Bioethanol production by Saccharomyces cerevisiae, Pichia stipitis and Zymomonas mobilis from delignified coconut fibre mature and lignin extraction according to biorefinery concept

In search to increase the offer of liquid, clean, renewable and sustainable energy in the world energy matrix, the use of lignocellulosic materials (LCMs) for bioethanol production arises as a valuable alternative. The objective of this work was to analyze and compare the performance of Saccharomyces cerevisiae, Pichia stipitis and Zymomonas mobilis in the production of bioethanol from coconut fibre mature (CFM) using different strategies: simultaneous saccharification and fermentation (SSF) and semi-simultaneous saccharification and fermentation (SSSF). The CFM was pretreated by hydrothermal pretreatment catalyzed with sodium hydroxide (HPCSH). The pretreated CFM was characterized by X-ray diffractometry and SEM, and the lignin recovered in the liquid phase by FTIR and TGA. After the HPCSH pretreatment (2.5% (v/v) sodium hydroxide at 180 °C for 30 min), the cellulose content was 56.44%, while the hemicellulose and lignin were reduced 69.04% and 89.13%, respectively. Following pretreatment, the obtained cellulosic fraction was submitted to SSF and SSSF. Pichia stipitis allowed for the highest ethanol yield 90.18% in SSSF, 91.17% and 91.03% were obtained with Saccharomyces cerevisiae and Zymomonas mobilis, respectively. It may be concluded that the selection of the most efficient microorganism for the obtention of high bioethanol production yields from cellulose pretreated by HPCSH depends on the operational strategy used and this pretreatment is an interesting alternative for add value of coconut fibre mature compounds (lignin, phenolics) being in accordance with the biorefinery concept.

Enzymatic hydrophobic modification of jute fibers via grafting to reinforce composites

Horseradish peroxidase (HRP)/H2O2 system catalyzes the free-radical polymerization of aromatic compounds such as lignins and gallate esters. In this work, dodecyl gallate (DG) was grafted onto the surfaces of lignin-rich jute fabrics by HRP-mediated oxidative polymerization with an aim to enhance the hydrophobicity of the fibers. The DG-grafted jute fibers and reaction products of their model compounds were characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). The results clearly indicated the grafting of DG to the jute fiber by HRP. Furthermore, the hydrophobicity of jute fabrics was determined by measuring the wetting time and static contact angle. Compared to the control sample, the wetting time and static contact angle of the grated fabrics changed from ~1 s to 1 h and from ~0° to 123.68°, respectively. This clearly proved that the hydrophobicity of jute fabrics improved considerably. Conditions of the HRP-catalyzed DG-grafting reactions were optimized in terms of the DG content of modified jute fabrics. Moreover, the results of breaking strength and elongation of DG-grafted jute/ polypropylene (PP) composites demonstrated improved reinforcement of the composite due to enzymatic hydrophobic modification of jute fibers.

Efeito do calor na atividade da polifenol oxidase e peroxidase em algumas frutas e hortaliças

O objetivo deste trabalho foi estudar o efeito de tratamentos térmicos na atividade da polifenol oxidase e da paroxidade em algumas frutas e hortaliças, bem como estudar a possível regeneração dessas enzimas após apertização. Em termos gerais, nas frutas a polifenol oxidase apresentou maior resistência à inativação pelo calor que a peroxidase e no caso das hortaliças ocorreu o inverso. Quanto à regeneração das enzimas após apertização, o fenômeno foi constatado somente no caso da peroxidase que mostrou assim grande estabilidade às condições adversas durante aquele tratamento térmico. A polifenol oxidase por sua vez, demonstrou ser uma enzima muito sensível, não se regenerando durante o tempo em que os produtos ficaram armazenados.

