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Whey is a highly polluting by-product of cheese processing. However, it has valuable nutritional properties since it is a rich and balanced source of proteins and amino acids. Therefore, it has a broad range of functional properties that can be exploited for diverse applications. Research has shown how the enzymatic hydrolysis of whey proteins releases bioactive peptides. In the present study, the hydrolysis of whey protein concentrate (WCP) was performed using purified Sporisorium reilianum aspartyl protease (Eap1), commercial enzymes chymotrypsin (C) and trypsin (T), as well as different enzymatic combinations in order to determine which enzyme or combination allowed for the release of peptides presenting the highest antioxidant and antihypertensive activity levels; our results indicated that hydrolysis with Eap1 releases the best-performing peptides in comparison with individual enzymes and their combinations.

The aim of this work was the isolation of endophytic cacti fungi producer of pigments. Twenty different endophytic fungi were isolated from Ferocactus latispinus using a standard method. One of these was an orange pigment producer and was identified with molecular biology techniques amplifying internal transcribe spacer region. The fungus identified was Fusarium solani; it is a producer of polyketides from naphthoquinones family. Experiments with different carbon and nitrogen sources were performed, and it was found that both glycerol and ammonia sulphate promote the naphthoquinones biosynthesis. It was also found that the carbon/nitrogen ratio and pH value had an effect on this biosynthesis phenomenon; increasing productivity being observed at the initial C: N ratio of 59.6, pH 6 and time of 113.3 h. This C: N ratio exhibited a stress condition for fungi in the pigment biosynthesis process in liquid media. The optimum value of total naphthoquinones (476 μmol/L) was obtained using a central rotable composite design.

Whey, a by-product of cheese processing, has important nutritional qualities, as it is a rich source of proteins balanced withamino acids. As such, it has a ...

Fungi provide a readily available alternative source of naturally derived pigmented molecules that may be utilized in different areas as foods, textile, medicine and cosmetics. These have been considered as potent biocompounds under two perspectives: for coloring to products, or as powerful chemical structures with biological functions. Both properties are also often found in their study and application in these areas. The present review presents an overview of diverse aspects that have been studied on the obtaining of fungal pigments, including pigment class, and metabolic pathway of biosynthesis, producer’s species of pigment, physicochemical condition of biosynthesis, extracting, isolation, purifying and storage. Commercial production and application of fungal pigments status is also described in this research as main findings.

Barley straw is a lignocellulosic biomass that can be used to obtain value-added products for industrial applications. Barley straw hydrolysis with sodium sulfite facilitates the production of lignosulfonates. In this work, the delignification process of barley straw by solubilizing lignin through sulfite method was studied. Response surface methodology and artificial neural network were used to develop predictive models for simulation and optimization of delignification process of barley straw. The influence of parameters over sulfite concentration (1.0 to 10.0%), particle size (8 to 20), and reaction time (30 to 90 min) on total percentage of solubilized material was investigated through a three level three factor (33) full factorial central composite design with the help of Matlab® ver. 8.1. The results show that particle size and sulfite concentration have the most significant effect on delignification process. Both techniques, response surface methodology and artificial neural networks, predicted the lignosulfonate yield adequately, although the artificial neural network technique produced a better fit (R2 = 0.9825) against the response surface methodology (R2 = 0.9290). Based on these findings, this study can be used as a guide to forecast the potential production of lignosulfonates from barley straw using different experimental conditions.

Depuration capacity of immobilized Phanerochaete chrysosporium was tested on food effluents which showed high COD, sugar, and nitrogen concentration. Comparative study in airlift reactor and agitated flasks was performed to determine an effective depuration treatment with suspended and immobilized biomass into alginate spheres. While enzymetic production was analyzed in these sytems in order to evaluate influence of the immobilization micro-organism, effluent composition, and aeration mechanism. In addition, the use of seed lettuce allowed evaluating the quality and effectiveness of the studied effluent treatment system. In particular, all tests showed that COD and color of the effluent was reduced by airlift cultures. Treatment achieved a decline in 85% of COD in 120 h. While experiments with 33 PCU color showed high efficiency (100%) in decolourization period of 5 h. Maximum laccase production was mainly found in airlift system with suspended biomass, whereas manganese peroxidase was detected on...

