Extraction, purification by cation exchange supermacroporous cryogel and physico-chemical characterization of γ-conglutin from lupin seeds (Lupinus albus L.).

This study focused on the extraction, purification, and physicochemical characterization of γ-conglutin, a protein present in lupin seeds with properties of reducing blood glucose levels. Total protein was extracted with an alkaline-saline solvent, followed by isoelectric precipitation. Chromatographic purification of the precipitated fraction was performed using a cation exchange supermacroporous cryogel column. Electrophoresis of the eluted fraction from chromatography presented a single band of ∼48 kDa under non-reducing conditions (two bands of ∼30 and ∼17 kDa, under reducing conditions) confirming the success of the purification protocol. Liquid chromatography-tandem mass spectrometry analysis confirmed the identity of the protein as γ-conglutin. The purified γ-conglutin had an isoelectric point of 7.51, ß-sheets prevailing as a secondary structure, and denaturation temperature close to 68°C. The outcome of this work showed that γ-conglutin was obtained with a high degree of purity. The proposed purification protocol is simple and can be easily scaled up.

Synbiotic VSL#3 and yacon-based product modulate the intestinal microbiota and prevent the development of pre-neoplastic lesions in a colorectal carcinogenesis model.

Colorectal cancer is a public health problem, with dysbiosis being one of the risk factors due to its role in intestinal inflammation. Probiotics and synbiotics have been used in order to restore the microbiota balance and to prevent colorectal carcinogenesis. We aimed to investigate the effects of the probiotic VSL#3® alone or in combination with a yacon-based prebiotic concentrate on the microbiota modulation and its influence on colorectal carcinogenesis in an animal model. C57BL/6J mice were divided into three groups: control (control diet), probiotic (control diet + VSL#3®), and synbiotic (yacon diet + VSL#3®). The diets were provided for 13 weeks and, from the third one, all animals were subjected to induction of colorectal cancer precursor lesions. Stool samples were collected to evaluate organic acids, feces pH, ß-glucuronidase activity, and microbiota composition. The colon was used to count pre-neoplastic lesions and to determine the cytokines. The microbiota composition was influenced by the use of probiotic and synbiotic. Modifications were also observed in the abundance of bacterial genera with respect to the control group, which confirms the interference of carcinogenesis in the microbiota. Pre-neoplastic lesions were reduced by the use of the synbiotic, but not with the probiotic. The protection provided by the synbiotic can be attributed to the modulation of the intestinal inflammatory response, to the inhibition of a pro-carcinogenic enzyme, and to the production of organic acids. The modulation of the composition and activity of the microbiota contributed to beneficial changes in the intestinal microenvironment, which led to a reduction in carcinogenesis. KEY POINTS: • Synbiotic reduces the incidence of colorectal cancer precursor lesions. • Synbiotic modulates the composition and activity of intestinal microbiota. • Synbiotic increases the abundance of butyrate-producing bacteria.

Impact of protein blocking on enzymatic saccharification of bagasse from sugarcane clones.

Lignin plays an important functional and structural role in plants, but also contributes to the recalcitrance of lignocellulosic biomass to hydrolysis. This study addresses the influence of lignin in hydrolysis of sugarcane bagasse from conventional bred lines (UFV260 and UFV204) that were selected from 432 field-grown clones. In addition to higher sugar production, bagasse clone UFV204 had a small, but statistically significant, lower insoluble lignin content compared with clone UFV260 (15.5% vs, 16.6%) and also exhibited a significantly higher cellulose conversion to glucose (81.3% vs. 63.3%) at a cellulase loading of 5 (filter paper unit) FPU/g of glucan or 3 FPU/g total solids for liquid hot water pretreated bagasse (200°C, 10 min). The enzyme loading was further decreased by 50% to 2.5 FPU/g glucan and resulted in a similar glucan conversion (88.5%) for clone UFV204 when the bagasse was preincubated with bovine serum albumin at pH 4.8 and nonproductive binding of cellulase components was blocked. Comparison of Langmuir adsorption isotherms and differential adsorption of the three major cellulolytic enzyme components endoglucanase, cellobiohydrolase, and ß-glucosidase help to explain differences due to lignin content.

