Moringa straw as cellulase production inducer and cellulolytic fungi source / Residuo de moringa como inductor de la producción de celulasas y como fuente de hongos celulolíticos

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.

Influence of iron and copper on the activity of laccases in Fusarium oxysporum f. sp. lycopersici

ABSTRACT Fusarium oxysporum f. sp. lycopersici is a phytopathogenic fungus that causes vascular wilt in tomato plants. In this work we analyze the influence of metal salts such as iron and copper sulphate, as well as that of bathophenanthrolinedisulfonic acid (iron chelator) and bathocuproinedisulfonic acid (copper chelator) on the activity of laccases in the intra (IF) and extracellular fractions (EF) of the wild-type and the non-pathogenic mutant strain (rho1::hyg) of F. oxysporum. The results show that laccase activity in the IF fraction of the wild and mutant strain increased with the addition of iron chelator (53.4 and 114.32%; respectively). With copper, it is observed that there is an inhibition of the activity with the addition of CuSO4 for the EF of the wild and mutant strain (reduction of 82 and 62.6%; respectively) and for the IF of the mutant strain (54.8%). With the copper chelator a less laccase activity in the IF of the mutant strain was observed (reduction of 53.9%). The results obtained suggest a different regulation of intracellular laccases in the mutant strain compared with the wild type in presence of CuSO4 and copper chelator which may be due to the mutation in the rho gene.

Negative correlation between phospholipase and esterase activity produced by Fusarium isolates

Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients’ blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified asFusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes.

Inoculum padronization for the production of cutinase by Fusarium oxysporum

Cutinase is a versatile enzyme showing several interesting properties for application in industrial processes. The widespread use of this enzyme depends on the development of an efficient and low-cost production system. One of the most important steps in a fermentation process is the standardization of the inoculum characteristics. In this study, the production of cutinase by Fusarium oxysporum showed a statistically significant relationship with both the inoculum size and the inoculum PDA pH. The greatest activities were 19.1 U/mL at PDA pH 7.0 and 22.72 U/mL using an aliquot of 12.72 x 10(7) spores/mL. The macroscopic characteristics of the colonies of Fusarium oxysporum changed according to the variation of the medium pH, with the best results recorded in those colonies presenting a cotton white aspect.

Cutinase é uma enzima versátil, que apresenta propriedades interessantes para aplicação em processos industriais. O uso desta enzima em larga escala depende do desenvolvimento de um sistema de produção eficiente e de baixo custo. Uma das etapas mais importantes em um processo de fermentação é a padronização do inóculo. Neste estudo, houve uma associação estatisticamente significativa entre a produção de cutinase por Fusarium oxysporum e tamanho do inóculo e pH do meio PDA. As maiores atividades de cutinase foram 19,1 U/mL em PDA com pH 7,0 e 22,72 U/mL empregando um inóculo de 12,72 x 10(7) esporos/mL. As características macroscópicas das colônias de Fusarium oxysporum mostraram alterações em função do pH do meio, com as maiores atividades sendo registradas em presença de colônias brancas com aspecto cotonoso.

Enzymatic characteristics of ligninperoxidases from Penicillium citrinum, Fusarium oxysporum and Aspergillus terreus using n-propanol as substrate.

The activities of ligninperoxidases from Penicillium citrinum MTCC 3565, Fusarium oxysporum MTCC 3379 and Aspergillus terreus MTCC 3374 have been assayed and the enzymatic characteristics like Km, pH and temperature optima using n-propanol as the substrate have been reported. The results suggest that n-propanol can substitute veratryl alcohol as substrate for assaying ligninperoxidase activities from different fungal strains, without affecting the enzymatic characteristics. The above strains were selected, as they were known to secrete ligninperoxidase in the liquid culture medium.

Secretion of ligninperoxidase by Penicillium citrinum, Fusarium oxysporum and Aspergillus terreus.

Secretion of ligninperoxidase [E.C.1.11.1.7] by Penicillium citrinum, Fusarium oxysporum and Aspergillus terreus in liquid culture growth medium has been demonstrated. Enzymatic characteristics like Km, pH and temperature optima using veratryl alcohol as the organic substrate of ligninperoxidases from above sources have been determined. Km values using veratryl alcohol as substrate for enzymes from P. citrinum, F. oxysporum and A. terreus were 69, 64 and 60 microM respectively. Km values using H2O2 as the variable substrate were 64, 72 and 80 microM.The pH optima were 4.0, 2.3 and 2.0 respectively. The values of temperature optima were 30 degrees, 25 degrees and 22 degrees C for the enzymes from P. citrinum, F. oxysporum and A. terreus respectively.

