Malbranchea Pulchella Research Articles

Homologous expression, purification, and characterization of a recombinant acetylxylan esterase from Aspergillus nidulans

Acetylxylan esterases (AXEs) are essential enzymes that break down the acetyl groups in acetylated xylan found in plant cell walls polysaccharides. They work synergistically with backbone-depolymerizing xylanolytic enzymes to accelerate the degradation of complex polysaccharides. In this study, we cloned the gene axeA, which encodes the acetylxylan esterase from Aspergillus nidulans FGSC A4 (AxeAN), into the pEXPYR expression vector and introduced it into the high protein-producing strain A. nidulans A773. The purified AxeAN, with a molecular weight of 33.5 kDa as confirmed by SDS-PAGE, was found to be active on ρ-nitrophenyl acetate (ρNPA), exhibiting a remarkably high specific activity (170 U mg−1) at pH 7.0 and 55 °C. AxeAN demonstrated stability over a wide pH range (5.5–9.0), retaining >80% of its initial activity after 24 h. The KM and Vmax were 0.098 mmol L−1 and 320 U mg−1, respectively, using ρNPA as a substrate. We also evaluated the synergistic effect of AxeAN with an endo-1,4-β-xylanase from Malbranchea pulchella (MpXyn10) in the hydrolysis of four different xylans (Birchwood, Beechwood, Oat spelt, and Arabinoxylan) to produce xylooligosaccharides (XOS). The best results were obtained using Birchwood xylan as substrate and MpXyn10-AxeAN as biocatalysts after 24 h of reaction (50 °C), with a XOS-yield of 91%, value 41% higher when compared to MpXyn10 (XOS-yield of 63%). These findings showed the potential of the application of AxeAN, together with other xylanases, to produce xylooligosaccharides with high purity and other products with high added value in the field of lignocellulosic biorefinery.

Immobilization and Application of the Recombinant Xylanase GH10 of Malbranchea pulchella in the Production of Xylooligosaccharides from Hydrothermal Liquor of the Eucalyptus (Eucalyptus grandis) Wood Chips.

Xylooligosaccharides (XOS) are widely used in the food industry as prebiotic components. XOS with high purity are required for practical prebiotic function and other biological benefits, such as antioxidant and inflammatory properties. In this work, we immobilized the recombinant endo-1,4-β-xylanase of Malbranchea pulchella (MpXyn10) in various chemical supports and evaluated its potential to produce xylooligosaccharides (XOS) from hydrothermal liquor of eucalyptus wood chips. Values >90% of immobilization yields were achieved from amino-activated supports for 120 min. The highest recovery values were found on Purolite (142%) and MANAE-MpXyn10 (137%) derivatives, which maintained more than 90% residual activity for 24 h at 70 °C, while the free-MpXyn10 maintained only 11%. In addition, active MpXyn10 derivatives were stable in the range of pH 4.0-6.0 and the presence of the furfural and HMF compounds. MpXyn10 derivatives were tested to produce XOS from xylan of various sources. Maximum values were observed for birchwood xylan at 8.6 mg mL-1 and wheat arabinoxylan at 8.9 mg mL-1, using Purolite-MpXyn10. Its derivative was also successfully applied in the hydrolysis of soluble xylan present in hydrothermal liquor, with 0.9 mg mL-1 of XOS after 3 h at 50 °C. This derivative maintained more than 80% XOS yield after six cycles of the assay. The results obtained provide a basis for the application of immobilized MpXyn10 to produce XOS with high purity and other high-value-added products in the lignocellulosic biorefinery field.

Increased Malbranchea pulchella β-glucosidase production and its application in agroindustrial residue hydrolysis: A research based on experimental designs

β-Glucosidases are a limiting factor in the conversion of cellulose to glucose for the subsequent ethanol production. Here, β-glucosidase production by Malbranchea pulchella was optimized using Composite Central Designs and Response Surface Methodologies from a medium designed. The coefficient of determination (R2) was 0.9960, F-value was very high, and the lack of fit was found to be non-significant. This indicates a statistic valid and predictive result. M. pulchella enzymatic extract was successfully tested as an enzymatic cocktail in a mixture design using sugarcane bagasse, soybean hull and barley bagasse. We proved that the optimization of the β-glucosidase production and the application in hydrolysis using unexpansive biomass and agricultural wastes can be accomplished by means of statistical methodologies. The strategy presented here can be useful for the improvement of enzyme production and the hydrolysis process, arising as an alternative for bioeconomy.