Engineered GH11 xylanases from Orpinomyces sp. PC-2 improve techno-functional properties of bread dough.

BACKGROUND: Endo-1,4-ß-xylanases have marked hydrolytic activity towards arabinoxylans. Xylanases (xynA) produced by the anaerobic fungus Orpinomyces sp. strain PC-2 have been shown to be superior in specific activity, which strongly suggests their applicability in the bakery industry for the processing of whole-wheat flour containing xylans. In the present study, two xylanases from this source, the small wild-type xylanase SWT and the small mutant xylanase SM2 (V108A, A199T), were expressed in Escherichia coli, purified, characterized, tested for their ability to hydrolyze whole-wheat flour and applied in dough processing. RESULTS: Both purified SM2 and SWT showed high specific activity against oat spelt xylan and wheat arabinoxylan, exhibiting maximum activity at pH 3-7 and 60 °C. SM2 was more thermostable than SWT, which suggests that the mutations enhanced its stability. Both SWT and SM2 were able to hydrolyze whole-wheat flour, and evaluation of their applicability in dough processing by the sponge method indicated that use of these enzymes increased dough volume by 60% and reduced texture hardness by more than 50%, while gumminess and chewiness were reduced by 40%. CONCLUSION: The recombinant xylanases showed potential for application in bakery processing and can improve techno-functional properties in sponges. © 2018 Society of Chemical Industry.

Comparative analysis of Chrysoporthe cubensis exoproteomes and their specificity for saccharification of sugarcane bagasse.

The phytopathogenic fungus Chrysoporthe cubensis is a relevant source of lignocellulolytic enzymes. This work aimed to compare the profile of lignocellulose-degrading proteins secreted by C. cubensis grown under semi-solid state fermentation using wheat bran (WB) and sugarcane bagasse (SB). The exoproteomes of the fungus grown in wheat bran (WBE) and sugarcane bagasse (SBE) were qualitative and quantitatively analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Data are available via ProteomeXchange with identifier PXD046075. Label-free proteomic analysis of WBE and SBE showed that the fungus produced a spectrum of carbohydrate-active enzymes (CAZymes) with exclusive characteristics from each extract. While SBE resulted in an enzymatic profile directed towards the depolymerization of cellulose, the enzymes in WBE were more adaptable to the degradation of biomass rich in hemicellulose and other non-lignocellulosic polymers. Saccharification of alkaline pre-treated sugarcane bagasse with SBE promoted glucose release higher than commercial cocktails (8.11 g L-1), while WBE promoted the higher release of xylose (5.71 g L-1). Our results allowed an in-depth knowledge of the complex set of enzymes secreted by C. cubensis responsible for its high lignocellulolytic activity and still provided the identification of promising target proteins for biotechnological applications in the context of biorefinery.

Effect of lactose on the in vitro development of sheep secondary follicles.

