RESUMO
Abstract An intronless endoglucanase from thermotolerant Aspergillus fumigatus DBINU-1 was cloned, characterized and expressed in the yeast Kluyveromyces lactis. The full-length open reading frame of the endoglucanase gene from A. fumigatus DBiNU-1, designated Cel7, was 1383 nucleotides in length and encoded a protein of 460 amino acid residues. The predicted molecular weight and the isoelectric point of the A. fumigatus Cel7 gene product were 48.19 kDa and 5.03, respectively. A catalytic domain in the N-terminal region and a fungal type cellulose-binding domain/module in the C-terminal region were detected in the predicted polypeptide sequences. Furthermore, a signal peptide with 20 amino acid residues at the N-terminus was also detected in the deduced amino acid sequences of the endoglucanase from A. fumigatus DBiNU-1. The endoglucanase from A. fumigatus DBiNU-1 was successfully expressed in K. lactis, and the purified recombinant enzyme exhibited its maximum activity at pH 5.0 and 60 °C. The enzyme was very stable in a pH range from 4.0 to 8.0 and a temperature range from 30 to 60 °C. These features make it suitable for application in the paper, biofuel, and other chemical production industries that use cellulosic materials.
Assuntos
Aspergillus fumigatus/enzimologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Expressão Gênica , Celulase/genética , Celulase/química , Clonagem Molecular , Aspergillus fumigatus/genética , Especificidade por Substrato , Estabilidade Enzimática , Kluyveromyces/genética , Kluyveromyces/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Fúngicas/metabolismo , Celulase/metabolismo , Temperatura Alta , Concentração de Íons de HidrogênioRESUMO
Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified:
Assuntos
Aspergillus fumigatus/enzimologia , Aspergillus niger/enzimologia , Celulose/metabolismo , /metabolismo , Kluyveromyces/enzimologia , Saccharum/microbiologia , Xilosidases/metabolismo , beta-Glucosidase/metabolismo , Aspergillus fumigatus/isolamento & purificação , Aspergillus fumigatus/metabolismo , Aspergillus niger/isolamento & purificação , Aspergillus niger/metabolismo , Sequência de Bases , Biomassa , Brasil , DNA Fúngico/genética , DNA Intergênico/genética , Fermentação , Kluyveromyces/isolamento & purificação , Kluyveromyces/metabolismo , Lignina/metabolismo , Tipagem Molecular , Técnicas de Tipagem Micológica , RNA Ribossômico/genética , Análise de Sequência de DNARESUMO
Low cost agro-waste was used as adsorption support for single-step purification of endoglucanase from the culture filtrate of A. fumigatus ABK-9. Among various agro-waste substrates, 1% NaOH pretreated rice bran was proved to be the best for adsorbing about 74.8 and 71.1% of endoglucanase at 4 °C and 10 °C respectively. Langmuir type adsorption isotherm at 4 °C showed maximum adsorption of enzyme at pH 5.0, which was in the range of optimum pH of the enzyme. The rice bran column bound enzyme was maximally eluted by a mixture of acetate buffer (0.05 M, pH 5.5) and ethanol (40%, v/v) at a ratio of 3:2 and a flow rate of 1 mL/min. A 5.52-fold purification of the enzyme was achieved from culture supernatant. The specific activity and recovery yield after purification were 294.0 U/mg and 40.15%, respectively, which were comparable with other contemporary protocols. The homogeneity of the enzyme was tested through sodium dodecyl sulphate polyacrylamide gel electrophoresis and a single band of 56.3 kDa was observed. Zymogram analysis finally confirmed the occurrence of endoglucanase in the single band.
Assuntos
Adsorção , Aspergillus fumigatus/enzimologia , Celulase/isolamento & purificação , Celulase/metabolismo , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Temperatura , TermodinâmicaRESUMO
Enzyme production varies in different fermentation systems. Enzyme expression in different fermentation systems yields important information for improving our understanding of enzymatic production induction. Comparative studies between solid-state fermentation (SSF) using agro-industrial waste wheat bran and submerged fermentation (SmF) using synthetic media were carried out to determinate the best parameters for peptidase production by the fungus Aspergillus fumigatus Fresen. Variables tested include: the concentration of carbon and protein nitrogen sources, the size of the inoculum, the pH of the media, temperature, and the length of the fermentation process. The best peptidase production during SSF was obtained after 96 hours using wheat bran at 30 ºC with an inoculum of 1 x 10(6) spores and yielded 1500 active units (UµmL). The best peptidase production using SmF was obtained after periods of 72 and 96 hours of fermentation in media containing 0.5% and 0.25% of casein, respectively, at a pH of 6.0 and at 30 ºC and yielded 40 UµmL. We also found examples of catabolite repression of peptidase production under SmF conditions. Biochemical characterization of the peptidases produced by both fermentative processes showed optimum activity at pH 8.0 and 50 ºC, and also showed that their proteolytic activity is modulated by surfactants. The enzymatic inhibition profile using phenylmethylsulfonyl fluoride (PMSF) in SmF and SSF indicated that both fermentative processes produced a serine peptidase. Additionally, the inhibitory effect of the ethylene-diaminetetraacetic acid (EDTA) chelating agent on the peptidase produced by SmF indicated that this fermentative process also produced a metallopeptidase.
