Differential Carbon Catabolite Repression and Hemicellulolytic Ability among Pathotypes of Colletotrichum lindemuthianum against Natural Plant Substrates.

Colletotrichum lindemuthianum is a phytopathogenic fungus that causes anthracnose in common beans (Phaseolus vulgaris) and presents a great diversity of pathotypes with different levels of virulence against bean varieties worldwide. The purpose of this study was to establish whether pathotypic diversity is associated with differences in the mycelial growth and secretion of plant-cell-wall-degrading enzymes (PCWDEs). We evaluated growth, hemicellulase and cellulase activity, and PCWDE secretion in four pathotypes of C. lindemuthianum in cultures with glucose, bean hypocotyls and green beans of P. vulgaris, and water hyacinth (Eichhornia crassipes). The results showed differences in the mycelial growth, hemicellulolytic activity, and PCWDE secretion among the pathotypes. Glucose was not the preferred carbon source for the best mycelial growth in all pathotypes, each of which showed a unique PCWDE secretion profile, indicating different levels of carbon catabolite regulation (CCR). The pathotypes showed a high differential hemicellulolytic capacity to degrade host and water hyacinth tissues, suggesting CCR by pentoses and that there are differences in the absorption and metabolism of different monosaccharides and/or disaccharides. We propose that different levels of CCR could optimize growth in different host tissues and could allow for consortium behavior in interactions with bean crops.

Obtención de celulasas bacterianas usando residuos orgánicos generados en plazas de mercado / Bacterial cellulases obtention from organic wastes generated in marketplaces

RESUMEN Los residuos agrícolas son una fuente de celulosa que puede ser aprovechada para producir enzimas hidrolíticas, como las celulasas, mediante acción microbiana. Estas celulasas son utilizadas en procesos extractivos de biomoléculas, en la producción de biogás, en la industria textil, detergente, alimentaria y del papel. El propósito del estudio es la obtención de celulasas bacterianas utilizando residuos orgánicos, generados en plazas de mercado. Se realizaron dos medios de cultivo (A1 y A2), a partir de ameros de mazorca y cáscaras de leguminosas, ajustando el pH del medio A1 a 7,0 y el de A2 a 5,0. Los medios fueron fermentados por 75 horas, mediante la cepa bacteriana C6M2, aislada de residuos de plazas de mercado, monitoreando la actividad enzimática, la concentración de azúcares reductores y la celulosa residual. La máxima actividad celulolítica se logró a las 56 horas de fermentación en A1 y a las 32 horas, en A2. El extracto enzimático se precipitó, dializó y ultrafiltró, obteniendo una actividad final de 9,07 ± 0,48 U/mL. Los ameros y las cáscaras de leguminosa se pueden aprovechar como sustratos en la producción de celulasas, con posibles aplicaciones en procesos donde requieran bajos grados de pureza.

ABSTRACT Agricultural wastes are a source of cellulose that could be used to produce hydrolytic enzymes such as cellulases by microbial action. Cellulases are used in the extractive process of biomolecules, in biogas production, textile, detergent, food, and paper industry. The study purpose is the obtention of bacterial cellulases using organic wastes generated in marketplaces. Two culture media were done (A1 and A2) with corn cob leaves and legume seed pods adjusting the A1 medium pH at 7.0 and the A2 pH at 5.0. The media were fermented for 75 hours by the bacterial strain C6M2, isolated from marketplace wastes, and the enzyme activity, reducing sugar concentration, and residual cellulose were monitored. The maximum cellulolytic activity was obtained at 56 hours of fermentation in A1 and in A2 it was at 32 hours. The enzyme extract was precipitated, dialyzed and ultra-filtrated obtaining a final activity of 9.07 ± 0.48 U/mL. The corn cob leaves and legume seed pods can be used as substrates in the production of cellulases which might be applied in processes with low requirements of enzyme purity.

Expression and characterization of a novel ß-1,4-endoglucanase from Bacillus subtilis strain isolated from a pulp and paper mill wastewater.