Considering that follicular development is an energy-dependent process, supplementation of the culture medium with energy substrates, such as lactose, would improve follicle viability and growth. Thus, the aim of this study was to evaluate the effect of lactose on morphology, development, glutathione (GSH) concentration, mitochondrial activity, DNA fragmentation, and meiotic resumption of oocytes from sheep secondary follicles cultured in vitro. Secondary follicles were isolated from the cortex of ovine ovaries and cultured individually for 18 days in α-MEM supplemented with bovine serum albumin (BSA), insulin, glutamine, hypoxanthine, transferrin, selenium and ascorbic acid (control medium: α-MEM+) or in α-MEM+ plus different concentrations of lactose (0.025, 0.05 and 0.1 M). After culture, some of the oocytes were subjected to TUNEL assay and in vitro maturation (IVM). Follicular morphology, glutathione (GSH) concentration and mitochondrial activity were evaluated at the end of the culture. At the day 18, the percentage of morphologically normal follicles was greater (P<0.05) in the treatment of 0.025 M lactose (92.5 %) compared to the control group (75.55 %). In addition, GSH concentrations increased (P<0.05) in treatment containing 0.025 M lactose compared to the other treatments. Furthermore, oocytes cultured in 0.025 M lactose had greater (P<0.05) mitochondrial activity levels than in α-MEM+ and 0.1 M lactose. The group α-MEM+ presented a increase of TUNEL-positive oocytes (35.09 %) compared to 0.025 lactose (9.09 %). The percentage of meiotic resumption was greater (P<0.05) in oocytes from secondary follicles cultured in 0.025 M lactose (54.5 %) than in α-MEM+ (45.5 %). In conclusion, 0.025 M lactose improved survival, GSH and active mitochondria levels and meiotic resumption of oocytes from in vitro cultured secondary follicles. Supplementation of the culture medium of preantral follicles with lactose can gradually provide energy to follicular cells, potentially enhancing the production of viable oocytes for biotechniques such as IVM and in vitro fertilization.

Exploring the impact of short daily haemodialysis on muscle strength and bone health in end-stage kidney disease patients.

BACKGROUND: Short-daily haemodialysis (SDH) has been strongly recommended over conventional haemodialysis (CHD) for end-stage kidney disease patients, though few studies have directly compared the effects of these two haemodialysis (HD) modalities on clinical variables related to patient's health. METHODS: We conducted a cross-sectional study in individuals undergoing HD, comparing epidemiological, clinical, metabolic, inflammatory, anthropometric, bone health/metabolism, and skeletal muscle function according to dialysis modality. One-hundred seventy-eight patients (20.8% females, 62 ± 2.5 years old), were analysed in this study, 86 (48%) of whom were undergoing CHD versus 92 (51%) who were undergoing SDH. RESULTS: SDH patients had significantly higher serum albumin levels (3.93 vs. 3.66 g/dL, P < 0.0001) and higher Kt/v (2.6 vs. 2.38, P < 0.0001). SDH group presented a significantly lower number of erythropoietin-stimulating agents compared with CHD group (percentage: 53.3 vs. 83.7%, P < 0.0001) and had lower levels of serum phosphate (4.9 vs. 5.3 mg/dL, P = 0.004) and parathyroid hormone (PTH) (398.4 vs. 480.4 pg/mL, P < 0.001) compared with CHD patients. In terms of bone health and metabolism, SDH patients had significantly higher total BMD, femur BMD, lumbar BMD, and femoral neck BMD compared with CHD patients (all P < 0.05). SDH patients also had lower anti-osteogenic and inflammatory biomarkers, including FGF23, sclerostin, TNF, IL-18, IL-17a, and C-reactive peptide (all P < 0.05). CHD modality was demonstrated to be a risk factor for low BMD (odds ratio: 4.02; 95% CI: 1.59-10.2, P = 0.003). In terms of skeletal muscle function, SDH patients had significantly higher 6-minute walking test (444.6 vs. 424.9 m, P = 0.04) and higher fat-free mass (52.3 vs. 51.68 kg, P = 0.02) compared with CHD patients. Higher fat-free mass and handgrip strength were associated with a 34% and 23% lower risk of low BMD, respectively. SDH patients had lower levels of the uremic toxin asymmetric dimethyl-l-arginine (ADMA) (1.8 vs. 2.07 µM, P = 0.002) and fasting blood glucose (132.6 vs. 141.7 mg/dL, P < 0.02) than CHD group. SDH patients also displayed higher levels of haemoglobin when compared with CHD group (11.9 vs. 10.2 g/dL, P < 0.0001). CONCLUSIONS: The present study improves our understanding of the relationship between dialysis modality and clinical variables that may influence HD patient's health. Grip strength and lean mass were positively correlated with bone mineral density in HD patients regardless of dialysis modality. SDH was associated with better bone mineral density, inflammatory profile, and skeletal muscle function when compared with CHD patients. These findings provide more evidence of the clinical benefits of SDH that should be explored in greater detail.