Assuntos
Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/isolamento & purificação , Azotobacter/enzimologia , Azotobacter/isolamento & purificação , Fermentação , Metaloexopeptidases/análise , Metaloexopeptidases/isolamento & purificação , Peptídeo Hidrolases/análise , Serina/análise , Ativação Enzimática , Métodos , Padrões de Referência , MétodosRESUMO
Aspergillus fumigatus contains a heat-stable phytase of great potential. To determine whether this phytase could be expressed in plants as a functional enzyme, we introduced the phytase gene from A. fumigatus (fphyA) in tobacco (Nicotiana tabacum L. cv. NC89) by Agrobacterium-mediated transformation. Phytase expression was controlled by the cauliflower mosaic virus (CaMV) 35S promoter. Secretion of recombinant phytase (tfphyA) to the extracellular fluid was established by use of the signal sequence from tobacco calreticulin. Forty-one independent transgenic plants were generated. Single-copy line A was selected based on segregation of T1 seeds for kanamycin resistance, phytase expression and Southern blotting analysis for use in further study. After 4-weeks of plant growth, the phytase was accumulated in leaves up to 2.3% of total soluble protein. tfphyA was functional and shared similar profiles of pH, temperature and thermal stability to the same enzyme expressed in Pichia pastoris (pfphyA). The expressed enzyme had an apparent molecular mass of 63 kDa and showed maximum activity at pH 5.5, and temperature, 55 degrees C. It had a high thermostability and retained 28.7% of the initial activity even after incubation at 90 degrees C for 15 min. The above results showed that the thermostable A. fumigatus phytase could be expressed in tobacco as a functional enzyme and thus has the potential of overexpressing it in other crop plants also.
Assuntos
6-Fitase/genética , Aspergillus fumigatus/enzimologia , Sequência de Bases , DNA Fúngico/genética , Estabilidade Enzimática , Expressão Gênica , Genes Fúngicos , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Nicotiana/enzimologiaRESUMO
A xylan-degrading enzyme (xylanase II) was purified to apparent homogeneity from solid-state cultures of Aspergillus fumigatus Fresenius. The molecular weight of xylanase II was found to be 19 and 8.5 kDa, as estimated by SDS-PAGE and gel filtration on FPLC, respectively. The purified enzyme was most active at 55 (degree)C and pH 5.5. It was specific to xylan. The apparent Km and Vmax values on soluble and indisoluble xylans from oat spelt and birchwood showed that xylanase II was most active on soluble birchwood xylan. Studies on hydrolysis products of various xylans and xylooligormers by xylanase II on HPLC showed that the enzyme released a range of products from xylobiose to xylohexaose, with a small amount of xylose from xylooligomers, and presented transferase activity.
Assuntos
Aspergillus fumigatus/enzimologia , Xilosidases/isolamento & purificação , Xilosidases/química , Xilosidases/metabolismo , Peso MolecularRESUMO
The steady state kinetics of ligninperoxidase catalysed reaction using n-propanol as the organic substrate and monitoring the formation of propanaldehyde at lambda = 300 nm spectrophotometerically as functions of different reaction parameters has been studied. It has been concluded that n-propanol can be used as a substrate for analysing the activity of ligninperoxidase. The turnover number of ligninperoxidase of Phanerochaete chrysosporium using n-propanol as substrate has been found to be higher approximately by a factor of 10(3) as compared to that using veratryl alcohol as the substrate. The method works in assaying the activity of ligninperoxidase produced by Aspergillus fumigatus indicating that it can be used for assaying the ligninperoxidase activities produced by other microorganisms also and is not limited to assaying the ligninase activity produced by Phanerochaete chrysosporium alone. Under identical experimental conditions, horseradish peroxidase does not show peroxidase activity using n-propanol as substrate indicating that the method does not interfere with the activities of other peroxidases.
Assuntos
1-Propanol , Aspergillus fumigatus/enzimologia , Álcoois Benzílicos , Concentração de Íons de Hidrogênio , Cinética , Peroxidases/análise , Phanerochaete/enzimologia , Especificidade por SubstratoRESUMO
A potential producer of extracellular phosphatase has been isolated and identified as A. fumigatus. The fungal phosphatase is active in pH range 5 to 8 and its temperature optimum is 65 degrees C. The mineralisation of organic phosphates present in Neem cake and press mud by this enzyme has been demonstrated.
Assuntos
Aspergillus fumigatus/enzimologia , Concentração de Íons de Hidrogênio , Cinética , Organofosfatos/metabolismo , Monoéster Fosfórico Hidrolases/biossíntese , Microbiologia do Solo , TemperaturaRESUMO
Treinta y cuatro cepas de Aspergillus fumigatus aisladas del aire, crín de caballo, suelo agrícola y del hombre, fueron examinadas con el fin de evaluar la producción de elastasa. Las cepas de Aspergillus fumigatus fueron cultivadas en un medio sólido con elastina, apreciándose en ella su amplio solubilización por la acción del hongo. Los aislamientos fúngicos provenientes de muestras aisladas del hobre y de suelos agrícolas fueron detectados como los más altos productores de elastasa. Ocho de las 34 cepas fueron desarrollas en 4 diferentes medios líquidos en las cuales se investigó la actividad proteolítica total y específica. Los resultados de este experimento sugieren que la producción de elastasa es inducida por la presencia de elastina como sustrato y que la primera es una enzima semejante a la quimiotripsina. El perfil inhibitorio comprobó que la elastina de A.fumigatus, es una serina-proteinasa