Traditional methods used for concentration of whey proteins have various levels of performance and effects on the nutritional properties and biological activities of the products. In this study, we showed that the greatest protein content was obtained using ultrafiltration and salt treatment methods. The effective concentration was approximately 40-53% (w/w) protein. Using electrophoresis and solubility tests, we also found that these methods offer the fundamental advantage of maintaining certain proteins in their native states. The products maintained key ABTS•+ radical scavenging activity; however, the antimicrobial activity was adversely affected by these separation methods.

Enzyme production by immobilized Phanerochaete chrysosporium was evaluated in airlift bioreactor and agitated cultures. Free mycelium and immobilized mycelium on alginate beads were tested in the decolourization of 50 and 500 mg/L of Remazol Brilliant Blue R. Dye concentration did not inhibit the fungi development in all tests. In addition, high decolourization percentage of dye was found with free mycelium (99%) in agitated flasks and with immobilized mycelium in airlift (98%). However, decolourization period by immobilized mycelium (120 h) was greater than that by the free mycelium (14 h). Important manganese peroxidase, lignine peroxidase and laccase activities were identified in decolourization process. Manganese peroxidase appeared to be promoted by high dye concentrations during the treatment with immobilized mycelium, but this enzyme was not detected with free mycelium in airlift. Bioreactor prompted also laccase and lignine peroxidase actions in both tests; free mycelium registered a maximum laccase action of 31.569 × 103 U/L in 70 h, whereas immobilized mycelium registered 1.680 × 103 U/L in 170 h, while lignine peroxidase secretion by free P. chrysosporium was higher (1.300 × 103 U/L) than immobilized mycelium (1.250 × 103 U/L). Maximum laccase activity coincided with the maximum percentage of decolourization, however, high peroxidase activity was identified from the start of dye treatment.

In this work, the extracellular protease Eap1 from Sporisorium reilianum was characterized in solid and liquid cultures using different culture media. The results showed that Eap1 was produced in all media and under all culture conditions, with the most activity in solid culture at an acidic pH of 3-5. Following purification, the 41 kDa protease demonstrated aspartyl protease activity. The enzyme was stable at a wide range of temperatures and pH values, but 45 C and pH 3 were optimal. The Km and Vmax values obtained were 0.69 mg/mL and 0.66 μmol/min, respectively, with albumin as the substrate. Eap1 degraded hemoglobin as well as proteins obtained from corn germ, roots, stems and slides at pH 3 and also had milk-clotting activity. Sequencing analysis showed that this protein has 100% similarity to the peptide sequence theoretically obtained from the sr11394 gene, which encodes an aspartyl protease secreted by S. reilianum. © 2013 Elsevier Ltd. All rights reserved., Fondo Mixto-Consejo Nacional de Ciencia y Tecnología; the government of the state of Hidalgo (FOMIXCONACyT); Fondo de Ciencia Básica SEP-CONACyT

The State of Hidalgo (Mexico) has a large area of forests known as the Huasteca Hidalguense, with a large variety of microorganisms inhabiting it. They represent an important resource from the ecological and technological point of view because they can be used in a broad variety of industrial processes. Due to the climatic conditions of this region, fungi inhabiting it must be thermophile or, at least, thermotolerant, as temperatures can be higher than 45°C in the summer, declining to 20°C in the winter. Use of ligninolytic fungi relies on their capacity to produce enzymes of industrial interest, a topic that has been under continuous research by academic and industrial investigators. Among the most important enzymes are proteases that are widely used due to their biotechnological applications with a high economic impact. Other enzymes, laccases, peroxidases, and lipases are of interest for the industries of the state of Hidalgo, especially in the textile industry, specifically in effluent processing. Fungi (n=156) were collected in the Huasteca Hidalguense, of which 100 were isolated in potato-dextrose-agar covered plates and maintained in tilted tubes. Afterwards, enzymatic activity (laccase, protease and lipase) was determined in the plates. The purpose was to select those fungi with the highest potential for biotechnological applications. Fungi generally grew at either 30°C or 37°C, and for some isolates enzymatic activities were detected at this higher temperature. Results are presented as the relation between enzymatic activity and growth rate: 60 fungi presented laccase activity, 49 had lipase activity, and none had protease activity. In most cases, enzymatic activity was higher than the growth rate, indicating that the isolated fungi have a great biotechnological potential. Statistical analysis revealed that isolates 31 (Trametes) and 8.1 (unidentified) have a larger potential to be studied as laccase-producing fungi. On the other hand, isolates 144.2 (Fomes), 154 (Trametes), and 147.2 (Pycnoporus) are of interest as lipase activity producers, an activity scarcely studied in this type of microorganisms.

