Cellulase immobilization on superparamagnetic nanoparticles for reuse in cellulosic biomass conversion

Current cellulosic biomass hydrolysis is based on the one-time use of cellulases. Cellulases immobilized on magnetic nanocarriers offer the advantages of magnetic separation and repeated use for continuous hydrolysis. Most immobilization methods focus on only one type of cellulase. Here, we report co-immobilization of two types of cellulases, β-glucosidase A (BglA) and cellobiohydrolase D (CelD), on sub-20 nm superparamagnetic nanoparticles. The nanoparticles demonstrated 100% immobilization efficiency for both BglA and CelD. The total enzyme activities of immobilized BglA and CelD were up to 67.1% and 41.5% of that of the free cellulases, respectively. The immobilized BglA and CelD each retained about 85% and 43% of the initial immobilized enzyme activities after being recycled 3 and 10 times, respectively. The effects of pH and temperature on the immobilized cellulases were also investigated. Co-immobilization of BglA and CelD on MNPs is a promising strategy to promote synergistic action of cellulases while lowering enzyme consumption

Biological Systems Engineering

The present work studied the optimization of aeration rate, agitation rate and oxygen transfer and the use of various batch fermentation strategies for xylanase production from a recombinant Aspergillus nidulans strain in a 3 L stirred tank reactor. Maximum xylanase production of 1250 U/mL with productivity of 313 U/mL/day was obtained under an aeration rate of 2 vvm and an agitation rate of 400 rpm using batch fermentation. The optimum volumetric oxygen transfer coefficient (kLa) for efficient xylanase production was found to be 38.6 h1. Fed batch mode and repeated batch fermentation was also performed with kLa was 38.6 h1. Xylanase enzyme productivity increased to 327 with fed batch fermentation and 373 U/mL/ day with repeated batch fermentation. Also, maximum xylanase activity increased to 1410 U/mL with fed batch fermentation and 1572 U/mL with repeated batch fermentation

Heterologous expression and functional characterization of a GH10 endoxylanase from \u3ci\u3eAspergillus fumigatus\u3c/i\u3e var. \u3ci\u3eniveus\u3c/i\u3e with potential biotechnological application

Xylanases decrease the xylan content in pretreated biomass releasing it from hemicellulose, thus improving the accessibility of cellulose for cellulases. In this work, an endo-β-1,4-xylanase from Aspergillus fumigatus var. niveus (AFUMN-GH10) was successfully expressed. The structural analysis and biochemical characterization showed this AFUMN-GH10 does not contain a carbohydrate-binding module. The enzyme retained its activity in a pH range from 4.5 to 7.0, with an optimal temperature at 60°C. AFUMN-GH10 showed the highest activity in beechwood xylan. The mode of action of AFUMNGH10 was investigated by hydrolysis of APTS-labeled xylohexaose, which resulted in xylotriose and xylobiose as the main products. AFUMN-GH10 released 27% of residual xylan from hydrothermally-pretreated corn stover and 14% of residual xylan from hydrothermally-pretreated sugarcane bagasse. The results showed that environmentally friendly pretreatment followed by enzymatic hydrolysis with AFUMN-GH10 in low concentration is a suitable method to remove part of residual and recalcitrant hemicellulose from biomass

Perspectivas emancipatórias sobre a saúde e o Bem Viver diante das limitações do processo de desenvolvimento brasileiro

RESUMO No contexto da Rio+20 e na perspectiva da garantia da saúde de todos, este ensaio discutiu um novo tipo de pensar e de realizar a participação dos povos, tradicionalmente explorados e excluídos por efeito dos modelos de desenvolvimento capitalistas e colonizadores da vida humana e da natureza. Para tanto, o ‘desenvolvimento brasileir o’foi analisado com foco no agronegócio e suas contradições, e criticado mediante a concepção do Bem Viver. Foi proposto aqui vincular a ideia do Bem Viver às demandas dos povos que lutam por outro mundo possível e que poderão ser concretizadas mediante dimensões contra-hegemônicas de poder, de saber e de direitos, no âmbito de um projeto emancipatório

Cloning, purification, and biochemical characterization of an esterase from Aspergillus nidulans