The production of fermentable sugars from lignocellulosic biomass is achieved by the synergistic action of a group of enzymes called cellulases. Cellulose is a long chain of chemically linked glucoses by ß-1,4 bonds. The enzyme ß-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A ß-1,4-endoglucanase enzyme with high activity was obtained from a Bacillus subtilis strain isolated from wastewater of a pulp and paper mill. Sequencing and bioinformatic analysis showed that the gene amplified by PCR consisting of 1407 nucleotides and coding for a ß-1,4-endoglucanase enzyme of approximately 55 kDa. The open reading frame (ORF) encoding the mature endoglucanase (eglS) was successfully inserted in a modified cloning plasmid (pITD03) and into the pYD1 plasmid used for its expression in yeast. Carboxymethylcellulose (CMC) plate assay, SDS-PAGE, and zymogram confirmed the production and secretion by the transformed E. coli BL21-SI strain of a 39 kDa ß-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated ß-1,4-endoglucanase had higher activity and stability.

Bioprospecting of endophytic fungi from semidesert candelilla (Euphorbia antisyphilitica Zucc): Potential for extracellular enzyme production.

Endophytic microbial communities colonize plants growing under various abiotic stress conditions. Candelilla (Euphorbia antisyphilitica Zucc.) is a shrub that develops functionally in arid and semi-arid zones of Mexico; these conditions generate an association between the plant and the microorganisms, contributing to the production of enzymes as a defense mechanism for resistance to abiotic stress. The objective of this research was to isolate and identify endophyte fungi of candelilla and bioprospection of these endophytic fungi for enzyme production using candelilla by-products. Fungi were isolated and identified using ITS1/ITS4 sequencing. Their potency index (PI) was evaluated in producing endoglucanase, xylanase, amylase, and laccase. Fermentation was carried out at 30°C for 8 days at 200 rpm, with measurements every 2 days, using candelilla by-products as substrate. All fungi exhibited higher cellulase, amylase, and laccase activities on the 2nd, 6th, and 8th day of fermentation, respectively, of fermentation. The fungus Aspergillus niger ITD-IN4.1 showed the highest amylase activity (246.84 U/mg), the genus Neurospora showed the highest cellulase activity, reaching up to 13.45 FPU/mg, and the strain Neurospora sp. ITD-IN5.2 showed the highest laccase activity (3.46 U/mg). This work provides the first report on the endophytic diversity of E. antisyphilitica and its potential role in enzyme production.

ß-glucosidases from Saccharomyces cerevisiae: production, protein precipitation, characterization, and application in the enzymatic hydrolysis of delignified sugarcane bagasse.

ß-glucosidase is an essential enzyme for the enzymatic hydrolysis of lignocellulosic biomass, as it catalyzes the final stage of cellulose breakdown, releasing glucose. This paper aims to produce ß-glucosidase from Saccharomyces cerevisiae and evaluate the enzymatic degradation of delignified sugarcane bagasse. S. cerevisiae was grown in yeast peptone dextrose medium. Partial purification of the enzyme was achieved through precipitating proteins with ethanol, and the optimal activity was measured by optimizing pH and temperature. The effects of ions, glucose tolerance, and heat treatment were evaluated. Delignified sugarcane bagasse was hydrolyzed by the enzyme. ß-glucosidase showed a specific activity of 14.0712 ± 0.0207 U mg-1. Partial purification showed 1.22-fold purification. The optimum pH and temperature were 6.24 and 54 °C, respectively. ß-glucosidase showed tolerance to glucose, with a relative activity of 71.27 ± 0.16%. Thermostability showed a relative activity of 58.84 ± 0.91% at 90 °C. The hydrolysis of delignified sugarcane bagasse showed a conversion rate of 87.97 ± 0.10% in the presence of Zn2+, an ion that promoted the highest increase in enzymatic activity. S. cerevisiae produced an extracellular ß-glucosidase with good stability at pH and temperatures conventionally applied in the hydrolysis of lignocellulosic biomass, showing viability for industrial application.

Overexpression of the Transcription Factor Azf1 Reveals Novel Regulatory Functions and Impacts ß-Glucosidase Production in Trichoderma reesei.

