RESUMO
Pleurotus sapidus monokaryotic strains (Mk) were screened as a novel source of mycelia to valorize rice straw (RS), rice husks (RH) and sunflower seed hulls (SSH) into value-added products through solid-state fermentation (SSF). P. sapidus Dk3174 basidiospores were cultured in the presence of Remazol Brillant Blue R for strain selection, revealing the ligninolytic ability of emerging colonies. Further screening demonstrated the intraspecific variability in dye degradation and enzyme production of 63 strains. Growth rate, biomass and enzyme production in plates containing RS, RH or SSH pointed at MkP6 as a suitable strain for pilot-scale SSF. MkP6 presented a similar laccase profile as the parental Dk3174, being greater in pasteurized substrates (300-1200 U/Kg) than in sterilized substrates (30-250 U/Kg). Peroxidase represented 25% of the total ligninolytic activity measured. The SSH fermented biomass with MkP6 obtained good yields of nanocellulose (67%) and the saccharide release for ethanol production increased by 3-4 times.
Assuntos
Fermentação , Helianthus/metabolismo , Oryza/metabolismo , Pleurotus/metabolismo , Biomassa , Lacase/metabolismo , Peroxidase/metabolismo , Peroxidases/metabolismoRESUMO
Whooping cough, which is caused by Bordetella pertussis and B. parapertussis, is a reemerging disease. New protective antigens are needed to improve the efficacy of current vaccines against both species. Using proteomic tools, it was here found that B. parapertussis expresses a homolog of AfuA, a previously reported new vaccine candidate against B. pertussis. It was found that this homolog, named AfuABpp , is expressed during B. parapertussis infection, exposed on the surface of the bacteria and recognized by specific antibodies induced by the recombinant AfuA cloned from B. pertussis (rAfuA). Importantly, the presence of the O-antigen, a molecule that has been found to shield surface antigens on B. parapertussis, showed no influence on antibody recognition of AfuABpp on the bacterial surface. The present study further showed that antibodies induced by immunization with the recombinant protein were able to opsonize B. parapertussis and promote bacterial uptake by neutrophils. Finally, it was shown that this antigen confers protection against B. parapertussis infection in a mouse model. Altogether, these results indicate that AfuA is a good vaccine candidate for acellular vaccines protective against both causative agents of whooping cough.
Assuntos
Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Infecções por Bordetella/prevenção & controle , Bordetella parapertussis/efeitos dos fármacos , Bordetella pertussis/genética , Vacina contra Coqueluche/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Infecções por Bordetella/imunologia , Bordetella parapertussis/imunologia , Bordetella parapertussis/patogenicidade , Bordetella pertussis/efeitos dos fármacos , Bordetella pertussis/imunologia , Bordetella pertussis/metabolismo , Modelos Animais de Doenças , Feminino , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/imunologia , Antígenos O/imunologia , Proteômica , Vacinação , Vacinas Acelulares/genética , Vacinas Acelulares/imunologia , Coqueluche/microbiologiaRESUMO
The application of cellulases in saccharification processes is restricted by its production cost. Consequently, new fungal strains able to elaborate higher cellulases titers and with special activity profiles are required to make the process economical. The aim of this investigation was to find a promising wild-type Trichoderma strain for cellulases production. The Trichoderma reesei strain 938 (CBS 836.91) was selected among twenty strains on the basis of cellulase-agar-plate screening. Evaluation of the selected strain on six solid substrates indicated the highest activities to be obtained from wheat bran. Statistical analyses of the experimental design indicated a significant effect of pH and moisture on the generation of endoglucanase (EGA) and filter-paper (FPA) activity. Furthermore, a central-composite design-based optimization revealed that pH values between 6.4 and 6.6 and moisture from 74 to 94% were optimal for cellulases production. Under these conditions, 8-10 IU gds(-1) of FPA and 15.6-17.8 IU gds(-1) of EGA were obtained. In addition, cultivation in a rotating-drum reactor under optimal conditions gave 8.2 IU gds(-1) FPA and 13.5 IU gds(-1) EGA. Biochemical characterization of T. reesei 938 cellulases indicated a substantially higher resistance to 4 mM Fe(+2) and a slightly greater tolerance to alkaline pH in comparison to Celluclast(®). These results suggest that T. reesei 938 could be a promising candidate for improved cellulases production through direct-evolution strategies.