Efeito do SO2 e do ácido ascórbico na atividade da polifenol oxidase e peroxidase em algumas frutas e hortaliças

No presente trabalho foi estudado o efeito do SO2 e do ácido ascórbico na atividade da polifenol oxidase e peroxidase em algumas frutas e hortaliças, visando determinar as melhores condições para o controle da reação de escurecimento. Para a peroxidase das frutas e hortaliças, o ácido ascórbico foi considerado o melhor método de inativação. Já para a polifenol oxidase das frutas e hortaliças, o SO2 foi mais eficiente, porém mostrou-se inadequado para controlar a atividade da peroxidase em quase todos os produtos estudados. Uma combinação dos compostos utilizados (SQ2 e ácido ascórbico) parece ser o metodo mais indicado para o controle simultâneo da atividade das duas enzimas.

Efeito comparativo do calor, S0(2) e ácido ascórbico na atividade da polifenol oxidase e peroxidase de algumas frutas e hortaliças

O objetivo deste trabalho foi o de comparar o efeito do calor, do SO2 e do ácido ascorbico, visando determinar qual o método mais eficiente para controlar o escurecimento enzímico de cada uma das frutas e hortaliças estudadas. Os resultados mostraram que para a banana, pêssego, maça, cenoura, couve-flor e palmito, o calor foi o melhor agente inativador do sistema enzímico responsável pelo escurecimento. O SO2 foi mais eficiente para a pera, e para o figo e batata, o melhor agente inibidor foi o ácido ascórbico.

Técnica de peroxidase-antiperoxidase (PAP) em biópsia renal: estudo comparativo

Os métodos de imunoperoxidase têm muito em comum com os da imunofluorescência para a demonstração de antígenos teciduais e celulares como no campo das doenças renais relacionadas a imunoglobinas e imunocomplexos. Neste trabalho os autores fazem um estudo comparativo da sensibilidade dos dois métodos (IF e PAP) em tecido de congelação e blocos parafinizados de material de biópsia renal. A análise estatística dos resultados mostrou uma concordãncia significativa entre a IF de congelação e a imunoperoxidase em tecido parafinizado com exceções para a detecção de frações de complemento e de fibrinogênio. Não houve concordância entre a IF em congelação e IF em tecido parafinizado.

Leishmania mexicana amazonensis: heterogeneity in 5-nucleotidase and peroxidase activities of mononuclear phagocytes during in vivo and in vitro infection

The degree of maturation of cells of the Mononuclear Phagocyte System (MPS), during in vivo and in vitro infection by Leishmania mexicana amazonenesis, was evaluated in this study. The macrophages' differentiation was assayed by cytochemical characterization at the ultrastrctural level, using two well-established markers: 5'-nucleotidase enzyme activity, for revealing the mature cells, and the peroxidase activity present in the cell granules to demonstrate immature mononuclear phagocytes. only a few mcrophages, demonstrating 5'-nucleotidase positive reaction in both the plasma membrane and within their cytoplasmic vesicles, were found scattered in the chronic inflammation at the L. m. amazonensis lesions in albino mice. However, by the peroxidase activity analysis, we were also able to demonstrate the presence of immature MPS cells, which predominate, together with parasitized vacuolated macrophages, in chronic lesions induced in this systemby L. m. amazonensis. The implications of these results on the pathogenesis of murine cutaneous leishmaniasis are discussed.

Trypanosoma cruzi: experimental Chagas' disease in Rhesus monkeys. II. Ultraestructural and cytochemical studies of peroxidase and acid phosphatase activities

Ultrastructural and cytochemical studies of peroxidase and acid phosphatase were performed in skin, lymph node and heart muscle tissue of thesus monkeys with experimental Chagas's disease. At the site of inoculation ther was a proliferative reaction with the presence of immature macrophages revealed by peroxidase technique. At the lymph node a difuse inflammatory exudate with mononuclear cells, fibroblasts and immature activated macrophages reproduces the human patrtern of acute Chagas' disease inflamatory lesions. The hearth muscle cells present different degrees of degenerative alterations and a striking increase in the number of lysosomal profiles that exhibit acid hydrolase reaction product. A strong inflammatory reaction was present due to lymphocytic infiltrate or due to eosinophil granulocytes associated to ruptured cells. The present study provides some experimental evidences that the monkey model could be used as a reliable model to characterize histopathological alterations of the human disease.

Dirigent domain-containing protein is part of the machinery required for formation of the lignin-based Casparian strip in the root.

