Characterization of the Solid-State and Liquid Fermentation for the Production of Laccases of Pleurotus ostreatus

In this chapter, the activity and isoenzymes number of laccases of Pleurotus ostreatus grown in solid-state and liquid fermentations are reported. An atypical behavior of this fungus with relation on enzyme production was observed, since the major laccase activity levels were observed in liquid fermentation, whereas the solid-state fermentation has been recognized as better system for enzyme production

Bioinformatics as a Tool for the Structural and Evolutionary Analysis of Proteins

This chapter deals with the topic of bioinformatics, computational, mathematics, and statistics tools applied to biology, essential for the analysis and characterization of biological molecules, in particular proteins, which play an important role in all cellular and evolutionary processes of the organisms. In recent decades, with the next generation sequencing technologies and bioinformatics, it has facilitated the collection and analysis of a large amount of genomic, transcriptomic, proteomic, and metabolomic data from different organisms that have allowed predictions on the regulation of expression, transcription, translation, structure, and mechanisms of action of proteins as well as homology, mutations, and evolutionary processes that generate structural and functional changes over time. Although the information in the databases is greater every day, all bioinformatics tools continue to be constantly modified to improve performance that leads to more accurate predictions regarding protein functionality, which is why bioinformatics research remains a great challenge

In silico Design of Laccase Thermostable Mutants From Lacc 6 of Pleurotus Ostreatus

Fungal laccase enzymes have a great biotechnological potential for bioremediation processes due to their ability to degrade compounds such as ρ-diphenol, aminophenols, polyphenols, polyamines, and aryldiamines. These enzymes have activity at different pH and temperature values, however, high temperatures can cause partial or total loss of enzymatic activity, so it is appropriate to do research to modify their secondary and/or tertiary structure to make them more resistant to extreme temperature conditions. In silico, a structure of the Lacc 6 enzyme of Pleurotus ostreatus was constructed using a laccase of Trametes versicolor as a template. From this structure, 16 mutants with possible resistance at high temperature due to ionic interactions, salt bridges and disulfide bonds were also obtained in silico. It was determined that 12 mutants called 4-DB, 3-DB, D233C-T310C, F468P, 3-SB, L132T, N79D, N372D, P203C, P203V, T147E, and W85F, presented the lowest thermodynamic energy. Based on the previous criterion and determining the least flexibility in the protein structures, three mutants (4-DB, 3-DB, and P203C) were selected, which may present high stability at high temperatures without affecting their active site. The obtained results allow the understanding of the molecular base that increase the structural stability of the enzyme Lacc 6 of Pleurotus ostreatus, achieving the in silico generation of mutants, which could have activity at high temperatures

Effect of textile dyes on activity and differential regulation of laccase genes from Pleurotus ostreatus grown in submerged fermentation

Additional file 1: Figure S1. Growth of P. ostreatus and pH profile in submerged fermentations in BMF (●black circle), BBF (■ black square) and AYF (♦black diamond) media. The error bars represent the standard deviation of three different fermentation runs. Figure S2. Genorm analysis of the expression stability of 4 reference genes. Figure S3. Variability of Cp values of 4 reference genes tested under the 3 different fermentation conditions using NormFinder. Table S1. Identifiers and product lengths of reference genes primers used in this study

GROWTH OF THE EDIBLE MUSHROOM PLEUROTUS OSTREATUS ON DIFFERENT CONCENTRATIONS OF DI (2-ETHYL HEXYL) PHTHALATE IN SOLID AND IN LIQUID MEDIA