Simultaneous saccharification and fermentation (SSF) of Jatropha curcas shells: utilization of co-products from the biodiesel production process.

Jatropha curcas has great potential as an oil crop for use in biodiesel applications, and the outer shell is rich in lignocellulose that may be converted to ethanol, giving rise to the concept of a biorefinery. In this study, two dilute pretreatments of 0.5% H(2)SO(4) and 1.0% NaOH were performed on Jatropha shells with subsequent simultaneous saccharification and fermentation (SSF) of the pretreated water-insoluble solids (WIS) to evaluate the effect of inhibitors in the pretreatment slurry. A cellulase loading of 15 FPU/g WIS, complimented with an excess of cellobiase (19.25 U/g), was used for SSF of either the washed WIS or the original slurry to determine the effect of inhibitors. Ethanol and glucose were monitored during SSF of 20 g of pretreated biomass. The unwashed slurry showed to have a positive effect on SSF efficiency for the NaOH-pretreated biomass. Maximum efficiencies of glucan conversion to ethanol in the WIS were 40.43% and 41.03% for the H(2)SO(4)- and NaOH-pretreated biomasses, respectively.

Penicillium Ochrochloron RLS11 Secretome Containing Carbohydrate-Active Enzymes Improves Commercial Enzyme Mixtures During Sugarcane Straw Saccharification.

Filamentous fungi are prolific producers of carbohydrate-active enzymes (CAZymes) and important agents that carry out plant cell wall degradation in natural environments. The number of fungal species is frequently reported in the millions range, with a huge diversity and genetic variability, reflecting on a vast repertoire of CAZymes that these organisms can produce. In this study, we evaluated the ability of previously selected ascomycete and basidiomycete fungi to produce plant cell wall-degrading enzyme (PCWDE) activities and the potential of the culture supernatants to increase the efficiency of the Cellic® CTec2/HTec2 for steam-exploded sugarcane straw saccharification. The culture supernatant of Penicillium ochrochloron RLS11 showed a promising supplementation effect on Cellic® CTec2/HTec2, and we conducted the whole-genome sequencing and proteomic analysis for this fungus. The size of the assembled genome was 38.06 Mbp, and a total of 12,015 protein-coding genes were identified. The repertoire of PCWDE-coding genes was comparatively high among Penicillium spp. and showed an expansion in important cellulases and xylanases families, such as GH3, GH6, GH7, and GH11. The proteomic analysis indicated cellulases that probably enhanced the biomass saccharification performance of the Cellic® CTec2/HTec2, which included enzymes from GH3, GH6, and GH7 families.

Comprehensive analysis of the GALACTINOL SYNTHASE (GolS) gene family in citrus and the function of CsGolS6 in stress tolerance.

Galactinol synthase (GolS) catalyzes the first and rate-limiting step in the synthesis of raffinose family of oligosaccharides (RFOs), which serve as storage and transport sugars, signal transducers, compatible solutes and antioxidants in higher plants. The present work aimed to assess the potential functions of citrus GolS in mechanisms of stress response and tolerance. By homology searches, eight GolS genes were found in the genomes of Citrus sinensis and C. clementina. Phylogenetic analysis showed that there is a GolS ortholog in C. clementina for each C. sinensis GolS, which have evolved differently from those of Arabidopsis thaliana. Transcriptional analysis indicated that most C. sinensis GolS (CsGolS) genes show a low-level tissue-specific and stress-inducible expression in response to drought and salt stress treatments, as well as to 'Candidatus Liberibacter asiaticus' infection. CsGolS6 overexpression resulted in improved tobacco tolerance to drought and salt stresses, contributing to an increased mesophyll cell expansion, photosynthesis and plant growth. Primary metabolite profiling revealed no significant changes in endogenous galactinol, but different extents of reduction of raffinose in the transgenic plants. On the other hand, a significant increase in the levels of metabolites with antioxidant properties, such as ascorbate, dehydroascorbate, alfa-tocopherol and spermidine, was observed in the transgenic plants. These results bring evidence that CsGolS6 is a potential candidate for improving stress tolerance in citrus and other plants.