Production of extracellular lipase by the phytopathogenic fungus "Fusarium solani" FS1

A Brazilian strain of "Fusarium solani" was tested for extracellular lipase production in peptone-olive oil medium. The fungus produced 10,500 U.L(E-1) of lipase after 72 hours of cultivation at 25ºC in shake-flask at 120rpm in a medium containing 3(per cent) (w/v) peptone plus 0.5(per cent)(v/v) olive oil. Glucose (1(per cent) w/v) was found to inhibit the inductive effect of olive oil. Peptone concentrations below 3(per cent)(w/v) resulted in a reduced lipase production while increased olive oil concentration (above o.5(per cent)) did not further stimulate lipase production. The optimum lipase activity was achieved at pH 8.6 and 30ºC and a good enzyme stability (80(per cent) activity retention) was observed at pH ranging from 7.6 to 8.6, and the activity rapidly dropped at temperatures above 50ºC. Lipase activity was stimulated by addition of n-hexane to the culture medium supernatatnts, in contrast to incubation with water-soluble solvents

Podredumbre post-cosecha del zapallo tipo butternut (cucurbita moschata duch) actividad enzimática de fusarium spp / Post-harvest rots of butternut type pumpkin (cucurbita moschata duch): enzymatic activity of fusarium spp

El zapallo tipo butternut (c moschata) es el de mayor consumo en la Argentina. Durante el almacenamiento, está expuesto a podredumbres ocasionadas por distintas especies de Fusarium con diferente virulencia. Para establecer alguna correlación entre la patogenicidad y la actividad enzimática, con 22 cepas de dichas especies, se evaluó la producción "in vitro" de enzimas pectinolíticas y celulolíticas. Para la determinación se consideró la disminución de la viscosidad que produjo el filtrado de los hongos sobre sus respectivos sustratos. Se comprobó que todas las cepas produjeron ambos grupos de enzimas pero no se pudo establecer la correlación entre la capacidad patogenética de las mismas y su actividad enzimática

Effect of heavy metals and vitamins on urease production and activity by aspergillus fumigatus and fusarium nivale

The effect of some microelements and vitamins supplied A either individually or in mixtures to the culture media of Aspergillus fumigatus and Fusarium nivale on their growth and ureolytic activity was studied. Zinc and nikel activated growth and ureolysis either in culture media or reaction mixtures of both fungi. Fe[2+] and Se[2+] activated ureolysis in A. fumigatus only whereas Mo[2+] activated ureolysis in F. nivale. The sequence of activation could be arranged in A. Fumigatus as Fe[2+] [200 microg /I] > Se[2+] [50 microg / L]> Ni[2+] [100 microg/l]> Zn[2+][200 microg / 1]. In F. nivale Mo[2+] [50 microg/l]> Ni[2]+ [200 microg/l]> Zn[2+] [200 microg]l. Hg[2]+, Cd[2+], Co[2+] Fe[3+] and Cu[2+]markedly inhibited growth and ureolysis in both fungal species. Thiamine - HC1 [5 mg/1] and biotin [20 mg/1] activated growth and ureolysis of the tested species, while riboflavin, ascorbic acid and nicotinic acid were inhibitory. Mixture of best microelements and mixture of best vitamins retarded growth and ureolysis in both species, while a mixture containing microelements and vitamins activated urease and growth in both fungi as compared to all other treatments

L-serine dehydratase in some filamentous fungi I Studies on the formation of L-serine dehydratase by cunninghamella elegans and fusarium oxysporum

Cunninghamella elegans and Fusarium oxysporum were selected as the most potent producers of L-serine dehydratase among 16 different fungi. Of the tested metal salts, FeSO4 was the best inducer for L- serine dehydratase synthesis by C. Elegans and F. oxysporum. The enzyme was produced during the logarithmic phase of growth of the two organisms and maximum production was obtained after 3 days incubation. The optimal pH range for L-serine dehydratase formation in F. oxysporum was 4-5, whereas for C. Elegans enzyme pH 5.0 was the optimal. L-serine dehydratase of both organisms was induced with L- serine, ammonium carbonate, some amino acids and amides, but L-serine was the best induced. L-serine concentration of 2.4 gm/I was the optimal for L-serine dehydratase synthesis by both cultures. The effect of different carbon sources on enzyme formation and growth of the two organisms was investigated