A large accumulation of agro-industrial waste from different segments is generated daily and is often not properly managed. There are now other fronts in research to give a destination to these residues; these studies are generally aimed at obtaining new and better enzymes and the formulation of enzymatic cocktails that contain (for example, cellulases and hemicellulases) responsible for the degradation of lignocellulosic material. The plant cell wall is mainly composed of cellulose, hemicellulose, and lignin, forming a complex structure. Xylan is one of the main constituents of hemicellulose. To degrade this structure, enzymatic hydrolysis must occur synergistically with xylanolytic enzymes, such as endo-beta-1,4-xylanases, -xylosidases, and acetyl xylan esterase (AXE). In the current work, we reported the purification and biochemical characterization of an acetyl xylan esterase (AxeCE3) from Aspergillus nidulans. The axeCE3 gene was cloned into the pEXPYR vector and transformed into A. nidulans A773 for protein expression. The enzyme AxeCE3 was purified and characterized for its biochemical properties. AxeCE3 showed activity over a wide range of pH (3.0-9.0) and temperature (30-70 °C), with maximum activity at 55 °C, pH 7.0. Regarding the stability at temperature, AxeCE3 showed values above 90% of residual activity after 24 h of incubation at 45 and 50 °C. In relation to stability at pH, AxeCE3 maintained more than 90% of its residual activity after being incubated at 25 °C for 24 h between the pH range 3.0 to 9.0. It was also verified the effect of possible inhibitors (ethylenediamine tetraacetic acid (EDTA), Furfural, and 5- Hydroxymethylfurfural (5-HMF)) on the enzyme activity. AxeCE3 maintained 88% of relative activity at 5 mM EDTA, 43% and 82% at 50 mM furfural and 5-HMF, respectively. The results showed that AxeCE3 has interesting properties to use in the development in the formulation of enzymatic cocktails for the hydrolysis of lignocellulosic residues.The work was supported by the following: FAPESP (São Paulo Research Foundation, grants: 2014/50884 and 2018/07522-6) and National Institute of Science and Technology of Bioethanol, INCT, CNPq (grant: 465319/2014-9) and process 301963/2017-7. Research scholarships were granted to RCA and DA by FAPESP (Grant No: 2020/00081-4 and No: 2020/15510-8), to GSA by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Finance Code 001).info:eu-repo/semantics/publishedVersio

The Genome of a Thermo Tolerant, Pathogenic Albino Aspergillus fumigatus

Biotechnologists are interested in thermo tolerant fungi to manufacture enzymes active and stable at high temperatures, because they provide improved catalytic efficiency, strengthen enzyme substrate interactions, accelerate substrate enzyme conversion rates, enhance mass transfer, lower substrate viscosity, lessen contamination risk and offer the potential for enzyme recycling. Members of the genus Aspergillus live a wide variety of lifestyles, some embrace GRAS status routinely employed in food processing while others such as Aspergillus fumigatus are human pathogens. A. fumigatus produces melanins, pyomelanin protects the fungus against reactive oxygen species and DHN melanin produced by the pksP gene cluster confers the gray-greenish color. pksP mutants are attenuated in virulence. Here we report on the genomic DNA sequence of a thermo tolerant albino Aspergillus isolated from rain forest composted floors. Unexpectedly, the nucleotide sequence was 95.7% identical to the reported by Aspergillus fumigatus Af293. Genome size and predicted gene models were also highly similar, however differences in DNA content and conservation were observed. The albino strain, classified as Aspergillus fumigatus var. niveus, had 160 gene models not present in A. fumigatus Af293 and A. fumigatus Af293 had 647 not found in the albino strain. Furthermore, the major pigment generating gene cluster pksP appeared to have undergone genomic rearrangements and a key tyrosinase present in many aspergilli was missing from the genome. Remarkably however, despite the lack of pigmentation A. fumigatus var. niveus killed neutropenic mice and survived macrophage engulfment at similar rates as A. fumigatus Af293

Heterologous expression and functional characterization of a GH10 endoxylanase from \u3ci\u3eAspergillus fumigatus\u3c/i\u3e var. \u3ci\u3eniveus\u3c/i\u3e with potential biotechnological application

