RESUMO
This study aimed to better characterize a recently purified stable extracellular alkaline peptidase produced by Penicillium aurantiogriseum (URM 4622) through fluorescence spectroscopy, far-UV circular dichroism, kinetic and thermodynamic models to understand its' structure-activity and denaturation. Fluorescence data showed that changing pH leads to tryptophan residues exposure to more hydrophilic environments at optimum activity pH 9.0 and 10.0. When thermally treated, it displayed less unfolding at these pH values, along with 4-fold less photoproducts formation than at neutral pH. Different pH CD spectra showed more ß-sheet (21.5-43.0%) than α-helix (1-6.2%). At pH9.0, more than 2-fold higher α-helix content than any other pH. The melting temperature (Tm) was observed between 50 and 60 °C at all pH studied, with lower Tm at pH 9.0-11.0 (54.9-50.3 °C). The protease displayed two phase transition, with two energies of denaturation, and a 4-fold higher thermal stability (ΔH°m) than reports for other microorganism's proteases. An irreversible folding transition occurs between 50 and 60 °C. It displayed energies of denaturation suggesting higher thermal stability than reported for other microorganism's proteases. These results help elucidating the applicability of this new stable protease.
Assuntos
Peptídeo Hidrolases , Dobramento de Proteína , Dicroísmo Circular , Endopeptidases , Concentração de Íons de Hidrogênio , Penicillium , Desnaturação Proteica , Espectrometria de Fluorescência , Temperatura , TermodinâmicaRESUMO
ß-Galactosidase production, partial purification and characterization by a new fungal were investigated. Partial purification was performed by aqueous two-phase system (ATPS) using polyethylene glycol (PEG) molar mass, PEG concentration, citrate concentration and pH as the independent variables. Purification factor (PF), partition coefficient (K) and yield (Y) were the responses. After identification by rDNA sequencing and classification as Cladosporium tenuissimum URM 7803, this isolate achieved a maximum cell concentration and ß-galactosidase activity of 0.48 g/L and 462.1 U/mL, respectively. ß-Galactosidase partitioned preferentially for bottom salt-rich phase likely due to hydrophobicity and volume exclusion effect caused in the top phase by the high PEG concentration and molar mass. The highest value of PF (12.94) was obtained using 24% (w/w) PEG 8000 g/mol and 15% (w/w) citrate, while that of Y (79.76%) using 20% (w/w) PEG 400 g/mol and 25% (w/w) citrate, both at pH 6. The enzyme exhibited optimum temperature in crude and ATPS extracts in the ranges 35-50 °C and 40-55 °C, respectively, and optimum pH in the range 3.0-4.5, with a fall of enzyme activity under alkaline conditions. Some metal ions and detergents inhibited, while others stimulated enzyme activity. Finally, C. tenuissimum URM 7803 ß-galactosidase showed a profile suitable for prebiotics production.
Assuntos
Cladosporium/enzimologia , Polietilenoglicóis/química , beta-Galactosidase/química , Biotecnologia , Citratos , DNA/análise , Detergentes/química , Fermentação , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Íons , Ferro/química , Lactose/química , Microscopia Eletrônica de Varredura , Filogenia , Reação em Cadeia da Polimerase , Prebióticos , Análise de Sequência de DNA , Temperatura , Água/química , beta-Galactosidase/isolamento & purificaçãoRESUMO
A new set of applications can be achieved when using high stability proteases. Industrially, high costs can be related to production medium and purification process. Magnetic nanoparticles have been successfully used for rapid and scalable purification. In this work, azocasein were immobilized on magnetite nanoparticles and applied in a single step purification of protease produced by Penicillium aurantiogriseum using soybean flour medium, and the new purified enzyme was characterized. Glutaraldehyde activated nanoparticles were used in azocasein immobilization and then incubated with dialyzed 60-80% saline precipitation fraction of crude extract for purification. Adsorbents were washed 7 times (0.1â¯M NaCl solution) and eluted 3 times (1â¯M NaCl solution), these final elutions contained the purified protease. This protease was purified 55.68-fold, retaining 46% of its original activity. Presented approximately 40â¯kDa on SDS-PAGE and optimum activity at 45⯰C and pH 9.0. Maintained over 60% of activity from pH 6.0 to 11.0. Kept more than 50% activity from 15 to 55⯰C, did not lose any activity over 48â¯hâ¯at 25⯰C. Inhibitors assay suggested a serine protease with aspartic residues on its active site. Results report a successful application of an alternative purification method and novel broad pH tolerant protease.