The fungus Trichoderma reesei is an essential producer of enzymes that degrade lignocellulosic biomass to produce value-added bioproducts. The cellulolytic system of T. reesei is controlled by several transcription factors (TFs) that efficiently regulate the production of these enzymes. Recently, a new TF named Azf1 was identified as a positive regulator of cellulase expression. Here, we investigated novel regulatory functions of Azf1 by its overexpression. In the mutant strain OEazf1, overexpression of azf1 was achieved under both repression and induction conditions. Although azf1 was more abundant in transcript and protein, overexpression of this TF did not activate transcription of the cellulase gene in the presence of the repressor glucose, suggesting that Azf1 may be subject to posttranslational regulation. In cellulose, the expression of swo, encoding the accessory protein swollenin, and the ß-glucosidases cel1a, cel1b, cel3b, and cel3g increases in the early stages of cultivation. The increased production of these ß-glucosidases increases the hydrolysis rate of cellobiose and sophorose, which activates carbon catabolite repression (CCR) and causes repression of cellulase genes and the regulator Xyr1 in the later stages of cultivation. Moreover, overexpression of azf1 led to increased cellulase activity in T. reesei during long-term cultivation in cellulose and sugarcane bagasse. Our results provide new insights into the mechanisms regulating Azf1 and novel genes that are important targets of this TF. This work contributes to a better understanding of the complex mechanisms regulating cellulase expression in T. reesei. It will contribute to the development of strains with higher production of these essential enzymes.

High-yield production of rod-like and spherical nanocellulose by controlled enzymatic hydrolysis of mechanically pretreated cellulose.

In this study, a simple and scalable mechanical pretreatment was evaluated as means of enhancing the accessibility of cellulose fibers, with the objective of improving the efficiency of enzymatic reactions for the production of cellulose nanoparticles (CNs). In addition, the effects of enzyme type (endoglucanase - EG, endoxylanase - EX, and a cellulase preparation - CB), composition ratio (0-200UEG:0-200UEX or EG, EX, and CB alone), and loading (0 U-200 U) were investigated in relation to CN yield, morphology, and properties. The combination of mechanical pretreatment and specific enzymatic hydrolysis conditions substantially improved CN production yield, reaching up to 83 %. The production of rod-like or spherical nanoparticles and their chemical composition were highly influenced by the enzyme type, composition ratio, and loading. However, these enzymatic conditions had minimal impact on the crystallinity index (approximately 80 %) and thermal stability (Tmax within 330-355 °C). Overall, these findings demonstrate that mechanical pretreatment followed by enzymatic hydrolysis under specific conditions is a suitable method to produce nanocellulose with high yield and adjustable properties such as purity, rod-like or spherical forms, high thermal stability, and high crystallinity. Therefore, this production approach shows promise in producing tailored CNs with the potential for superior performance in various advanced applications, including, but not limited to, wound dressings, drug delivery, thermoplastic composites, 3D (bio)printing, and smart packaging.

Molecular insight into cellulose degradation by the phototrophic green alga Scenedesmus.

Lignocellulose is the most abundant natural biopolymer on earth and a potential raw material for the production of fuels and chemicals. However, only some organisms such as bacteria and fungi produce enzymes that metabolize this polymer. In this work we have demonstrated the presence of cellulolytic activity in the supernatant of Scenedesmus quadricauda cultures and we identified the presence of extracellular cellulases in the genome of five Scenedesmus species. Scenedesmus is a green alga which grows in both freshwater and saltwater regions as well as in soils, showing highly flexible metabolic properties. Sequence comparison of the different identified cellulases with hydrolytic enzymes from other organisms using multisequence alignments and phylogenetic trees showed that these proteins belong to the families of glycosyl hydrolases 1, 5, 9, and 10. In addition, most of the Scenedesmus cellulases showed greater sequence similarity with those from invertebrates, fungi, bacteria, and other microalgae than with the plant homologs. Furthermore, the data obtained from the three dimensional structure showed that both, their global structure and the main amino acid residues involved in catalysis and substrate binding are well conserved. Based on our results, we propose that different species of Scenedesmus could act as biocatalysts for the hydrolysis of cellulosic biomass produced from sunlight.

The improvement of guava (Psidium guajava) juice quality using crude multi-enzymatic extracts obtained from yeasts.