A Highly Glucose Tolerant \xdf-Glucosidase from Malbranchea pulchella (MpBg3) Enables Cellulose Saccharification

β-glucosidases catalyze the hydrolysis β-1,4, β-1,3 and β-1,6 glucosidic linkages from non-reducing end of short chain oligosaccharides, alkyl and aryl β-D-glucosides and disaccharides. They catalyze the rate-limiting reaction in the conversion of cellobiose to glucose in the saccharification of cellulose for second-generation ethanol production, and due to this important role the search for glucose tolerant enzymes is of biochemical and biotechnological importance. In this study we characterize a family 3 glycosyl hydrolase (GH3) β-glucosidase (Bgl) produced by Malbranchea pulchella (MpBgl3) grown on cellobiose as the sole carbon source. Kinetic characterization revealed that the MpBgl3 was highly tolerant to glucose, which is in contrast to many Bgls that are completely inhibited by glucose. A 3D model of MpBgl3 was generated by molecular modeling and used for the evaluation of structural differences with a Bgl3 that is inhibited by glucose. Taken together, our results provide new clues to understand the glucose tolerance in GH3 β-glucosidases.

Efficient hydrolysis of wine and grape juice anthocyanins by Malbranchea pulchella β-glucosidase immobilized on MANAE-agarose and ConA-Sepharose supports

β-glucosidases (BGLs) hydrolyze short-chain cellulooligosaccharides. Some BGLs can hydrolyze anthocyanins and be applied in the clarification process of food industries, especially grape juice and wine. Enzyme immobilization is a valuable tool to increase enzyme stabilization. In this work, Malbranchea pulchella BGL was immobilized on Monoaminoethyl-N-ethyl-agarose ionic support, MANAE-agarose, and Concanavalin A-Sepharose affinity support, Con-A-Sepharose. The formed biocatalysts, denominated BLG-MANAE and BLG-ConA, were applied in the grape juice and red wine clarification. BGL-MANAE and BGL-ConA hyperactivated M. pulchella BGL 10- and 3-fold, respectively. Both biocatalysts showed at least 70% activity at pH range 2–11, until 24 h incubation. BGL-MANAE and BGL-ConA showed activity of 60% and 100%, respectively, at 50 °C, up to 24 h. Both biocatalysts were efficiently reused 20-fold. They were stable in the presence of up to 0.1 M glucose for 24 h incubation, and with 5%, 10% and 15% ethanol kept up to 70% activity. BGL-MANAE biocatalyst was 11% and 25% more efficient than BGL-ConA in clarification of concentrate and diluted wines, respectively. Likewise, BGL-MANAE biocatalysts were 14% and 33% more efficient than the BGL-ConA in clarification of diluted and concentrated juices, respectively. Therefore, the BGL-MANAE biocatalyst was especially effective in red wine and grape juice clarification.

A contribution to an investigation of nitrogen metabolism of some thermophilic and thermophilous soil fungi

The author has examined the assimilation of nitrogen on the basis of different sources of nitrogen and the secretion of nitrite to the substrate by some soil fungi. The following species of thermophilic and thermophilous fungi have been investigated: <i>Malbranchea pulchella</i> Saccardo et Penzig var. <i>sulfurea</i> Miehe, <i>Humicola lanuginosa</i> (Griffon et Maublanc) Bunce. <i>Sporotrichum thermophile</i> Apinis, <i>Thielavia sepedonium</i> Emmons, <i>Paecilomyces varioti</i> Bainier, <i>Aspergillus fumigatus</i> Fresenius, A. terreus Thom.