ABSTRACT Di (2-ethylhexyl) phthalate (DEHP) is one of the most widely used plasticizers, giving flexibility to the plastics. Specific growth rate (µ), maximum biomass (X max ), laccase and esterase activities, pH profiles and enzymatic kinetic parameters were evaluated in Pleurotus ostreatus grown in DEHP in flasks. Radial growth rate (u r ), laccase and esterase activities and mycelial biomass (X) of P. ostreatus grown in agar plates containing DEHP were also evaluated. Flasks of 125 ml and agar plates containing 0, 750, 1200 and 1500 mg of DEHP/l were used. All media were added with 10 g of glucose/l. Flasks containing 50 ml culture medium were inoculated and incubated at 25 o C for 16 days on a rotary shaker (120 rpm). Petri dishes were inoculated and incubated at 25 o C for 7 days. X max , µ, and u r were evaluated using the logistic and lineal equations, respectively. X was determined by dry weight method. Laccase and esterase activities were evaluated using 2, 6-dimethoxyphenol and p-nitrophenyl butyrate as substrates, respectively. The highest X max was observed in media containing 1500 mg of DEHP/l and the esterase activity was much higher than the laccase activity at the beginning of the stationary phase in medium containing 1000 mg of DEHP/l in flasks. In agar plates, the laccase activity was higher than the esterase activity in all the media containing DEHP. These results suggest that there was no catabolite repression (glucose effect) and that DEHP was used as carbon and energy source by this fungus

Enzymatic, Antioxidant, Antimicrobial, and Insecticidal Activities of Pleurotus pulmonarius and Pycnoporus cinnabarinus Grown Separately in an Airlift Reactor

Crude extract samples of Pleurotus pulmonarius and Pycnoporus cinnabarinus were taken during growth in liquid broth in an airlift reactor. Growth was monitored indirectly by sugar consumption and pH profile. During growth Pleurotus pulmonarius consumed glucose more slowly than Pycnoporus cinnabarinus, reaching a final pH of 8.0. In contrast, Pycnoporus cinnabarinus started consuming glucose faster from the beginning to the end with a pH of 3.6, suggesting the production of different metabolites while they grow in the same culture broth. Additionally, antioxidant activity, polyphenol and flavonoid contents, as well as laccase and hydrolase activities were quantified in the culture extracts during the fermentation. Pleurotus pulmonarius showed higher antioxidant activity than Pycnoporus cinnabarinus. Both fungi have a very low polyphenol and flavonoid content. Values of amylase and pectinase activities were similar in crude extracts of both fungi; however, cellulase, xylanase, invertase, and laccase activities showed higher levels in crude extract of Pleurotus pulmonarius. Antimicrobial and insecticidal activities were also evaluated in each crude extract. In fact, Pycnoporus cinnabarinus presented a very strong bacteriostatic and bactericidal effect against Escherichia coli and Staphylococcus aureus and reliably killed Diatraea magnifactella larvae, while Pleurotus pulmonarius did not showed any negative effect on the growth of these bacteria or larvae

Integral use of amaranth starch to obtain cyclodextrin glycosyltransferase, by Bacillus megaterium, to produce β-cyclodextrin.

Cyclodextrin glycosyltransferase (CGTase) is an enzyme that produces cyclodextrins (CDs) from starch and related carbohydrates, producing a mixture of α-, β-, and γ-CDs in different amounts. CGTase production, mainly by Bacillus sp., depends on fermentation conditions such as pH, temperature, concentration of nutrients, carbon and nitrogen sources, among others. Bacillus megaterium CGTase produces those three types of CDs, however, β-CD should prevail. Although waxy corn starch (CS) is used industrially to obtain CGTase and CDs because of its high amylopectin content, alternative sources such as amaranth starch (AS) could be used to accomplish those purposes. AS has high susceptibility to the amylolytic activity of CGTase because of its 80% amylopectin content. Therefore, the aim of this work was evaluate the AS as carbon source for CGTase production by B. megaterium in a submerged fermentation. Afterwards, the CGTase was purified partially and its activity to synthesize α-, β- and γ-CDs was evaluated using 1% AS as substrate. B. megaterium produced a 66 kDa CGTase (Topt=50°C; pHopt=8.0), from the early exponential growth phase which lasted 36 h. The maximum CGTase specific activity (106.62±8.33 U/mg protein) was obtained after 36 h of culture. CGTase obtained with a Km=0.152 mM and a Vmax=13.4 µM/min yielded 40.47% total CDs using AS which was roughly twice as much as that of corn starch (CS; 24.48%). High costs to produce CDs in the pharmaceutical and food industries might be reduced by using AS because of its higher α-, β- and γ-CDs production (12.81%, 17.94% and 9.92%, respectively) in a shorter time than that needed for CS