First report on the enzymatic profile of Kretzschmaria zonata.

Enzymes from phytopathogenic fungi are desirable for biotechnological applications and a highly virulent phytopathogen shows great appeal for enzymes production. To assess the biotechnological potential of Kretzschmaria zonata, a plant pathogenic fungus, we analyzed its enzymatic profile after growth on six different types of lignocellulosic biomasses. The fungus was able to produce a wide variety of enzymes with superior xylanase activity. The corn cob induced the highest specific activity of xylanase, 56.30 U/mg of protein, as well as other important enzymatic activities such as endoglucanase, 11.20 U/mg of protein; pectinase, 4.52 U/mg of protein; and ß-glucosidase, 2.77 U/mg of protein. The highest release of xylose, 0.88 g/L, was observed after saccharification of 10% of alkaline pretreated sugarcane bagasse by a commercial cocktail supplemented with the crude extract from K. zonata after growth on corn cob. The fungus extract is rich in hemicellulases and accessory enzymes and the result showed synergism between the enzymes present in the commercial mixture and in the K. zonata extract. This is the first report concerning the biotechnological potential of the fungus K. zonata, especially regarding to its ability to produce plant biomass degrading enzymes related to second generation ethanol production.

Secretomic insight into the biomass hydrolysis potential of the phytopathogenic fungus Chrysoporthe cubensis.

The phytopathogenic fungus Chrysoporthe cubensis has a great capacity to produce highly efficient enzymes for the hydrolysis of lignocellulosic biomass. The bioinfosecretome of C. cubensis was identified by computational predictions of secreted proteins combined with protein analysis using 1D-LC-MS/MS. The in silico secretome predicted 562 putative genes capable of encoding secreted proteins, including 273 CAZymes. Proteomics analysis confirmed the existence of 313 proteins, including 137 CAZymes classified as Glycosyl Hydrolases (GH), Polysaccharide Lyases (PL), Carbohydrate Esterases (CE) and Auxiliary Activities enzymes (AA), which indicates the presence of classical and oxidative cellulolytic mechanisms. The enzymes diversity in the extract shows fungal versatility to act in complex biomasses. This study provides an insight into the lignocellulose-degradation mechanisms by C. cubensis and allows the identification of the enzymes that are potentially useful in improving industrial process of bioconversion of lignocellulose. SIGNIFICANCE: Chrysoporthe cubensis is an important deadly canker pathogen of commercially cultivated Eucalyptus species. The effective depolymerisation of the recalcitrant plant cell wall performed by this fungus is closely related to its high potential of lignocellulolytic enzymes secretion. Since the degradation of biomass occurs in nature almost exclusively by enzyme secretion systems, it is reasonable to suggest that the identification of C. cubensis lignocellulolytic enzymes is relevant in contributing to new sustainable alternatives for industrial solutions. As far as we know, this work is the first accurate proteomic evaluation of the enzymes secreted by this species of fungus. The integration of the gel-based proteomic approach, the bioinformatic prediction of the secretome and the analyses of enzymatic activity are powerful tools in the evaluation of biotechnological potential of C. cubensis in producing carbohydrate-active enzymes. In addition, analysis of the C. cubensis secretome grown in wheat bran draws attention to this plant pathogen and its extracellular enzymatic machinery, especially regarding the identification of promising new enzymes for industrial applications. The results from this work allowed for explanation and reinforce previous research that revealed C. cubensis as a strong candidate to produce enzymes to hydrolyse sugarcane bagasse and similar substrates.