A novel thermostable xylanase GH10 from Malbranchea pulchella expressed in Aspergillus nidulans with potential applications in biotechnology.

BackgroundThe search for novel thermostable xylanases for industrial use has intensified in recent years, and thermophilic fungi are a promising source of useful enzymes. The present work reports the heterologous expression and biochemical characterization of a novel thermostable xylanase (GH10) from the thermophilic fungus Malbranchea pulchella, the influence of glycosylation on its stability, and a potential application in sugarcane bagasse hydrolysis.ResultsXylanase MpXyn10A was overexpressed in Aspergillus nidulans and was active against birchwood xylan, presenting an optimum activity at pH 5.8 and 80°C. MpXyn10A was 16% glycosylated and thermostable, preserving 85% activity after 24 hours at 65°C, and deglycosylation did not affect thermostability. Circular dichroism confirmed the high alpha-helical content consistent with the canonical GH10 family (β/α)8 barrel fold observed in molecular modeling. Primary structure analysis revealed the existence of eight cysteine residues which could be involved in four disulfide bonds, and this could explain the high thermostability of this enzyme even in the deglycosylated form. MpXyn10A showed promising results in biomass degradation, increasing the amount of reducing sugars in bagasse in natura and in three pretreated sugarcane bagasses.ConclusionsMpXyn10A was successfully secreted in Aspergillus nidulans, and a potential use for sugarcane bagasse biomass degradation was demonstrated.

Screening of Filamentous Fungi for Xylanases and Cellulases Not Inhibited by Xylose and Glucose

Aims: Screening different filamentous fungi for thermostable xylanases and cellulases that would not be inhibited by xylose and glucose, respectively. Methodology: Samples of fungi collected in the Atlantic forest region, Minas Gerais, Brazil, and some fungi from our Culture Collection were used in this screening. All fungi were grown in liquid media containing 1% sugar cane bagasse (SCB). After that, an aliquot of the crude broth was incubated at different temperatures (from 4 to 60 °C) in carboxymethyl cellulose (CMC) or xylan-media plates, for 12 hours. After this period, the plates were stained with Congo Red. Fungi that presented the best results (larger halos) were tested for the effect of adding xylose and glucose in the xylanase and cellulases activities, respectively. Crude extracts obtained from fungi grown in SCB were used for laccase and lichenase assay. Results: The screening on agar plates with CMC/xylan presented halos of different sizes. From all tested fungi, the best cellulase producer was Malbranchea pulchella, which also Original Research Article British Biotechnology Journal, 4(1): 30-39, 2014 31 presented the most thermostable xylanase. Penicillium griseofulvum presented bigger halos at all temperatures tested, but the xylanase lost almost 14% of its stability in higher temperatures. The effect of xylose and glucose on the enzymatic activities recorded dosedependent. It was observed that 20% activation of the enzymes produced by M. pulchella with 30 mM glucose or 20 mM xylose to cellulase and xylanase, respectively. It was observed a loss of less than 20% for P. griseofulvum xylolytic activity using 50 mM xylose. Lichenase was detected in some fungi prospected but laccase was not detected. Conclusion: Malbranchea pulchella was a good producer of xylanase and cellulase tolerant to xylose and glucose, respectively. Other studies must be performed with this fungus so that it can be used in the future for biotechnological purposes.

Biochemical properties of an extracellular trehalase from Malbranchea pulchella var. Sulfurea

The thermophilic fungus Malbranchea pulchella var. sulfurea produced good amounts of extracellular trehalase activity when grown for long periods on starch, maltose or glucose as the main carbon source. Studies with young cultures suggested that the main role of the extracellular acid trehalase is utilizing trehalose as a carbon source. The specific activity of the purified enzyme in the presence of manganese (680 U/mg protein) was comparable to that of other thermophilic fungi enzymes, but many times higher than the values reported for trehalases from other microbial sources. The apparent molecular mass of the native enzyme was estimated to be 104 kDa by gel filtration and 52 kDa by SDS-PAGE, suggesting that the enzyme was composed by two subunits. The carbohydrate content of the purified enzyme was estimated to be 19 % and the pi was 3.5. The optimum pH and temperature were 5.0-5.5 and 55° C, respectively. The purified enzyme was stimulated by manganese and inhibited by calcium ions, and insensitive to ATP and ADP, and 1 mM silver ions. The apparent K(M) values for trehalose hydrolysis by the purified enzyme in the absence and presence of manganese chloride were 2.70 ± 0.29 and 2.58 ± 0.13 mM, respectively. Manganese ions affected only the apparent V(max), increasing the catalytic efficiency value by 9.2-fold. The results reported herein indicate that Malbranchea pulchella produces a trehalase with mixed biochemical properties, different from the conventional acid and neutral enzymes and also from trehalases from other thermophilic fungi.