Guava juice is cloudy and viscous, which hinders filtration, decreases yield, and causes the loss of quality after its processing and during storage. This study aimed to evaluate enzymatic treatment effects using crude multi-enzymatic extracts (CME) obtained from Rhodotorula mucilaginosa, Rhodotorula orizycola, and Pseudozyma sp. produced by submerse fermentation in the extraction of juice guava. Mixtures of 100 ml of guava pulp and multi-enzymatic extracts proposed by Doehlert planning were incubated under constant agitation at 150 rpm and 50°C, and a Doehlert design was applied as a multivariate optimization strategy. The optimal conditions using the multi-enzymatic extract were: 0.4% (v/v) of CME for 131 min for the multi-enzymatic treatment using Pseudozyma sp.; 3.0% (v/v) of CME for 154 min using the R. mucilaginosa CME; and 5.0% (v/v) of CME for 90 min using R. oryzicola. The maximum viscosity reduction values for the juices treated with the CME of yeasts were 10.33%, 86.38%, and 13.33% for the juices treated with the CME of Pseudozyma sp., R. mucilaginosa, and R. orizycola, respectively. The physical-chemical properties were improved after treatment with CMEs, yielding a reduction of clarity, increase of total soluble solids and reducing sugars, and decreasing the acidity (pH) for all treatments with enzymatic extracts of all strains. The yeasts studied showed a potential for CME production to be applied to juice, improving the quality of the juice, and R. mucilaginosa was the most prominent yeast due to most significant reduction of viscosity in guava juice.

Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico / Identificação por técnicas moleculares de bactérias halofílicas que produzem enzimas importantes em área intocada de Campeche, México

Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.(AU)

Isla Arena está localizada na coordenada 20°70N - 90°45W, de Campeche, México. Nesses estuários, o oceano se mistura com a água doce e os ecossistemas se concentram onde proliferam petenos e flamingos rosa. Crustáceos e moluscos abundam no mar. Apesar de sua enorme riqueza marinha, não há estudos realizados sobre a presença de microrganismos halofílicos nessas águas. Neste trabalho, a diversidade e estrutura da comunidade microbiana foram investigadas através de uma abordagem metagenômica e corroboradas para o sequenciamento de genes 16S rRNA. Verificou-se que o filo Fimicutes predomina com mais de 50%, quase na mesma proporção da classe Bacilli e com quase 41% de abundância relativa da ordem Bacillales. Os resultados do sequenciamento mostraram que uma das amostras apresentou alto percentual de similaridade (99,75%) pelo programa Nucleotide BLAST com um microrganismo peculiar: Bacillus subtilis. Nossos resultados demonstram que B. subtilis pode ser uma fonte eficiente de proteases, lipases e celulases, provenientes de comunidades microbianas halofílicas localizadas em áreas pouco exploradas.(AU)

Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico / Identificação por técnicas moleculares de bactérias halofílicas que produzem enzimas importantes em área intocada de Campeche, México

Abstract Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.

Resumo Isla Arena está localizada na coordenada 20°70'N - 90°45'W, de Campeche, México. Nesses estuários, o oceano se mistura com a água doce e os ecossistemas se concentram onde proliferam petenos e flamingos rosa. Crustáceos e moluscos abundam no mar. Apesar de sua enorme riqueza marinha, não há estudos realizados sobre a presença de microrganismos halofílicos nessas águas. Neste trabalho, a diversidade e estrutura da comunidade microbiana foram investigadas através de uma abordagem metagenômica e corroboradas para o sequenciamento de genes 16S rRNA. Verificou-se que o filo Fimicutes predomina com mais de 50%, quase na mesma proporção da classe Bacilli e com quase 41% de abundância relativa da ordem Bacillales. Os resultados do sequenciamento mostraram que uma das amostras apresentou alto percentual de similaridade (99,75%) pelo programa Nucleotide BLAST com um microrganismo peculiar: Bacillus subtilis. Nossos resultados demonstram que B. subtilis pode ser uma fonte eficiente de proteases, lipases e celulases, provenientes de comunidades microbianas halofílicas localizadas em áreas pouco exploradas.

Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico / Identificação por técnicas moleculares de bactérias halofílicas que produzem enzimas importantes em área intocada de Campeche, México

Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.