Assuntos
Celulases/biossíntese , Fibras na Dieta/metabolismo , Proteínas Fúngicas/biossíntese , Trichoderma/crescimento & desenvolvimentoRESUMO
BACKGROUND: In the food industry, the use of pectinase preparations with high pectin esterase (PE) activity leads to the release of methanol, which is strictly regulated in food products. Herein, a pectin-degrading enzyme (PDE) complex exhibiting low PE activity of three Aspergillus sojae ATCC 20235 mutants (M3, DH56 and Guserbiot 2.230) was investigated. Production of exo-/endo-polygalacturonase (PG), exo-polymethylgalacturonase (PMG) and pectin lyase (PL) by mutant M3 and A. sojae using two different carbon sources was evaluated in solid-state fermentation. Finally, experimental preparations obtained from the mutants and commercial pectinases standardized to the same potency were screened for PDEs. RESULTS: Mutant M3 grown on sugar beet was found to be the best producer of exo-PG, endo-PG, exo-PMG and PL, with maximum yields of 1111, 449, 130 and 123 U g(-1), respectively. All experimental preparations exhibited low PE activity, at least 21.5 times less than commercial pectinases, and higher endo-PG (40 U mL(-1)). CONCLUSION: Mutant M3 was the best PDE producer using sugar beet. Mutant strains presented a PDE complex featuring high endo-PG and very low PE activities. This novel complex with low de-esterifying activity can be exploited in the food industry to degrade pectin without releasing methanol.
Assuntos
Aspergillus niger/enzimologia , Beta vulgaris , Fermentação , Complexos Multienzimáticos/metabolismo , Mutação , Pectinas/metabolismo , Poligalacturonase/metabolismo , Aspergillus niger/genética , Aspergillus niger/crescimento & desenvolvimento , Meios de Cultura , Esterases/metabolismo , Esterificação , Humanos , Liases/biossíntese , Liases/metabolismo , Metanol/metabolismoRESUMO
Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13.
Assuntos
Cromatografia de Afinidade/métodos , Cisteína/química , Proteínas de Fluorescência Verde/isolamento & purificação , Histidina/química , Paládio/química , Fragmentos de Peptídeos/química , Proteínas Recombinantes/isolamento & purificação , Cisteína/metabolismo , Escherichia coli , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Histidina/metabolismo , Humanos , Paládio/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
In recent years, affinity fusion-tag systems have become a popular technique for the purification of recombinant proteins from crude extracts. However, several drawbacks including the high expense and low stability of ligands, their leakage during operation, and difficulties in immobilization, make it important to further develop the method. The present work is concerned with the utilization of a ceramic fluorapatite (CFT)-based chromatographic matrix to overcome these drawbacks. A heptapeptide library exhibiting a range of properties have been synthesized and subjected to ceramic fluorapatite (CFT) chromatography to characterize their retention behavior as a function of pH and composition of the binding buffer. The specific binding and elution behavior demonstrates the possible application of CFT-binding peptides as tags for enhancing the selective recovery of proteins by CFT chromatography. To materialize this strategy, a phage-derived CFT-specific sequence KPRSVSG (Tag1) with/without a consecutive hexalysine sequence, KKKKKKKPRSVSG (Tag2), were fused at the C-terminus of an enhanced green fluorescent protein (eGFP). The resulting gene constructs H-eGFP, H-eGFP-Tag1 and H-eGFP-Tag2 were expressed in Escherichia coli strain BL-21, and the clarified cell lysate was applied to the CFT column equilibrated with binding buffer (20-50mM sodium phosphate, pH 6-8.4). Sodium phosphate (500mM) or 1M NaCl in the respective binding buffer was used to elute the fused proteins, and the chromatographic fractions were analyzed by gel electrophoresis. Both the yield and purity were over 90%, demonstrating the potential application of the present strategy.
Assuntos
Marcadores de Afinidade/química , Apatitas/química , Proteínas de Fluorescência Verde/química , Peptídeos/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Cerâmica , Cromatografia de Afinidade/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Peptídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Hydrophobic interaction chromatography (HIC) is an important tool in the industrial purification of proteins from various sources. The HIC separation behavior of individual (or model) proteins has been widely researched by others. On the contrary, this study focused on the fractionation ability of HIC when it is challenged with whole proteomes. The impact of the nature of three different proteomes, that is, yeast, soybean, and Chinese hamster ovary cells, on HIC separation was investigated. In doing so, chromatography fractions obtained under standardized conditions were evaluated in terms of their overall hydrophobicity--as measured by fluorescence dye binding. This technique allowed for the calculation of an average protein surface hydrophobicity (S(0)) for each fraction; a unique correlation between S(0) and the observed chromatographic behavior was established in each case. Following a similar strategy, the effect of three different ligands (polypropylene glycol, phenyl, and butyl) and two adsorbent particle sizes (65 and 100 µm) on the chromatographic behavior of the yeast proteome was evaluated. As expected, the superficial hydrophobicity of the proteins eluted is correlated with the salt concentration of its corresponding elution step. The findings reveled how--and in which extent--the type of ligand and the size of the beads actually influenced the fractionation of the complex biological mixture. Summarizing, the approach presented here can be instrumental to the study of the performance of chromatography adsorbents under conditions close to industrial practice and to the development of downstream processing strategies.