Straw is an agricultural byproduct that can be utilized to obtain bioethanol without affecting animal or human sustinence. This process involves recovering the sugars and reducing the lignin content present through the use of ligninolytic fungi such as the basidiomycete Trametes sp. 44. Fermentation was carried out using particle sizes 4 (4.76 mm, No. 4 sieve) and 8 (2.30 mm, No. 8 sieve), and two velocities of airflow (100 and 200 mL/min). Study results showed that particle size affected the production of hydrolytic enzymes, as particle size 8 favored the expression of cellulases and hemicellulases. In addition, both aeration and particle size affected the expression of ligninolytic enzymes, as it was observed that with particle size 8 and airflow of 200 mL/min, the study detected 63 AU/mL of LiP and 11 AU/mL of MnP. In the case of laccase, the enzymatic activity detected reached 220 AU/mL using particle size 8 and an airflow velocity of 200 mL/min. Statistical analysis indicated that the treatment that produced the highest biological delignification occurred when Trametes sp. 44 was grown on corn straw at particle size 4 and airflow of 100 mL/min, conditions that yielded 34% delignification at day 12 of fermentation.

The extracellular protease APSm1 was purified to homogeneity from Stenocarpella maydis that was grown in acidic minimal media with glucose and ammonium sulfate. The purification procedure consisted of ion exchange chromatography coupled to an FPLC (Fast Protein Liquid Chromatography) system, resulting in a 15.3% recovery and a 2.3-fold increase in specific activity. The molecular weight of the purified enzyme was estimated to be 56.8 kDa by SDS-PAGE. Enzymatic activity toward hemoglobin was optimal at pH 2.0 and at 25 °C. The effects of six protease inhibitors on APSm1 activity were tested. Pepstatin A inhibited APSm1 activity, as the protein is in fact an aspartyl protease. The pure enzyme degraded hemoglobin, albumin and proteins obtained from corn germ at pH 3 but did not have any milk-clotting activities. The Km and Vmax values obtained were 0.514 mg/mL and 0.222 μmol/min, respectively, using hemoglobin as the substrate. This work is the first to report the purification of a secreted aspartyl protease from S. maydis.

Laccase gene IV from Trametes versicolor was inserted by REMI in Aspergillus niger C28B25. One selected transformant (C28eco3) was subjected to a second round of transformation and a new transformant was isolated (C28eco3–13). Laccase production was compared for these strains in submerged fermentation (SmF) and solid-state fermentation (SSF) using glucose or maltodextrin at different concentrations (10, 50 and 100 g/L). Enzymes yields were higher with glucose as a carbon source. However, repression of laccase was evident for SmF after increasing glucose from 10 to 50 g/L. In contrast, in SSF glucose at 50 g/L allowed the production of the highest laccase activity for strain C28eco3 (362 U/L) and C28eco3–13 (592 U/L). Advantages of SSF as an expression and production system over SmF are also discussed.

Nine phenolic compounds (p-coumaric acid, ferulic acid, guaiacol, syringol, p-methoxyphenol, pyrocatechol, phloroglucinol, 3,5-dihydroxybenzoic acid, and syringaldazine) were tested for their ability to increase laccase production in the ligninolytic basidiomycete Trametes sp. I-62. All these compounds resulted in increases in laccase activity, with the highest levels being detected in the presence of p-coumaric acid (273-fold) and guaiacol (73-fold). The three laccase isozyme genes in this fungus lcc1, lcc2, and lcc3 are differentially expressed in the presence of some of these aromatics with total lcc transcript levels differing markedly depending on the aromatic compound tested. Guaiacol (the best inducer of lcc gene transcription) and p-coumaric acid selectively induced expression of lcc1 and lcc2; ferulic acid induced lcc3 expression, while 3,5-dihydroxybenzoic acid had no marked effect on laccase gene transcription. The results demonstrate that close-related aromatic compounds appear to have different effects on both laccase activity levels and lcc gene expression in this basidiomycete. © 2004 Elsevier Inc. All rights reserved.

Laccase enzymatic activity in biological samples is usually detected spectrophotometrically through its capacity to oxidize several specific aromatic compounds. One of the most commonly used substrates is the compound 2-2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), which becomes green-blue coloured when it is oxidized by laccase. In this work we study the interference of tannic acid with the spectrophotometric assay to detect laccase by using ABTS as the substrate. Our data show that under the normal reaction conditions of this assay, but in the absence of any catalyst, tannic acid is able to carry out the chemical reduction of the oxidized specie of ABTS, thus decreasing the overall detectable laccase-activity values observed when this enzyme is present in the reaction mixture. Therefore, our results represent an important warning concerning a commonly used method for measuring, detecting or screening laccases in biological samples that may content tannic acid or structural-related molecules.