Isla Arena está localizada na coordenada 20°70’N - 90°45’W, de Campeche, México. Nesses estuários, o oceano se mistura com a água doce e os ecossistemas se concentram onde proliferam petenos e flamingos rosa. Crustáceos e moluscos abundam no mar. Apesar de sua enorme riqueza marinha, não há estudos realizados sobre a presença de microrganismos halofílicos nessas águas. Neste trabalho, a diversidade e estrutura da comunidade microbiana foram investigadas através de uma abordagem metagenômica e corroboradas para o sequenciamento de genes 16S rRNA. Verificou-se que o filo Fimicutes predomina com mais de 50%, quase na mesma proporção da classe Bacilli e com quase 41% de abundância relativa da ordem Bacillales. Os resultados do sequenciamento mostraram que uma das amostras apresentou alto percentual de similaridade (99,75%) pelo programa Nucleotide BLAST com um microrganismo peculiar: Bacillus subtilis. Nossos resultados demonstram que B. subtilis pode ser uma fonte eficiente de proteases, lipases e celulases, provenientes de comunidades microbianas halofílicas localizadas em áreas pouco exploradas.

Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico

Abstract Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.

Resumo Isla Arena está localizada na coordenada 20°70N - 90°45W, de Campeche, México. Nesses estuários, o oceano se mistura com a água doce e os ecossistemas se concentram onde proliferam petenos e flamingos rosa. Crustáceos e moluscos abundam no mar. Apesar de sua enorme riqueza marinha, não há estudos realizados sobre a presença de microrganismos halofílicos nessas águas. Neste trabalho, a diversidade e estrutura da comunidade microbiana foram investigadas através de uma abordagem metagenômica e corroboradas para o sequenciamento de genes 16S rRNA. Verificou-se que o filo Fimicutes predomina com mais de 50%, quase na mesma proporção da classe Bacilli e com quase 41% de abundância relativa da ordem Bacillales. Os resultados do sequenciamento mostraram que uma das amostras apresentou alto percentual de similaridade (99,75%) pelo programa Nucleotide BLAST com um microrganismo peculiar: Bacillus subtilis. Nossos resultados demonstram que B. subtilis pode ser uma fonte eficiente de proteases, lipases e celulases, provenientes de comunidades microbianas halofílicas localizadas em áreas pouco exploradas.

Cellulolytic activity of anaerobic facultative fungi from the digestive tract of sheep fed with banana leaf hay / Atividade celulolítica de fungos aeróbios facultativos do trato digestório de ovinos alimentados com feno da folha de bananeira

The addition of cellulolytic fungi, or their enzymes, in diets containing high levels of fiber are promising strategy for improving the performance. In this study, the aims were to select cellulolytic fungi from the digestive tract of sheep fed different concentrations of banana leaf hay. Thirty lambs raised in a feedlot were evaluated, distributed in a completely randomized design, with diets containing 0, 125, 250, 375, or 500 g/Kg of dry matter and six replications. Approximately 15 mL of ruminal fluid and swabs from the rectal ampulla were collected. The cultures were carried out in a medium containing microcrystalline cellulose (C medium). The mycelial fungi isolates were identified through the microculture technique. Among the fungi from the ruminal fluid, 23 isolates corresponded to the genus Aspergillus and three to Paecilomyces spp. Among the isolates from the rectal ampulla, seven were A. spp., and three were P. spp. The A. genus predominated among the isolates from both evaluated sites (p <0.05). Fragments of these fungi were inoculated in triplicate in medium C at 37 °C and the cellulolytic activity index (CAI) was determined after 24, 48, and 72 hours of incubation. There was no difference in the CAI of Aspergillus spp. from animals fed different diets or of different evaluated sites (P > 0.05). However, 22 isolates of Aspergillus spp. and three of Paecilomyces spp. showed a CAI > 1, indicating biotechnological potential for cellulase production. These selected isolates could be selected for the elaboration of microbial additives in ruminant diets.(AU)

