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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.

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Abstract Currently, the valorization of agroindustrial waste is of great interest. Moringa oleifera is a multipurpose tree whose softwood residues could be used as raw material for low-cost cellulase production. The aim of this study was to isolate, identify, and characterize microorganisms with cellulolytic activity in different carbon sources. We isolated and puri-fied 42 microorganisms from M. oleifera biomass. Fungi presenting the largest hydrolytic halos in carboxymethylcellulose as a substrate were molecularly identified as Penicillium funiculosum (FG1), Fusarium verticillioides (FG3) and Cladosporium cladosporioides (FC2). The ability of these fungal strains to break down cellulose was assessed in a submerged fermentation using either amorphous CMC or crystalline form (Avicel). P. funiculosum and C. cladosporioides displayed similar endoglucanase (606 U/l) and exoglucanase (205 U/l) activities in the Avicel-containing medium, whereas F. verticillioides showed the highest level of p-glucosidase activity (664 U/l) in the carboxymethylcellulose medium. In addition, the effect of three culture media (A, B, and C) on cellulase production was evaluated in P. funiculosum using moringa straw as a carbon source. The results showed a volumetric productivity improvement of cellulases that was 2.77-, 8.26-, and 2.30-fold higher for endoglucanase, exoglucanase and p-glucosidase, respectively when medium C containing moringa straw was used as a carbon source. The enzymatic extracts produced by these fungi have biotechnological potential especially for second-generation bioethanol production (2G) from moringa straw. This is the first report on the use of M. oleifera biomass to induce the production of various cellulases in P. funiculosum. © 2019 Asociación Argentina de Microbiología. Published by Elsevier Espana, S.L.U. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Resumen Actualmente, la valorización de los residuos agroindustriales es de gran interés. En este trabajo se emplearon residuos de madera blanda de Moringa oleifera para la producción de celulasas de bajo costo. El objetivo fue aislar, identificar y caracterizar microorganismos con actividad celulolítica en diferentes fuentes de carbono. A partir de la biomasa de M. oleifera, se aislaron e identificaron 42 microorganismos productores de celulasas. Los hongos que presentaron los mayores halos de hidrólisis en carboximetilcelulosa como sustrato fueron identificados molecularmente como Penicillium funiculosum (FG1), Fusarium verticillioides (FG3) y Cladosporium cladosporioides (FC2). Mediante fermentación sumergida, se evaluó la capacidad de estas cepas en la producción de celulasas utilizando celulosa cristalina (Avicel) y amorfa (CMC) como fuentes de carbono. P. funiculosum y C. cladosporioides presentaron las mayores actividades de endoglucanasa (606 U/l) y exoglucanasa (205 U/l) en medio Avicel, mientras que F. verticillioides mostró la mayor actividad de p-glucosidasa (664 U/l) en medio CMC. Además, se evaluó el efecto de tres medios de cultivo (A, B y C) sobre la producción de celulasas en P. funiculosum empleando residuos de moringa como fuente de carbono. Los resultados mostraron que en el medio C, la productividad volumétrica de celulasas se incrementó en 2,77; 8,26 y 2,30 veces para las actividades de endoglucanasa, exoglucanasa y p-glucosidasa, respectivamente. Los extractos enzimáticos producidos tienen gran potencial para su utilización biotecnológica, especialmente en la sacarificación de residuos de moringa y la producción de bioetanol de segunda generación. Este es el primer estudio del uso de la biomasa de M. oleifera para inducir la producción de diversas celulasas en P. funiculosum.

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Currently, the valorization of agroindustrial waste is of great interest. Moringa oleifera is a multipurpose tree whose softwood residues could be used as raw material for low-cost cellulase production. The aim of this study was to isolate, identify, and characterize microorganisms with cellulolytic activity in different carbon sources. We isolated and purified 42 microorganisms from M. oleifera biomass. Fungi presenting the largest hydrolytic halos in carboxymethylcellulose as a substrate were molecularly identified as Penicillium funiculosum (FG1), Fusarium verticillioides (FG3) and Cladosporium cladosporioides (FC2). The ability of these fungal strains to break down cellulose was assessed in a submerged fermentation using either amorphous CMC or crystalline form (Avicel). P. funiculosum and C. cladosporioides displayed similar endoglucanase (606U/l) and exoglucanase (205U/l) activities in the Avicel-containing medium, whereas F. verticillioides showed the highest level of ß-glucosidase activity (664U/l) in the carboxymethylcellulose medium. In addition, the effect of three culture media (A, B, and C) on cellulase production was evaluated in P. funiculosum using moringa straw as a carbon source. The results showed a volumetric productivity improvement of cellulases that was 2.77-, 8.26-, and 2.30-fold higher for endoglucanase, exoglucanase and ß-glucosidase, respectively when medium C containing moringa straw was used as a carbon source. The enzymatic extracts produced by these fungi have biotechnological potential especially for second-generation bioethanol production (2G) from moringa straw. This is the first report on the use of M. oleifera biomass to induce the production of various cellulases in P. funiculosum.