Assuntos
Proteínas Fúngicas/isolamento & purificação , Nanopartículas de Magnetita/química , Penicillium/enzimologia , Serina Proteases/isolamento & purificação , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Caseínas/química , Domínio Catalítico , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Peso Molecular , Serina Proteases/química , Serina Proteases/metabolismoRESUMO
Abstract Specific proteases capable of degrading native triple helical or denatured collagen have been required for many years and have a large spectrum of applications. There are few complete reports that fully uncover production, characterization and purification of fungi collagenases. In this review, authors searched through four scientific on line data bases using the following keywords (collagenolytic OR collagenase) AND (fungi OR fungus OR fungal) AND (production OR synthesis OR synthesize) AND (characterization). Scientific criteria were adopted in this review to classify found articles by score (from 0 to 10). After exclusion criteria, 21 articles were selected. None obtained the maximum of 10 points defined by the methodology, which indicates a deficiency in studies dealing simultaneously with production, characterization and purification of collagenase by fungi. Among microorganisms studied the non-pathogenic fungi Penicillium aurantiogriseum and Rhizoctonia solani stood out in volumetric and specific collagenase activity. The only article found that made sequencing of a true collagenase showed 100% homology with several metalloproteinases fungi. A clear gap in literature about collagenase production by fungi was verified, which prevents further development in the area and increases the need for further studies, particularly full characterization of fungal collagenases with high specificity to collagen.
Assuntos
Colágeno/metabolismo , Colagenases/metabolismo , Fungos/metabolismo , Especificidade por Substrato , Colágeno/química , Colagenases/isolamento & purificação , Colagenases/biossíntese , Colagenases/química , Meios de Cultura , Ativação Enzimática , Proteólise , Fungos/classificaçãoRESUMO
Specific proteases capable of degrading native triple helical or denatured collagen have been required for many years and have a large spectrum of applications. There are few complete reports that fully uncover production, characterization and purification of fungi collagenases. In this review, authors searched through four scientific on line data bases using the following keywords (collagenolytic OR collagenase) AND (fungi OR fungus OR fungal) AND (production OR synthesis OR synthesize) AND (characterization). Scientific criteria were adopted in this review to classify found articles by score (from 0 to 10). After exclusion criteria, 21 articles were selected. None obtained the maximum of 10 points defined by the methodology, which indicates a deficiency in studies dealing simultaneously with production, characterization and purification of collagenase by fungi. Among microorganisms studied the non-pathogenic fungi Penicillium aurantiogriseum and Rhizoctonia solani stood out in volumetric and specific collagenase activity. The only article found that made sequencing of a true collagenase showed 100% homology with several metalloproteinases fungi. A clear gap in literature about collagenase production by fungi was verified, which prevents further development in the area and increases the need for further studies, particularly full characterization of fungal collagenases with high specificity to collagen.(AU)
Assuntos
Fungos/isolamento & purificação , Fungos/enzimologia , Colágeno/genéticaRESUMO
Specific proteases capable of degrading native triple helical or denatured collagen have been required for many years and have a large spectrum of applications. There are few complete reports that fully uncover production, characterization and purification of fungi collagenases. In this review, authors searched through four scientific on line data bases using the following keywords (collagenolytic OR collagenase) AND (fungi OR fungus OR fungal) AND (production OR synthesis OR synthesize) AND (characterization). Scientific criteria were adopted in this review to classify found articles by score (from 0 to 10). After exclusion criteria, 21 articles were selected. None obtained the maximum of 10 points defined by the methodology, which indicates a deficiency in studies dealing simultaneously with production, characterization and purification of collagenase by fungi. Among microorganisms studied the non-pathogenic fungi Penicillium aurantiogriseum and Rhizoctonia solani stood out in volumetric and specific collagenase activity. The only article found that made sequencing of a true collagenase showed 100% homology with several metalloproteinases fungi. A clear gap in literature about collagenase production by fungi was verified, which prevents further development in the area and increases the need for further studies, particularly full characterization of fungal collagenases with high specificity to collagen.
Assuntos
Colágeno/metabolismo , Colagenases/metabolismo , Fungos/metabolismo , Colágeno/química , Colagenases/biossíntese , Colagenases/química , Colagenases/isolamento & purificação , Meios de Cultura , Ativação Enzimática , Fungos/classificação , Proteólise , Especificidade por SubstratoRESUMO
Enzymatic hydrolysis of pretreated sugarcane bagasse was performed to investigate the production of ethanol. The sugarcane bagasse was pretreated in a process combining steam explosion and alkaline delignification. The lignin content decreased to 83%. Fed-batch enzymatic hydrolyses was initiated with 8% (w/v) solids loading, and 10 FPU/g cellulose. Then, 1% solids were fed at 12, 24 or 48 h intervals. After 120 h, the hydrolysates were fermented with Saccharomyces cerevisiae UFPEDA 1238, and a fourfold increase in ethanol production was reached when fed-batch hydrolysis with a 12-h addition period was used for the steam pretreated and delignified bagasse.