A adição de fungos celulolíticos, ou suas enzimas, em dietas contendo elevados teores de fibras são estratégias pro-missoras para melhorar o desempenho. Neste estudo os objetivos foram selecionar fungos celulolíticos do trato digestório de ovinos alimentados com diferentes concentrações do feno da folha da bananeira (FBH). Foram avaliados 30 borregos criados em sistema intensivo, distribuídos em delineamento inteiramente ao acaso, com cinco dietas contendo 0, 125, 250, 375 ou 500 g/KG de matéria seca em seis repetições. Foram coletados aproximadamente 15 mL de fluido ruminal e swabs da ampola retal. Os cultivos foram realizados em meio de cultura contendo celulose microcristalina (meio C). Os fungos micelianos foram identificados após a técnica de microcultivo. Entre os fungos provenientes do fluido ruminal, 23 isolados corresponderam ao gênero Aspergillus e três a Paecilomyces spp.. Foram identificados nas fezes dos animais sete Aspergillus spp. e três Paecilomyces spp.. O gênero Aspergillus predominou entre os isolados de ambos os sítios avaliados (p =0,013). Fragmentos desses fungos foram inoculados em triplicada em meio C a 37 °C e determinou-se o índice de atividade celulolítica(IAC) após 24, 48 e 72 horas de incubação. Não houve diferença entre CAI de isolados de Aspergillus spp. provenientes dos animais em diferentes dietas ou sítios avaliados (P > 0.05). Entretanto, 22 isolados de Aspergillus spp. e três de Paecilomyces spp. apresentaram IAC >1, indicando potencial biotecnológico para produção de celulases.(AU)

Taxonomy, comparative genomics and evolutionary insights of Penicillium ucsense: a novel species in series Oxalica.

The genomes of two Penicillium strains were sequenced and studied in this study: strain 2HH was isolated from the digestive tract of Anobium punctatum beetle larva in 1979 and the cellulase hypersecretory strain S1M29, derived from strain 2HH by a long-term mutagenesis process. With these data, the strains were reclassified and insight is obtained on molecular features related to cellulase hyperproduction and the albino phenotype of the mutant. Both strains were previously identified as Penicillium echinulatum and this investigation indicated that these should be reclassified. Phylogenetic and phenotype data showed that these strains represent a new Penicillium species in series Oxalica, for which the name Penicillium ucsense is proposed here. Six additional strains (SFC101850, SFCP10873, SFCP10886, SFCP10931, SFCP10932 and SFCP10933) collected from the marine environment in the Republic of Korea were also classified as this species, indicating a worldwide distribution of this new taxon. Compared to the closely related strain Penicillium oxalicum 114-2, the composition of cell wall-associated proteins of P. ucsense 2HH shows five fewer chitinases, considerable differences in the number of proteins related to ß-D-glucan metabolism. The genomic comparison of 2HH and S1M29 highlighted single amino-acid substitutions in two major proteins (BGL2 and FlbA) that can be associated with the hyperproduction of cellulases. The study of melanin pathways shows that the S1M29 albino phenotype resulted from a single amino-acid substitution in the enzyme ALB1, a precursor of the 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis. Our study provides important knowledge towards understanding species distribution, molecular mechanisms, melanin production and cell wall biosynthesis of this new Penicillium species.

Aspergillus niger LBM 134 isolated from rotten wood and its potential cellulolytic ability.

Aspergillus is a genus of filamentous and cosmopolitan fungi that includes important species for medical mycology, food, basic research and agro-industry areas. Aspergillus section Nigri are efficient producers of hydrolytic enzymes such as cellulases that are employed in the cellulose conversion. Hence, the search of new cellulolytic isolates and their correct identification is important for carrying out safe biotechnological processes. This study aimed to characterise the cellulolytic potential of Aspergillus sp. LBM 134, isolated from the Paranaense rainforest (Argentina) and to identify the isolate through a polyphasic approach. The fungus was identified as Aspergillus niger and its cellulolytic potential was evaluated by using Congo red technique and fluorescence plate assays for carboxymethyl cellulase, ß-glucosidase and cellobiohydrolase, respectively. All three cellulase activities were positive; this bio-prospective positioned A. niger LBM 134 as a promising alternative for industries that require organisms capable of carrying out cellulosic biomass processing.

Cellulolytic enzymes production guided by morphology engineering.

Endoglucanase and xylanase are critical enzymes for liquefaction and enzyme hydrolysis of high solids lignocellulosic biomass to facilitate its transport and production of desired derived products. Here is reported how combinations of different spore concentrations and pH influence microbial morphology, and how this may be used to direct expression and secretion of enzymes by Aspergillus niger. While xylanase production is not affected by A. niger morphology changes, endoglucanase production is enhanced under conditions of lower stress and by morphology that results in pellets. ß-glucosidase production is enhanced under dispersed morphology, which results in up to fourfold increase of this enzyme production under the tested experimental conditions. A morphologic scale (Y) is proposed based on a form factor that considers the size and frequency of each morphology class, and that points to conditions that result in high selectivity for either endoglucanase or ß-glucosidase production. An equation proposed to relate enzyme activity to morphology provides a useful tool for tuning enzyme production of A. niger, where morphology is a first indication of relative enzyme activities in a fermentation broth.