Inhibición micelial de Trichoderma spp. (Hypocreaceae) aislado del cultivo de Pleurotus ostreatus (Pleurotaceae) con un extracto de Pycnoporus sp. (Polyporaceae)

Background and Aims: The production of edible fungi is affected by bacterial, fungal and viral diseases, which very often cause large losses. In the production of mushrooms of the genus Pleurotus, the fungi of Trichoderma spp. represent a serious problem of contamination and although there are some chemical com-pounds that control the infection, they are not entirely safe for human consumption. As a consequence, alternatives are being searched for through biotechnol-ogy, such as the one presented in this paper.Methods: Strains of fungi of the genus Trichoderma were isolated from the substrate where Pleurotus ostreatus was being cultivated. These were identified morphologically and molecularly, followed by tests to inhibit the growth of Trichoderma strains in both agar and wheat straw, using a cetonic extract of the dehydrated fruiting body of Pycnoporus sp.Key results: Two strains of Trichoderma (T. pleuroti and T. atrobrunneum, belonging to the clade of T. harzianum) were isolated from infected substrate obtained in production modules of Pleurotus ostreatus located in Tlaquitenango and Cuernavaca, in the state of Morelos, Mexico. The effect of a cetonic extract of the fruiting body of Pycnoporus sp. on the mycelial growth of the isolated strains of Trichoderma was also evaluated, observing decrease in mycelial growth rate in Petri dish up to 72% and on lignocellulosic substrate both mycelial growth and sporulation were delayed up to 10 days. Conclusions: The extract of Pycnoporus sp. could be an alternative to control the infection by Trichoderma spp. in mushroom cultures of the genus Pleurotus.Antecedentes y Objetivos: La producción de hongos comestibles se ve afectada por enfermedades bacterianas, fúngicas y virales, que a menudo causan grandes pérdidas. En la producción de hongos del género Pleurotus, los hongos de Trichoderma spp. representan un grave problema de contaminación y, aunque hay algunos compuestos químicos que controlan la infección, no son completamente seguros para el consumo humano. Como consecuencia, se están buscando alternativas a través de la biotecnología, como la que se presenta en este manuscrito. Métodos: Cepas de hongos del género Trichoderma se aislaron del sustrato donde se cultivaba Pleurotus ostreatus. Estos se identificaron morfológicamente y molecularmente; posteriormente se hicieron pruebas para inhibir el crecimiento de las cepas de Trichoderma tanto en agar como en paja de trigo, utilizando un extracto cetónico del cuerpo fructífero deshidratado de Pycnoporus sp. Resultados clave: Se aislaron dos cepas de Trichoderma (T. pleuroti y T. atrobrunneum, perteneciente al clado de T. harzianum) del sustrato infectado obtenido en los módulos de producción de Pleurotus ostreatus ubicados en Tlaquitenango y Cuernavaca, en el estado de Morelos, México. También se evaluó el efecto de un extracto cetónico del cuerpo fructífero de Pycnoporus sp. En el crecimiento micelial de las cepas aisladas de Trichoderma, observando una disminución en la tasa de crecimiento del micelio en la placa de Petri hasta en 72%, y en el sustrato lignocelulósico, tanto el crecimiento micelial como la esporulación se retrasaron hasta 10 días. Conclusiones: El extracto de Pycnoporus sp. podría ser una alternativa para controlar la infección por Trichoderma spp. en cultivos de hongos del género Pleurotus.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA

Fungal Melanins and their Potential Applications: A Review

Melanins are complex molecules found in many organisms including bacteria, fungi, plants, and animals that have protective functions against stress. In fungi there are several biochemical routes for their synthesis, and their diversity and structural complexity have made their analysis and characterization difficult. However, the possible specific functions of melanins in organisms have been determined. Based on their physicochemical properties, their potential in the application in various areas of interest and benefit of the human being have been visualized, such as in health and medicine, in bioremediation, and in the food industry. In this review, the type of melanins produced by fungi are discussed, as well as their main biological functions, the main biochemical routes involved in their synthesis, and potential applications in various areas. An important research area is visualized to find the best melanin-producing fungi as well as the conditions in which their production is maximized, in order to continue investigating the relationship of the structure of melanin with its biological functions as well as the determination of its physicochemical properties to establish its applications