A hybrid glucose microfluidic fuel cell composed of an enzymatic cathode (Laccase/ABTS/C) and an inorganic anode (AuAg/C) was developed and tested. The enzymatic cathode was prepared by adsorption of 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and Laccase on Vulcan XC-72, which act as a redox mediator, enzymatic catalyst and support, respectively. The Laccase/ABTS/C composite was characterised by Fourier Transform Infrared (FTIR) Spectroscopy, streaming current measurements (Zeta potential) and cyclic voltammetry. The AuAg/C anode catalyst was characterised by Transmission electron microscopy (TEM) and cyclic voltammetry. The hybrid microfluidic fuel cell exhibited excellent performance with a maximum power density value (i.e., 0.45 mW cm−2) that is the highest reported to date. The cell also exhibited acceptable stability over the course of several days. In addition, a Mexican endemic Laccase was used as the biocathode electrode and evaluated in the hybrid microfluidic fuel cell generating 0.5 mW cm−2 of maximum power density.

Sporisorium reilianum is a phytopathogenic fungus that produces the maize head smut. Hydrolytic enzymes such as xylanases have not been studied in this basidiomycete, although these enzymes are widely used within the industry. In this study, the characterization of a xylanase produced by S. reilianum grown in submerged and solid-state fermentation using different culture media was performed. Submerged fermentation with a medium containing birch xylan and yeast extract showed the highest xylanase activity (12.6 U/mL). The enzyme, purified through ion exchange chromatography, had a molecular weight of 42 kDa, as determined by SDS-PAGE, and a Km and Vmax of 1.72 mg/mL and 2.48 μmol/mL/min, respectively. The optimal values of pH and temperature were 5.0 and 70 °C, respectively. The enzyme showed activity over a broad range of temperatures and pHs. Zn2+, Fe3+, and Mn2+ ions increased xylanase activity. Sequence protein analysis showed 100% similarity with the theoretical protein encoded by the sr14403 gene of S. reilianum, encoding a putative endo-Beta-1,4-xylanase. This is the first report on the production and purification of a xylanase from this fungus, which has interesting biochemical characteristics for application in biotechnological processes.

Stenocarpella maydis and Stenocarpella macrospora are the causal agents of ear rot in corn, which is one of the most destructive diseases in this crop worldwide. These fungi are important mycotoxin producers that cause different pathologies in farmed animals and represent an important risk for humans. In this work, 160 strains were isolated from soil of corn crops of which 10 showed antifungal activity against these phytopathogens, which, were identified as: Bacillus subtilis, Pseudomonas spp., Pseudomonas fluorescens, and Pantoea agglomerans by sequencing of 16S rRNA gene and the phylogenetic analysis. From cultures of each strain, extracellular filtrates were obtained and assayed to determine antifungal activity. The best filtrates were obtained in the stationary phase of B. subtilis cultures that were stable to the temperature and extreme pH values; in addition they did not show a cytotoxicity effect against brine shrimp and inhibited germination of conidia. The bacteria described in this work have the potential to be used in the control of white ear rot disease.

Cellulose pulp bleaching is one of the main biotechnological applications of fungal laccases due to their capacity to degrade lignin from the unbleached pulp. For this application, not only a low cost enzyme production, but also higher enzyme concentrations than those obtained by its natural producers are needed. Heterologous expression of laccase in yeasts is an option for producing these enzymes at industrial scale. In this work the heterologous expression of the cglcc1 gene, responsible for laccase production in the basidiomicetous fungus Coriolopsis gallica, in the yeast Kluyveromyces lactis has been tested. In order to know if the transformed yeast obtains delignificant capability, a Pinus radiata kraft pulp has been incubated with it. After the treatment, an important decrease in kappa number (13%) and in lignin content (22%) has been observed. These results showed the delignificant capacity of this transformed yeast. It can be concluded that the employment of genetically modified microorganisms, without cellulolitic activity, to produce high laccase levels could be a feasible option to delignify cellulose pulps with a potential application in cellulose pulp bleaching., Una posible aplicación biotecnológica de las lacasas fúngicas es el blanqueo de pasta de papel mediante la degradación de la lignina en la pasta cruda. Para ello es necesaria su producción a bajo coste y a concentraciones superiores a las secretadas por los hongos de forma natural. La expresión heteróloga en levaduras es una alternativa para su producción a mayor escala. En el presente trabajo se ha realizado la expresión heteróloga del gen cglcc1, responsable de la producción de lacasa en el hongo basidiomiceto Coriolopsis gallica, en la levadura Kluyveromyces lactis. Posteriormente y para verificar si la capacidad deslignificante se mantiene en la levadura, se ha tratado una pasta kraft de Pinus radiata con la cepa recombinante. Tras el tratamiento, el contenido en lignina disminuyó en un 22% (aunque el número kappa solo disminuyó en un 13%, posiblemente por interferencias con el microorganismo), corroborando así el efecto deslignificador. Se concluye que el empleo de microorganismos carentes de actividades celulolíticas y genéticamente modificados para producir altos niveles de actividad lacasa constituye una opción para deslignificar aplicable a procesos de blanqueo de pasta de celulosa.