Statistical optimization of cellulases by Talaromyces thermophilus utilizing Saccharum spontaneum, a novel substrate

BACKGROUND: At present, cellulases are the most important enzymes worldwide, and their demand has been increasing in the industrial sector owing to their notable hydrolysis capability. RESULTS: In the present study, contrary to conventional techniques, three physical parameters were statistically optimized for the production of cellulase by thermophilic fungi by using response surface methodology (RSM). Among all the tested thermophilic strains, the best cellulase producing fungus was identified as Talaromyces thermophilus ­ both morphologically and molecularly through 5.8S/ITS rDNA sequencing. The central composite design (CCD) was used to evaluate the interactive effect of the significant factors. The CCD was applied by considering incubation period, pH, and temperature as the model factors for the present investigation. A second-order quadratic model and response surface method revealed that the independent variables including pH 6, temperature 50 C, and incubation period 72 h significantly influenced the production of cellulases. The analysis of variance (ANOVA) indicated that the established model was significant (P 0.05) and showed the high adequacy of the model. The actual and predicted values of CMCase and FPase activity showed good agreement with each other and also confirmed the validity of the designed model. CONCLUSIONS: We believe the present findings to be the first report on cellulase production by exploiting Kans grass (Saccharum spontaneum) as a substrate through response surface methodology by using thermophilic fungus, Talaromyces thermophilus.

Immobilization of endoglucanase on kaolin by adsorption and covalent bonding.

In the current research, endoglucanase, one of the enzymes of the cellulolytic complex, was immobilized on kaolin by two different techniques, adsorption, and covalent bonding. A comparative study was conducted between free, adsorbed, and covalently immobilized endoglucanase. For the covalent bonding, the kaolin particles were functionalized with 3-aminopropyltriethoxysilane (APTES) and activated with glutaraldehyde. Immobilization by adsorption was performed using the kaolin without any treatment. Recovered activities after the endoglucanase immobilization by adsorption and covalent bonding were found to be 60 ± 2.5 and 65 ± 3.5%, respectively. The studies of optima pH and temperature, as well as thermal stability, showed that the catalytic characteristic of the enzyme was maintained after the immobilization by both adsorption and covalent bonding. Even after 8 cycles of use, the endoglucanase immobilized by the two techniques retained about 86% of its initial activity. The results showed that the adsorption was as effective as covalent bonding for the immobilization of endoglucanase on kaolin. However, the adsorption technique seems to have a greater potential for use in future studies, as it is simpler, cheaper, and faster than covalent immobilization. Therefore, in this work it was demonstrated that endoglucanases can be immobilized efficiently on kaolin through a very simple immobilization protocol, offering a promising strategy for performing repeated enzymatic hydrolysis reactions.

Protective effects of non-catalytic proteins on endoglucanase activity at air and lignin interfaces.

The manner in which added non-catalytic proteins during enzymatic hydrolysis of lignocellulosic substrates enhances hydrolysis mechanisms is not completely understood. Prior research has indicated that a reduction in the non-specific adsorption of enzymes on lignin, and deactivation of enzymes exposed to air-liquid interface provide rationale. This work investigated root causes including effects of the air-liquid interface on non-catalytic proteins, and effects of lignin on endoglucanase. Three different experimental designs and three variables (air-liquid interfacial area, the types of lignin (acid or enzymatic lignin), and the presence of non-enzymatic protein (bovine serum albumin [BSA] or soy proteins ) were used. The results showed that acid isolated lignin adsorbed almost all endoglucanase activity initially present in supernatant, independent of air interface conditions (25 or 250 ml flasks) with the presence of BSA preventing this effect. Endoglucanase lost 30%-50% of its activity due to an air-liquid interface in the presence of lignin while addition of non-enzymatic protein helped to preserve this enzyme's activity. Langmuir and Freundlich models applied to experimental data indicated that the adsorption increases with increasing temperature for both endoglucanase and BSA. Adsorption of the enzyme and protein were endothermic with an increase in entropy. These results, combined, show that hydrophobicity plays a strong role in the adsorption of both endoglucanase and BSA on lignin.