The endodermis acts as a "second skin" in plant roots by providing the cellular control necessary for the selective entry of water and solutes into the vascular system. To enable such control, Casparian strips span the cell wall of adjacent endodermal cells to form a tight junction that blocks extracellular diffusion across the endodermis. This junction is composed of lignin that is polymerized by oxidative coupling of monolignols through the action of a NADPH oxidase and peroxidases. Casparian strip domain proteins (CASPs) correctly position this biosynthetic machinery by forming a protein scaffold in the plasma membrane at the site where the Casparian strip forms. Here, we show that the dirigent-domain containing protein, enhanced suberin1 (ESB1), is part of this machinery, playing an essential role in the correct formation of Casparian strips. ESB1 is localized to Casparian strips in a CASP-dependent manner, and in the absence of ESB1, disordered and defective Casparian strips are formed. In addition, loss of ESB1 disrupts the localization of the CASP1 protein at the casparian strip domain, suggesting a reciprocal requirement for both ESB1 and CASPs in forming the casparian strip domain.

The level of ascorbate peroxidase is enhanced in benznidazole-resistant populations of Trypanosoma cruzi and its expression is modulated by stress generated by hydrogen peroxide

Ascorbate peroxidases (APX) are class I heme-containing enzymes that convert hydrogen peroxide into water molecules. The gene encoding APX has been characterized in 11 strains of Trypanosoma cruzi that are sensitive or resistant to benznidazole (BZ). Bioinformatic analysis revealed the presence of two complete copies of the T. cruzi APX (TcAPX) gene in the genome of the parasite, while karyotype analysis showed that the gene was present in the 2.000-kb chromosome of all of the strains analyzed. The sequence of TcAPX exhibited greater levels of similarity to those of orthologous enzymes from Leishmania spp than to APXs from the higher plant Arabidopsis thaliana. Northern blot and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed no significant differences in TcAPX mRNA levels between the T. cruzi strains analyzed. On the other hand, Western blots showed that the expression levels of TcAPX protein were, respectively, two and three-fold higher in T. cruzi populations with in vitro induced (17 LER) and in vivo selected (BZR) resistance to BZ, in comparison with their corresponding susceptible counterparts. Moreover, the two BZ-resistant populations exhibited higher tolerances to exogenous hydrogen peroxide than their susceptible counterparts and showed TcAPX levels that increased in a dose-dependent manner following exposure to 100 and 200 µM hydrogen peroxide.

The use of proteomics to identify markers associated with metastasis in "Leishmania Viannia" species the role of tryparedoxin peroxidase