Involvement of PTEN and FOXO3a Proteins in the Protective Activity of Protocatechuic Acid Against Cisplatin-Induced Ovarian Toxicity in Mice.

The present study evaluated the effects of protocatechuic acid (PCA) after cisplatin-induced ovarian toxicity in mice and if PTEN and FOXO3a proteins are involved in PCA action. The mice were divided into five experimental groups (five animals per group) and treated once a day for 3 days as follows: (1) the control group was pretreated with oral administration (o.p.) of saline solution, followed by an intraperitoneal (i.p.) injection of saline solution. The other groups were pretreated (o.p.) with (2) saline solution (cisplatin group), (3) N-acetylcysteine (150 mg/kg of body weight), or with (4) 20 or (5) 50 mg/kg body weight of PCA, followed by 5 mg/kg body weight (i.p.) of cisplatin. Next, the ovaries were destined to histological (morphology and activation), immunohistochemical (PCNA and cleaved caspase-3 expression), and fluorescence (reactive oxygen species [ROS], glutathione [GSH], and active mitochondria levels) analyses. Moreover, the immunoreactivity for p-PTEN and p-FOXO3a was evaluated to investigate a potential mechanism by which PCA could prevent the cisplatin-induced ovarian damage. Pretreatment with N-acetylcysteine or 20 mg/kg PCA before cisplatin preserved the percentage of normal follicles and cell proliferation as observed in the control, reduced apoptosis and ROS levels, and showed higher active mitochondria and GSH levels than the cisplatin treatment (P < 0.05). Moreover, pretreatment with 20 mg/kg PCA decreased cisplatin-induced p-PTEN and increased (P < 0.05) nuclear export of p-FOXO3a. In conclusion, PCA at 20 mg/kg reduced apoptosis, maintained cell proliferation and mitochondrial function, reduced ROS production, and increased GSH expression likely through the involvement of PTEN and FOXO3a proteins.

Genome-Scale Characterization of Fungal Phytases and a Comparative Study Between Beta-Propeller Phytases and Histidine Acid Phosphatases.

This work intended to prospect new phytase-producing organisms. In silico genomic analyses allowed the selection of twelve potential phytase-producing fungi. Based on gene sequence, it was possible to identify four well-defined groups of phytate-degrading enzymes: esterase-like, ß-propeller phytases (ßPP), phosphoglycerate mutase-like, and phytases of the histidine acid phosphatases (HAP) family. Analysis of the predicted genes encoding phytases belonging to the HAP family and ßPP phytases and in silico characterization of these enzymes indicated divergence among the catalytic activities. Predicted fungal ßPP phytases exhibited higher molecular mass (around 77 kDa) probably due to the epidermal growth factor-like domain. Twelve sequences of phytases contained signal peptides, of which seven were classified as HAP and five as ßPP phytases, while ten sequences were predicted as phytases secreted by non-classical pathways. These fungi were grown in liquid or semi-solid medium, and the fungal enzymatic extracts were evaluated for their ability to hydrolyze sodium phytate at 50 °C and pH ranging from 2.0 to 9.0. Seven fungi were identified as phytase producers based on phosphate release under enzyme assay conditions. Results obtained from in silico analyses combining experimental enzymatic activities suggest that some selected fungi could secrete ßPP phytases and HAP phytases.

Lactococcus lactis expressing either Staphylococcus aureus fibronectin-binding protein A or Listeria monocytogenes internalin A can efficiently internalize and deliver DNA in human epithelial cells.