DERMATOPHYTES, RELATED KERATINOPHILIC AND OPPORTUNISTIC FUNGI IN INDOOR DUST OF HOUSES AND HOSPITALS

Dermatophytes, related keratinophilic and opportunistic fungi were isolated from indoor dust samples of 46 hospitals and 47 houses in Kanpur. A total of 19 fungi represented by 11 genera were isolated by the hair-baiting technique from 230 and 235 samples from hospitals and houses respectively. The isolated fungi are Acremonium implicatum (Indian Type Culture Collection) ITCC 5266, A. strictum (Germplasm Centre for Keratinophilic Fungi) GPCK 1137, Aphanoascus fulvescens GPCK 1081, Arthroderma simii GPCK 1275, Chrysosporium queenslandicum ITCC 5270, C. indicum ITCC 5269, C. pannicola GPCK 1022, C. tropicum GPCK 1269, Ctenomyces serratus ITCC 5267, Gymnoascus reessii ITCC 5265, Malbranchea fulva GPCK 1075, Malbranchea pulchella ITCC 5268, Micosporum gypseum GPCK 1038, Microsporum cookei GPCK 2001, M. fulvum GPCK 2002, Paecilomyces lilacinum GPCK 1080, Penicillium expansum GPCK 1082, Trichophyton mentagrophytes GPCK 2003 and T. terrestre GPCK 2004. In hospitals, the minimum frequency was of Ctenomyces serratus ITCC 5267 while the maximum frequency was of Arthroderma simii GPCK 1275. In houses, Chrysosporium queenslandicum ITCC 5270 and C. tropicum GPCK 1269 were with minimum and maximum frequencies respectively. This makes the first report of these fungi with keratinolytic ability in the indoor dust of hospitals and houses.

Keratinophilic fungi on feathers of pigeon in Maharashtra, India.

Results of a preliminary survey of keratinophilic fungi associated with feathers of pigeon on high rise buildings in Thane district of Maharashtra (India) are reported. A total of 100 samples were examined, of which 67 samples were positive for keratinophilic fungi. Altogether 67 fungal strains belonging to 10 species of seven genera were isolated viz. Chrysosporium indicum (24%), Chrysosporium sp. (2%), Chr. tropicum (8%), Chrysosporium state of Arthroderma tuberculatum (3%), Chrysosporium state of Ctenomyces serratus (15%), Malbranchea pulchella (3%), Malbranchea sp. (1%), Microsporum gypseum (5%), Myriodontium keratinophilum (2%) and Trichophyton terrestre (4%).

Malbranchea pulchella fungal sinusitis

Fungal sinus disease is being reported with increasing frequency. We could find no previously reported cases of sinonasal infections with Malbranchea pulchella and only two previously reported cases of human infection of any kind. We now report on a case of suspected sinonasal Malbranchea pulchella in a patient with aspirin-sensitive triad asthma.