RESUME En Amérique Centrale et en Amérique du Sud, la leishmaniose cutanéo-muqueuse (LCM) est provoquée par le protozoaire Leishmania du sous-genre Viannia dont font partie L. (V.) braziliensis, L. (V.) panamensis et L. (V.) guyanensis. Dans la LCM, après guérison apparente de la lésion primitive, des lésions secondaires peuvent apparaître dues à la migration de l'infection à partir du site d'inoculation vers les muqueuses de l'ororhino-pharynx. Ce type de dissémination, communément appelé métastase, peut se produire plusieurs années après la guérison de la lésion cutanée initiale, et est un facteur majeur contribuant à la morbidité associée à la LCM. L'expression reproductible de l'activité métastatique au sein de populations discrètes de leishmanies chez le hamster fournit un modèle expérimental permettant d'étudier le degré de virulence du parasite. Nous avons utilisé des clones de L. (V.) guyanensis présentant des phénotypes stables allant d'un caractère hautement métastatique (M+) à non-métastatique (M-) comme outils pour mettre en évidence des facteurs spécifiques liés à la métastase chez les leishmanies du Nouveau Monde. Des analyses protéomiques comparatives utilisant l'électrophorèse bidimensionnelle sur gel de polyacrylamide couplée à de la spectrométrie de masse ont permis l'identification de plusieurs formes de la tryparedoxine peroxidase (TXNPx) en tant que polypeptides associés au phénotype métastatique. TXNPx, une enzyme de la famille des peroxiredoxines (Prxs), protéines antioxydantes, fonctionne comme la dernière peroxydase d'une cascade d'oxydoréductases qui réduit le peroxyde d'hydrogène aux dépens de NADPH. Toutes les Prxs sont caractérisées par un (1-Cys Prx) ou par deux résidus cystéines (2-Cys Prx), respectivement placés dans un environnement structurel conservé de la protéine et sont centrales dans la réaction catalytique. Des immuno-empreintes (« immunoblotting ») ont révélé que TXNPx est présente sous forme dimérique dans les promastigotes (M+) alors que dans les promastigotes, (M-) TXNPx est présente sous forme monomérique et dimérique. Cette caractéristique spécifique de dimérisation pourrait expliquer les différentes activités enzymatiques observées entre les deux promastigotes (M+) et (M-) en présence de peroxyde d'hydrogène ainsi que leur différence de survie et de charge parasitaire à l'intérieur des macrophages. Par conséquent, le processus métastatique pourrait être lié à la capacité du parasite à échapper efficacement aux défenses microbicides de la cellule hôte. ABSTRACT In South and Central America, protozoan parasites of the Leishmania Viannia subgenus including L. (V.) braziliensis, L. (V.) guyanensis and L. (V). panamensis cause mucocutaneous leishmaniasis (MCL). In MCL, after apparent cure of the primary lesion, secondary lesions may appear in the nasopharyngeal tissues of the infected host due to dissemination of the infection from the inoculation site. This type of dissemination, known as metastasis, can occur several years after healing of the original cutaneous lesion, and is a major contributory factor to the morbidity associated with MCL. The reproducible expression of metastasis by discrete populations of Leishmania parasites in hamsters provides an experimental model to examine the expression of parasite virulence. We used laboratory clones of L. (V.) guyanensis with stable phenotypes ranging from highly metastatic (M+) to non-metastatic (M-) as tools for the discovery of specific factors associated with metastasis in New World Leishmania species. Comparative proteome analyses via 2D-electrophoresis (2-DE) coupled with mass spectrometry (MS) enabled the identification of various isoforms of tryparedoxin peroxidase (TXNPx) as polypeptides associated with the metastatic phenotype. TXNPx, an enzyme related to the antioxidant peroxiredoxin family (Prx) functions as the terminal peroxidase of a redox cascade that reduces hydroperoxides by NADPH. All Prxs are characterized by one (1-Cys Prx) or two cysteine residue(s) (2-Cys Prx), respectively, located in a conserved structural environment of the protein which are central for the catalytic reaction. Immunoblotting analysis revealed that, under non-reducing denaturing conditions, TXNPx is present in dimeric forms in (M+) promastigotes, whereas in (M-) promastigotes, both monomeric and dimeric forms are found. This specific dimerization feature may explain the different enzymatic activities of both (M+) and (M-) promastigote parasites in the presence of H2O2 and their difference in survival and parasite load inside macrophages. Therefore, the metastatic process could be related to the ability of the parasite to efficiently evade the microbicidal effect of the host cell.

AtABCG29 is a monolignol transporter involved in lignin biosynthesis.

Lignin is the defining constituent of wood and the second most abundant natural polymer on earth. Lignin is produced by the oxidative coupling of three monolignols: p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. Monolignols are synthesized via the phenylpropanoid pathway and eventually polymerized in the cell wall by peroxidases and laccases. However, the mechanism whereby monolignols are transported from the cytosol to the cell wall has remained elusive. Here we report the discovery that AtABCG29, an ATP-binding cassette transporter, acts as a p-coumaryl alcohol transporter. Expression of AtABCG29 promoter-driven reporter genes and a Citrine-AtABCG29 fusion construct revealed that AtABCG29 is targeted to the plasma membrane of the root endodermis and vascular tissue. Moreover, yeasts expressing AtABCG29 exhibited an increased tolerance to p-coumaryl alcohol by excreting this monolignol. Vesicles isolated from yeasts expressing AtABCG29 exhibited a p-coumaryl alcohol transport activity. Loss-of-function Arabidopsis mutants contained less lignin subunits and were more sensitive to p-coumaryl alcohol. Changes in secondary metabolite profiles in abcg29 underline the importance of regulating p-coumaryl alcohol levels in the cytosol. This is the first identification of a monolignol transporter, closing a crucial gap in our understanding of lignin biosynthesis, which could open new directions for lignin engineering.