Lactococci are noninvasive bacteria frequently used as protein delivery vectors and, more recently, as in vitro and in vivo DNA delivery vehicles. We previously showed that a functional eukaryotic enhanced green fluorescent protein (eGFP) expression plasmid vector was delivered in epithelial cells by Lactococcus lactis producing Listeria monocytogenes internalin A (L. lactis InlA(+)), but this strategy is limited in vivo to transgenic mice and guinea pigs. In this study, we compare the internalization ability of L. lactis InlA(+) and L. lactis producing either the fibronectin-binding protein A of Staphylococcus aureus (L. lactis FnBPA(+)) or its fibronectin binding domains C and D (L. lactis CD(+)). L. lactis FnBPA(+) and L. lactis InlA(+) showed comparable internalization rates in Caco-2 cells, while the internalization rate observed with L. lactis CD(+) was lower. As visualized by conventional and confocal fluorescence microscopy, large clusters of L. lactis FnBPA(+), L. lactis CD(+), and L. lactis InlA(+) were present in the cytoplasm of Caco-2 cells after internalization. Moreover, the internalization rates of Lactobacillus acidophilus NCFM and of an NCFM mutant strain with the gene coding for the fibronectin-binding protein (fbpA) inactivated were also evaluated in Caco-2 cells. Similar low internalization rates were observed for both wild-type L. acidophilus NCFM and the fbpA mutant, suggesting that commensal fibronectin binding proteins have a role in adhesion but not in invasion. L. lactis FnBPA(+), L. lactis CD(+), and L. lactis InlA(+) were then used to deliver a eukaryotic eGFP expression plasmid in Caco-2 cells: flow cytometry analysis showed that the highest percentage of green fluorescent Caco-2 cells was observed after coculture with either L. lactis FnBPA(+) or L. lactis InlA(+). Analysis of the in vivo efficiency of these invasive recombinant strains is currently in progress to validate their potential as DNA vaccine delivery vehicles.

A new plasmid vector for DNA delivery using lactococci.

BACKGROUND: The use of food-grade lactococci as bacterial carriers to DNA delivery into epithelial cells is a new strategy to develop live oral DNA vaccine. Our goal was to develop a new plasmid, named pValac, for antigen delivery for use in lactococci. The pValac plasmid was constructed by the fusion of: i) a eukaryotic region, allowing the cloning of an antigen of interest under the control of the pCMV eukaryotic promoter to be expressed by a host cell and ii) a prokaryotic region allowing replication and selection of bacteria. In order to evaluate pValac functionality, the gfp ORF was cloned into pValac (pValac:gfp) and was analysed by transfection in PK15 cells. The applicability of pValac was demonstrated by invasiveness assays of Lactococcus lactis inlA+ strains harbouring pValac:gfp into Caco-2 cells. RESULTS: After transfection with pValac:gfp, we observed GFP expression in PK15 cells. L. lactis inlA+ were able to invade Caco-2 cells and delivered a functional expression cassette (pCMV:gfp) into epithelial cells. CONCLUSION: We showed the potential of an invasive L. lactis harbouring pValac to DNA delivery and subsequent triggering DNA expression by epithelial cells. Further work will be to examine whether these strains are able to deliver DNA in intestinal cells in vivo.

Inhibitors Compounds on Sugarcane Bagasse Saccharification: Effects of Pretreatment Methods and Alternatives to Decrease Inhibition.

Considering bioethanol production, extensive research has been performed to decrease inhibitors produced during pretreatments, to diminish energy input, and to decrease costs. In this study, sugarcane bagasse was pretreated with NaOH, H2SO4, and water. The higher concentration of phenols, 3.3 g/L, was observed in biomass liquid fraction after alkaline pretreatment. Acid pretreatment was responsible to release considerable acetic acid concentration, 2.3 g/L, while water-based pretreatment was the only to release formic acid, 0.02 g/L. Furans derivatives were not detected in liquid fractions regardless of pretreatment. Furthermore, washing step removed most of the phenols from pretreated sugarcane bagasse. Saccharification of alkali-pretreated biomass plus polyethylene glycol (PEG) at 0.4% (w/v) enhanced 8 and 26% the glucose and the xylose release, respectively, while polyvinylpyrrolidone (PVP) also at 0.4% (w/v) increased the release by 10 and 31% of these sugars, respectively, even without washing and filtration steps. Moreover, these polymers cause above 50% activation of endoglucanase and xylanase activities which are crucial for biomass hydrolysis.