Saccharification of Leptochloa fusca (kallar grass straw) using thermostable cellulases

Saccharifying ability of thermostable cellulases on kallar grass straw at 50 and 60°C was evaluated. Enzyme filtrate from Chaetomium thermophile saccharified 5% kallar grass straw to 69% reducing sugars (quantitatively) at 50°C. Culture filtrates from all the thermophilic fungi showed a considerable saccharification rate up to 40 h. Freeze-dried enzyme of Trichoderma reesei VTT-D-79125 (mesophile), even at higher enzyme concentration resulted in 60% reducing sugars yield (quantitatively). Glucose concentration in the hydrolysates from the fungi was in the order of C. thermophile > T. reesei > Sporotrichum thermophile > Aspergillus fumigatus > Torula thermophila > Humicola grisea > Malbranchea pulchella. At 60°C, thermostable enzymes hydrolysed kallar grass straw at a maximum rate for the initial 20 h, after which the rate essentially stopped increasing. However, at elevated temperature C. thermophile enzymes saccharified kallar grass straw up to 20 h to almost the same yield in half the time as compared to that at 50°C. The overall decline in saccharification yield (11%) of C. thermophile enzymes was significantly lower than T. reesei enzymes (34%) at 60°C.

Thermophilic and thermotolerant fungi isolated from cultivated and desert soils, exposed continuously to cement dust particles in Egypt

Summary Thermophilic and thermotolerant fungi of forty soil samples collected from cultivated and desert soils, exposed continuously to cement dust particles were studied. Ten genera and 16 species in addition to two varieties of Aspergillus flavus and Malbranchia pulchella were recovered from all soil samples, on three types of media at 45 °C. From cultivated soils, 11 species and 2 varieties representing 7 genera were collected on yeast starch agar; on glucose- (3 genera and 3 species) and on cellulose-Czapek's agar (2 genera and 2 species). From desert soils, 12 species and 1 variety which belong to 8 genera were isolated on yeast starch agar and on both glucose- and cellulose Czapek's agar (1 genus and 1 species). In this investigation, the most dominant species was A. fumigatus . Truly thermophilic fungi were encountered and these were: Chaetomium thermophile, Malbranchea pulchella var. sulfurea, Rhizomucor pusillus, Myriococcum albomyces, Talaromyces thermophilus and Torula thermophila . Yeast starch agar was the best medium for isolation of thermophilic and thermotolerant fungi in the present study.

Fungal flora associated with combine harvester wheat and sorghum dusts from Egypt

107 species and 8 species varieties belonging to 44 genera were collected from combine harvester wheat and sorghum dusts (35 genera and 91 species + 4 varieties) and from the atmosphere of their hay sites (26 genera and 69 species + 4 varieties) on glucose- and cellulose-Czapek's Dox agar at 28 degrees C and 45 degrees C. The mycoflora of wheat and sorghum dusts were basically similar on the two types of media and the most common fungi were: Alternaria alternata, Aspergillus flavus, A. fumigatus, A. niger, A. ochraceus, A. sydowii, A. terreus, Cochliobolus spicifer, Emericella nidulans, Fusarium moniliforme, Penicillium chrysogenum, P. duclauxii, P. funiculosum and P. oxalicum. Truly thermophilic species were frequently encountered from the two substrates: Chaetomium thermophilum, Humicola grisea var. thermoidae, H. insolens, Malbranchea pulchella var., sulphurea, Rhizomucor pusillus, Sporotrichum thermophilum, Talaromyces thermophilus, Thermoascus thermophilus and Thermomyces lanuginosus. The airborne fungi in the two atmospheres were basically similar and the most prevalent species were members of Alternaria (1 species), Aspergillus (18 species + 2 varieties), Chaetomium (2 species), Cochliobolus(3 species), Emericella (3 species + 2 varieties), Fusarium (3 species), Mucor (1 species), Penicillium (14 species) and Stachybotrys (1 species).

Hydrolysis of para-substituted phenyl-.BETA.-D-xylosides by .BETA.-xylosidase from Malbranchea pulchella var. sulfurea No. 48.