Xylanases decrease the xylan content in pretreated biomass releasing it from hemicellulose, thus improving the accessibility of cellulose for cellulases. In this work, an endo-β-1,4-xylanase from Aspergillus fumigatus var. niveus (AFUMN-GH10) was successfully expressed. The structural analysis and biochemical characterization showed this AFUMN-GH10 does not contain a carbohydrate-binding module. The enzyme retained its activity in a pH range from 4.5 to 7.0, with an optimal temperature at 60°C. AFUMN-GH10 showed the highest activity in beechwood xylan. The mode of action of AFUMNGH10 was investigated by hydrolysis of APTS-labeled xylohexaose, which resulted in xylotriose and xylobiose as the main products. AFUMN-GH10 released 27% of residual xylan from hydrothermally-pretreated corn stover and 14% of residual xylan from hydrothermally-pretreated sugarcane bagasse. The results showed that environmentally friendly pretreatment followed by enzymatic hydrolysis with AFUMN-GH10 in low concentration is a suitable method to remove part of residual and recalcitrant hemicellulose from biomass

Transcriptional profiling reveals the expression of novel genes in response to various stimuli in the human dermatophyte Trichophyton rubrum

<p>Abstract</p> <p>Background</p> <p>Cutaneous mycoses are common human infections among healthy and immunocompromised hosts, and the anthropophilic fungus <it>Trichophyton rubrum </it>is the most prevalent microorganism isolated from such clinical cases worldwide. The aim of this study was to determine the transcriptional profile of <it>T. rubrum </it>exposed to various stimuli in order to obtain insights into the responses of this pathogen to different environmental challenges. Therefore, we generated an expressed sequence tag (EST) collection by constructing one cDNA library and nine suppression subtractive hybridization libraries.</p> <p>Results</p> <p>The 1388 unigenes identified in this study were functionally classified based on the Munich Information Center for Protein Sequences (MIPS) categories. The identified proteins were involved in transcriptional regulation, cellular defense and stress, protein degradation, signaling, transport, and secretion, among other functions. Analysis of these unigenes revealed 575 <it>T. rubrum </it>sequences that had not been previously deposited in public databases.</p> <p>Conclusion</p> <p>In this study, we identified novel <it>T. rubrum </it>genes that will be useful for ORF prediction in genome sequencing and facilitating functional genome analysis. Annotation of these expressed genes revealed metabolic adaptations of <it>T. rubrum </it>to carbon sources, ambient pH shifts, and various antifungal drugs used in medical practice. Furthermore, challenging <it>T. rubrum </it>with cytotoxic drugs and ambient pH shifts extended our understanding of the molecular events possibly involved in the infectious process and resistance to antifungal drugs.</p

Two structurally discrete GH7-cellobiohydrolases compete for the same cellulosic substrate fiber

Background: Cellulose consisting of arrays of linear beta-1,4 linked glucans, is the most abundant carbon-containing polymer present in biomass. Recalcitrance of crystalline cellulose towards enzymatic degradation is widely reported and is the result of intra- and inter-molecular hydrogen bonds within and among the linear glucans. Cellobiohydrolases are enzymes that attack crystalline cellulose. Here we report on two forms of glycosyl hydrolase family 7 cellobiohydrolases common to all Aspergillii that attack Avicel, cotton cellulose and other forms of crystalline cellulose.Results: Cellobiohydrolases Cbh1 and CelD have similar catalytic domains but only Cbh1 contains a carbohydrate-binding domain (CBD) that binds to cellulose. Structural superpositioning of Cbh1 and CelD on the Talaromyces emersonii Cel7A 3-dimensional structure, identifies the typical tunnel-like catalytic active site while Cbh1 shows an additional loop that partially obstructs the substrate-fitting channel. CelD does not have a CBD and shows a four amino acid residue deletion on the tunnel-obstructing loop providing a continuous opening in the absence of a CBD. Cbh1 and CelD are catalytically functional and while specific activity against Avicel is 7.7 and 0.5 U.mg prot-1, respectively specific activity on p NPC is virtually identical. Cbh1 is slightly more stable to thermal inactivation compared to CelD and is much less sensitive to glucose inhibition suggesting that an open tunnel configuration, or absence of a CBD, alters the way the catalytic domain interacts with the substrate. Cbh1 and CelD enzyme mixtures on crystalline cellulosic substrates show a strong combinatorial effort response for mixtures where Cbh1 is present in 2:1 or 4:1 molar excess. When CelD was overrepresented the combinatorial effort could only be partially overcome. CelD appears to bind and hydrolyze only loose cellulosic chains while Cbh1 is capable of opening new cellulosic substrate molecules away from the cellulosic fiber.Conclusion: Cellobiohydrolases both with and without a CBD occur in most fungal genomes where both enzymes are secreted, and likely participate in cellulose degradation. The fact that only Cbh1 binds to the substrate and in combination with CelD exhibits strong synergy only when Cbh1 is present in excess, suggests that Cbh1 unties enough chains from cellulose fibers, thus enabling processive access of CelD.Peer reviewedMicrobiology and Molecular GeneticsBiochemistry and Molecular Biolog