Production of Fungal Phytases from Agroindustrial Byproducts for Pig Diets.

The application of phytases for animal feed in developing countries is limited due to the high cost of these enzymes, determined by the importation fees and the expensive substrates used for their production. In this work, we have used agroindustrial byproducts for the production of extracts containing phytases, which were accessed for their stability focusing on the conditions found in the gastrointestinal tract of pigs. The fungus Acremonim zeae presented higher phytase production in medium containing cornmeal, while the yeast Kluyveromyces marxianus produced 10-fold more phytase when cultivated on rice bran. Process optimization increased the difference in productivity to more than 300 fold. The phytase from A. zeae was thermostable, with higher activity at neutral pH and 50 °C, but was inhibited at pH 2.5 and by various ions. The phytase activity in the K. marxianus extract was stable at a wide range of conditions, which indicates the presence of at least two enzymes. As far as we know, this manuscript describes for the first time the phytase production and the characteristics of the extracts produced by both these microbial species. These enzymes could be produced at low cost and have potential to replace enzymes currently imported for this purpose.

Characterization and biotechnological application of an acid alpha-galactosidase from Tachigali multijuga Benth. seeds.

Tachigali multijuga Benth. seeds were found to contain protein (364 mg g(-1)dwt), lipids (24 mg g(-1)dwt), ash (35 mg g(-1)dwt), and carbohydrates (577 mg g(-1)dwt). Sucrose, raffinose, and stachyose concentrations were 8.3, 3.0, and 11.6 mg g(-1)dwt, respectively. alpha-Galactosidase activity increased during seed germination and reached a maximum level at 108 h after seed imbibition. The alpha-galactosidase purified from germinating seeds had an M(r) of 38,000 and maximal activity at pH 5.0-5.5 and 50 degrees C. The enzyme was stable at 35 degrees C and 40 degrees C, but lost 79% of its activity after 30 min at 50 degrees C. The activation energy (E(a)) values for p-nitrophenyl-alpha-d-galactopyranoside (pNPGal) and raffinose were 13.86 and 4.75 kcal mol(-1), respectively. The K(m) values for pNPGal, melibiose, raffinose, and stachyose were 0.45, 5.37, 39.62 and 48.80 mM, respectively. The enzyme was sensitive to inhibition by HgCl(2), SDS, AgNO(3), CuSO(4), and melibiose. d-Galactose was a competitive inhibitor (K(i)=2.74 mM). In addition to its ability to hydrolyze raffinose and stachyose, the enzyme also hydrolyzed galactomannan.

Impact of the removal of N-terminal non-structured amino acids on activity and stability of xylanases from Orpinomyces sp. PC-2.

Xylanases catalyze the random hydrolysis of xylan backbone from plant biomass and thus, they have application in the production of biofuels, Kraft pulps biobleaching and feed industry. Here, xylanases derived from Orpinomyces sp. PC-2 were engineered guided by molecular dynamics methods to obtain more thermostable enzymes. Based on these models, 27 amino acid residues from the N-terminal were predicted to reduce protein stability and the impact of this removal was validated to two enzyme constructs: small xylanase Wild-Type (SWT) obtained from Wild-Type xylanase (WT) and small xylanase Mutant (SM2) generated from M2 mutant xylanase (V135A, A226T). The tail removal promoted increase in specific activity of purified SWT and SM2, which achieved 5,801.7 and 5,106.8Umg-1 of protein, respectively, while the WT activity was 444.1Umg-1 of protein. WT, SWT and SM2 showed half-life values at 50°C of 0.8, 2.3 and 29.5h, respectively. Overall, in view of the results, we propose that the presence of non-structured amino acid in the N-terminal leads to destabilization of the xylanases and may promote less access of the substrate to the active site. Therefore, its removal may promote increased stability and enzymatic activity, interesting properties that make them suitable for biotechnological applications.