The rates of hydrolysis of arylβ-D-xylosides (e.g. p-NO2-phenyl-, p-Cl-phenyl-, p-CH3-phenyl- and p-CH3O-phenyl-β-D-xylosides) by Malbranchea β-xylosidase were investigated. The Michaelis constants, Km, were almost independent of aglycone, whereas maximum velocity, Vmax, showed a marked dependence. In all hydrolysis reactions studied, the molecular activities, k0 (Vmax/e0, where e0 is the molar concentration of enzyme), were markedly increased by introducing both electron-withdrawing and electron-donating substituents in the para position of the phenyl ring. Between the values of log k0and inductive sigma constants, σ, V-shaped Hammett plots were obtained. When electron-withdrawing substituents were introduced, the Hammett reaction constant, p, was +1.27, when electron-donating substituents were introduced, its value was –2.07. On enzymic hydrolysis of p-nitrophenyl- and phenyl-β-D-xyloside, the reaction products, in both cases, were found to be α-D-xylose with inversion of configuration.

Lipoamide dehydrogenase from Malbranchea pulchella: isolation and characterization.

Lipoamide dehydrogenase (EC 1.6.4.3) has been isolated from a total homogenate of frozen mycelium of the thermophilic fungus Malbranchea pulchella var. sulfurea by a three-step procedure involving ammonium sulfate fractionation, Procion Brilliant Blue M-R--Sepharose 4B chromatography, and hydroxylapatite chromatography. The second step is the key purification step with the Procion Brilliant Blue M-R dye acting as an affinity ligand for the enzyme. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The enzyme is a dimer of molecular weight 102 000, and each monomer of 51 000 molecular weight binds one molecule of flavin adenine dinucleotide. Other properties determined include a pH optimum of 8.2, a strong specificity for the substrates dihydrolipoamide and nicotinamide adenine dinucleotide, the apparent lack of multiple enzymic forms, the presence of diaphorase activity, and resistance to temperature denaturation up to 60 degrees C. The amino acid composition and absorption spectrum of the enzyme were also determined. The properties of lipoamide dehydrogenase from this source are very similar to those reported for the enzyme from serveral other sources.

Cellulase activity, degradation of cellulose and lignin, and humus formation by thermophilic fungi

Several species of thermophilic fungi exhibited cellulase activity when grown on wheat straw but only Humicola insolens -1 and Aspergillus fumigatus exhibited significant cellulase activity when grown on cellulose powder. Humicola insolens -1 and 2 and Torula thermophila degraded cellulose and lignin and reduced weight of the inoculated material. H. lanuginosa, Mucor miehei and Talaromyces dupontii degraded lignin but not cellulose whereas A. fumigatus and Malbranchea pulchella degraded more cellulose and less lignin. Humus formation was greatest with A. fumigatus and H. insolens -1.

Amylase and cellulase activities of thermophilic fungi causing deterioration of tobacco products in Nigeria

The conditions necessary for the production and activities of extracellular enzymes of thermophilic fungi obtained from tobacco products were determined. While amylases were produced in extractable amounts by all the fungi used in this study, extracellular cellulases were produced only by Chaetomium thermophile La Touche, Humicola insolens Cooney and Emerson, Malbranchea pulchella Sacc. et Penzig var. sulfurea (Miehe) Cooney and Emerson, and Mucor pusillus Lindt. A narrow range of pH 6–7 was best for the activities of the amylases of all the fungi used while a lower range of pH 5–6 was optimal for their cellulase activities. Peak activities were recorded for both enzymes at a temperature range of 45 °–50 °C. The possible use of the exo-enzymes of these fungi in industrial fermentation processes is suggested.

Studies on xylanase system of thermophilic fungi. II. Purification and some properties of .BETA.-xylosidase from Malbranchea pulchella var. sulfurea No. 48.

A β-xylosidase of a thermophilic fungus, Malbranchea pulchella var. sulfurea No. 48, was purified 99-fold from the culture filtrate after ammonium sulfate fractionation, DEAE-cellulose column chromatography, column electrophoresis and gel filtration on Sephadex G–200. The purified enzyme was found to be homogeneous upon ultracentrifugal analysis, disc electrophoresis and gel filtration. The molecular weight of the enzyme was estimated to be 26,000 by gel filtration, and the sedimentation coefficient was calculated to be 2.78S. at 280 nm in phosphate buffer (pH 6.7) was 13.2. The optimum pH was found to be in the range of 6.2~6.8, and the optimum temperature was 50°C.