Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the prevalent systemic mycosis in Latin America, paracoccidioidomycosis. We present here a survey of expressed genes in the yeast pathogenic phase of P. brasiliensis. We obtained 13,490 expressed sequence tags from both 5' and 3' ends. Clustering analysis yielded the partial sequences of 4,692 expressed genes that were functionally classified by similarity to known genes. We have identified several Candida albicans virulence and pathogenicity homologues in P. brasiliensis. Furthermore, we have analyzed the expression of some of these genes during the dimorphic yeast-mycelium-yeast transition by real-time quantitative reverse transcription-PCR. Clustering analysis of the mycelium-yeast transition revealed three groups: (i) RBT, hydrophobin, and isocitrate lyase; (ii) malate dehydrogenase, contigs Pb1067 and Pb1145, GPI, and alternative oxidase; and (iii) ubiquitin, delta-9-desaturase, HSP70, HSP82, and HSP104. The first two groups displayed high mRNA expression in the mycelial phase, whereas the third group showed higher mRNA expression in the yeast phase. Our results suggest the possible conservation of pathogenicity and virulence mechanisms among fungi, expand considerably gene identification in P. brasiliensis, and provide a broader basis for further progress in understanding its biological peculiarities.