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Numerous endoxylanases from mesophilic fungi have been purified and characterized. However, endoxylanases from cold-adapted fungi, especially those from Antarctica, have been less studied. In this work, a cDNA from the Antarctic fungus Cladosporium sp. with similarity to endoxylanases from glycosyl hydrolase family 10, was cloned and expressed in Pichia pastoris. The pure recombinant enzyme (named XynA) showed optimal activity on xylan at 50 °C and pH 6-7. The enzyme releases xylooligosaccharides but not xylose, indicating that XynA is a classical endoxylanase. The enzyme was most active on xylans with high content of arabinose (rye arabinoylan and wheat arabinoxylan) than on xylans with low content of arabinose (oat spelts xylan, birchwood xylan and beechwood xylan). Finally, XynA showed a very low thermostability. After 20-30 min of incubation at 40 °C, the enzyme was completely inactivated, suggesting that XynA would be the most thermolabile endoxylanase described so far in filamentous fungi. This is one of the few reports describing the heterologous expression and characterization of a xylanase from a fungus isolated from Antarctica.

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Cladosporium cladosporioides is a dematiaceous fungus with coloured mycelia and conidia due to the presence of dark pigments. The purpose of this study was to characterize the dark pigments synthetized by Cladosporium sp. LPSC no. 1088 and also to identify the putative polyketide synthase (pks) gene that might be involved in the pigment biosynthesis. Morphological as well as molecular features like the ITS sequence confirmed that LPSC 1088 is Cladosporium cladosporioides. UV-visible, Fourier Transform Infrared (FTIR) and Electron Spin Resonance (ESR) spectroscopy analysis as well as melanin inhibitors suggest that the main dark pigment of the isolate was 1,8 dihydroxynaphthalene (DHN)-melanin-type compound. Two commercial fungicides, Difenoconazole and Chlorothalonil, inhibited fungal growth as well as increased pigmentation of the colonies suggesting that melanin might protect the fungus against chemical stress. The pigment is most probably synthetized by means of a pentaketide pathway since the sequence of a 651 bp fragment, coding for a putative polyketide synthase, is highly homologous to pks sequences from other fungi.

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The enzyme known as invertase (E.C. 3.2.1.26 - beta-D-fructofuranosidase) catalyzes the sucrose hydrolysis producing an equimolar mixture of glucose and fructose named inverted sugar. The fungus Cladosporium cladosporioides has invertase as its constituent. Hence, its use as a natural immobilized support for the invertase produces interesting results for the enzyme. The present work has the objective of determining the optimum operational conditions of auto-immobilized invertase, as well as its kinetic parameters (K M and Vmax). A complete 2³ factorial planning was done for the evaluation of such parameters. Temperature, pH and agitation level were the studied variables. The hydrolysis percentage was the monitored result. Batch tests in optimum conditions were done to determine the kinetic parameters. Temperature of 70ºC, pH 6 and agitation of 170 rpm were the established conditions for the hydrolysis process. The auto-immobilized invertase presented a K M of 447 mM and Vmax of 2,805 mmol/min.

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Thermostable invertase (E.C. 3.2.1.26) and inulinase 2,1-beta-D-fructan fructanohydrolase (E.C. 3.2.1.7) activities were produced by Cladosporium cladosporioides grown on sucrose, inulin, yam extract, or Jerusalem artichoke. The ratio I (inulinase)/S(invertase) activity was between 0.31 and 0.36. Both activities had high temperature optima (60 degrees C) and were stable during pretreatment for 4.5 h at this temperature. Whole cells of C. cladosporioides were used for batch fructose production from Jerusalem artichoke extract at several concentrations. With the highest extract concentration used (260 g total sugars/L), total hydrolysis was achieved in 150 min at 60 degrees C. Thin-layer chromatography of the enzymatic hydrolysis of inulin and Jerusalem artichoke extract showed that from the beginning of the reaction, fructose was the only product released. This suggests an exoaction mechanism, beta-D-fructofuranoside fructohydrolase [E.C. 3.2.1.2.6].

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Spherical and osmotically sensitive protoplasts were released from cultures of the yeast-like form of Paracoccidioides basilienisis strain IVIC Pb9 through the action of a mixture of crude enzyme preparations: alpha and beta-glucanases and chitinase, obtained from culture filtrates of Cladosporium resinae, Basidiomycete QM 806 and Streptomyces sp respectively. The highest efficiency of protoplast liberation was achieved when each crude enzyme preparation was used at 1 mg/ml.

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