Exploring glycoside hydrolases and accessory proteins from wood decay fungi to enhance sugarcane bagasse saccharification

Abstract\ud \ud Background\ud Glycoside hydrolases (GHs) and accessory proteins are key components for efficient and cost-effective enzymatic hydrolysis of polysaccharides in modern, biochemically based biorefineries. Currently, commercialized GHs and accessory proteins are produced by ascomycetes. However, the role of wood decay basidiomycetes proteins in biomass saccharification has not been extensively pursued. Wood decay fungi degrade polysaccharides in highly lignified tissues in natural environments, and are a promising enzyme source for improving enzymatic cocktails that are designed for in vitro lignocellulose conversion.\ud \ud \ud Results\ud GHs and accessory proteins were produced by representative brown- and white-rot fungi, Laetiporus sulphureus and Pleurotus ostreatus, respectively. Concentrated protein extracts were then used to amend commercial enzymatic cocktails for saccharification of alkaline-sulfite pretreated sugarcane bagasse. The main enzymatic activities found in the wood decay fungal protein extracts were attributed to endoglucanases, xylanases and β-glucosidases. Cellobiohydrolase (CBH) activities in the L. sulphureus and P. ostreatus extracts were low and nonexistent, respectively. The initial glucan conversion rates were boosted when the wood decay fungal proteins were used to replace half of the enzymes from the commercial cocktails. L. sulphureus proteins increased the glucan conversion levels, with values above those observed for the full load of commercial enzymes. Wood decay fungal proteins also enhanced the xylan conversion efficiency due to their high xylanase activities. Proteomic studies revealed 104 and 45 different proteins in the P. ostreatus and L. sulphureus extracts, respectively. The enhancement of the saccharification of alkaline-pretreated substrates by the modified enzymatic cocktails was attributed to the following protein families: GH5- and GH45-endoglucanases, GH3-β-glucosidases, and GH10-xylanases.\ud \ud \ud Conclusions\ud The extracellular proteins produced by wood decay fungi provide useful tools to improve commercial enzyme cocktails that are currently used for the saccharification of alkaline-pretreated lignocellulosic substrates. The relevant proteins encompass multiple glycoside hydrolase families, including the GH5- and GH45-endoglucanases, GH3-β-glucosidases, and GH10-xylanases.The authors thank J.M. Silva and J.C. Tavares for technical assistance. This work\ud was supported by FAPESP (contract numbers 08/56256-5 and 2014/06923-6),\ud CNPq (contract numbers 442333/2014-5; 310186/2014-5 and 140796/2013-\ud 4), and CAPES. We gratefully acknowledge the provision of time on the MAS\ud and NGS facilities (LNBio and CTBE, respectively) at the National Center for\ud Research in Energy and Materials (CNPEM).\ud The work was supported by Fundação de Amparo à Pesquisa do Estado de\ud São Paulo (FAPESP), contract numbers 08/56256-5 and 2014/06923-6, and by\ud Conselho Nacional de Pesquisa (CNPq), contract numbers 442333/2014-5;\ud 310186/2014-5, 140796/2013-4