Study of chemical modification of alkaline lignin by the glyoxalation reaction

In this study, glyoxalated alkaline lignins with a non-volatile and non-toxic aldehyde, which can be obtained from several natural resources, namely glyoxal, were prepared and characterized for its use in wood adhesives. The preparation method consisted of the reaction of lignin with glyoxal under an alkaline medium. The influence of reaction conditions such as the molar ratio of sodium hydroxide-to-lignin and reaction time were studied relative to the properties of the prepared adducts. The analytical techniques used were FTIR and 1H-NMR spectroscopies, gel permeation chromatography (GPC), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Results from both the FTIR and 1H-NMR spectroscopies showed that the amount of introduced aliphatic hydroxyl groups onto the lignin molecule increased with increasing reaction time and reached a maximum value at 10 h, and after they began to decrease. The molecular weights remained unchanged until 10 h of reaction time, and then started to increase, possibly due to the repolymerization reactions. DSC analysis showed that the glass transition temperature (Tg) decreased with the introduction of glyoxal onto the lignin molecule due to the increase in free volume of the lignin molecules. TGA analysis showed that the thermal stability of glyoxalated lignin is not influenced and remained suitable for wood adhesives. Compared to the original lignin, the improved lignin is reactive and a suitable raw material for adhesive formula

The suitability of steam exploded vitis vinifera and alkaline lignin for the manufacture of fiberboard

The main objective of this study was to explore the suitability of Vitis vinifera as a raw material and alkaline lignin as a natural binder for fiberboard manufacturing. In the first step, Vitis vinifera was steam- exploded through a thermo-mechanical vapor process in a batch reactor, and the obtained pulp was dried, ground, and pressed to produce the boards. The effects of pretreatment factors and pressing conditions on the chemical composition of the fibers and the physico-mechanical properties of binderless fiberboards were evaluated, and the conditions that optimize these properties were found. A response surface method based on a central composite design and multiple-response optimization was used. The variables studied and their respective variation ranges were: pretreatment temperature (Tr: 190-210ºC), pretreatment time (tr: 5-10 min), pressing temperature (Tp: 190-210ºC), pressing pressure (Pp: 8-16MPa), and pressing time (tp: 3-7min). The results of the optimization step show that binderless fiberboards have good water resistance and weaker mechanical properties. In the second step, fiberboards based on alkaline lignin and Vitis vinifera pulp produced at the optimal conditions determined for binderless fiberboards were prepared and their physico-mechanical properties were tested. Our results show that the addition of about 15% alkaline lignin leads to the production of fiberboards that fully meet the requirements of the relevant standard specifications

Synthesis and characterization of kraft lignin-based epoxy resins

Epoxidization is an interesting way to develop a new application of lignin and therefore to improve its application potential. In this work, kraft lignin-based epoxy resins were obtained by the epoxidization reaction, using the kraft lignin recovered directly from pulping liquor and modified by a methylolation reaction. The methylolated lignins were obtained by the reaction of original kraft lignin with formaldehyde and glyoxal, which is a less volatile and less toxic aldehyde. 1H-NMR spectroscopy showed that methylolated kraft lignin has more hydroxymethyl groups than glyoxalated kraft lignin. For the epoxidization reaction we studied the influence of the lignin:NaOH (w/w) ratio, temperature, and time of the reaction on the properties of the prepared epoxidized lignins. The structures of lignin-based epoxy resins were followed by epoxy index test and FTIR spectroscopy. Optimal conditions were obtained for lignin-based epoxy resin produced at lignin/NaOH = 1/3 at 70 ºC for 3h. Thermogravimetry analysis (TGA) revealed that the epoxidization enhances the thermal stability of lignins and may allow a wider temperature range for applications with lignin epoxy-PF blends