Wastewaters generated from the production of ethanol from sugar cane molasses may have detrimental effects on the environment due to their high chemical oxygen demand and dark brown color. The color is mainly associated with the presence of melanoidins, which are highly recalcitrant to biodegradation. We report here the induction of laccases by molasses wastewaters and molasses melanoidins in the basidiomycetous fungus Trametes sp. I-62. The time course of effluent decolorization and laccase activity in the culture supernatant of the fungus were correlated. The expression of laccase genes lcc1 and lcc2 increased as a result of the addition of complete molasses wastewater and its high molecular weight fraction to fungal cultures. This is the first time differential laccase gene expression has been reported to occur upon exposure of fungal cultures to molasses wastewaters and their melanoidins. © 2007 Elsevier Masson SAS. All rights reserved.

This work investigated the identification and selection of fungi isolated from sugarcane bagasse and their application for phenanthrene (Phe) removal from soil. Fungi were identified by PCR amplification of ITS regions as Aspergillus terrus, Aspergillus fumigatus and Aspergillus niger, Penicillium glabrum and Cladosporium cladosporioides. A primary selection of fungi was accomplished in plate, considering Phe tolerance of every strain in two different media: potato dextrose agar (PDA) and mineral medium (MM). The radial extension rate (r(r)) in PDA exhibited significant differences (p

The study of isozymes has to date been successfully applied in the identification of a number of different microorganisms, particularly fungi. Trametes sp. I-62 (CECT20197) is a white-rot fungus with potential biotechnological applications. It has been identified as Trametes on the basis of the mycelium morphology, but the features observed are not sufficient for its classification at the species level. Given that this strain produces a very characteristic laccase isozyme pattern, in the present work we compared its isozyme profile with the profiles produced by ten other very closely phylogenetically related fungi from the Polyporaceae family. The data indicates that Trametes sp. I-62 could not be included in any of the taxonomically related species which were analysed in the present work. Extracellular laccases were detected in nine of the fungi studied and in four of them namely Trametes subectypus, Trametes pavonia, Trametes ochracea and Trametes gallica, this is the first report of such activity. Given that phylogenetically related fungi growing in the same culture conditions exhibit different laccase patterns, these isozymes could be used as an additional criterion for fungal identification. (© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

A biocathode based on the physical absorption on carbon surfaces of crude extract with laccase activity (ExLac) obtained of C. polyzona, a Mexican endemic fungus that uses barley straw as substrate, is prepared and evaluated toward oxygen reduction reaction. The biocathode electrode consists in a mixture of ExLac with 10 mM ABTS {2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)} diammonium salt and vulcan XC-72. Electrochemical evaluation of the biocathode consisted of cyclic voltammetry CV and linear sweep voltammetry in a standard three-electrode glass cell containing a neutral aqueous media phosphate buffer solution (pH 7.4 near to blood pH) in the absence and presence of oxygen. The biocathode working electrode is constructed as follows: an ink of ExLac-ABTS/Vulcan is prepared by dispersion in deionized water and 5% Nafion solution in a ratio of 1:56:5 in a vortex and subsequently deposited onto a PVC chip and dried at room temperature. The results showed that the biocathode catalyzes the oxygen reduction reaction better than the platinum in a neutral media in glucose presence. Further, the electrocatalyst activity of ExLac-ABTS/C is compared with that achieved by using of a biocathode prepared with commercial laccase. One of the advantages of this type of bioelectrode array is the utilization of barley straw as a substrate in the crude extract production which is an inexpensive material that may be a good alternative for great-scale production.