Xylanases from Aspergillus niger, Aspergillus niveus and Aspergillus ochraceus produced under solid-state fermentation and their application in cellulose pulp bleaching

This study describes the production of xylanases from Aspergillus niveus, A. niger, and A. ochraceus under solid-state fermentation using agro-industrial residues as substrates. Enzyme production was improved using a mixture of wheat bran and yeast extract or peptone. When a mixture of corncob and wheat bran was used, xylanase production from A. niger and A. ochraceus increased by 18%. All cultures were incubated at 30 A degrees C at 70-80% relative humidity for 96 h. For biobleaching assays, 10 or 35 U of xylanase/g dry cellulose pulp were incubated at pH 5.5 for 1 or 2 h, at 55 A degrees C. The delignification efficiency was 20%, the brightness (percentage of ISO) increased two to three points and the viscosity was maintained confirming the absence of cellulolytic activity. These results indicated that the use of xylanases could help to reduce the amount of chlorine compounds used in cellulose pulp treatment.

Heterologous expression of an Aspergillus niveus xylanase GH11 in Aspergillus nidulans and its characterization and application

A xylanase was cloned from Aspergillus niveus and successfully expressed in Aspergillus nidulans (XAN). The full-length gene consisted of 890 bp and encoded 275 mature amino acids with a calculated mass of 31.3 kDa. The deduced amino acid sequence was highly homologous with the xylanase belonging to family 11 of the glycoside hydrolases. The recombinant protein was purified to electrophoretic homogeneity by anion-exchange chromatography and gel filtration. The optima of pH and temperature for the recombinant enzyme were 5.0 and 65 degrees C, respectively. The thermal stability of the recombinant xylanase was extremely improved by covalent immobilization on glyoxyl agarose with 91.4% of residual activity after 180 min at 60 degrees C, on the other hand, the free xylanase showed a half-life of 9.9 min at the same temperature. Affinity chromatography on Concanavalin A- and Jacalin-agarose columns followed by SDS-PAGE analyses showed that the XAN has O- and N-glycans. XAN promotes hydrolysis of xylan resulting in xylobiose, xylotriose and xylotetraose. Intermediate degradation of xylan resulting in xylo-oligomers is appealing for functional foods as the beneficial effect of oligosaccharides on gastrointestinal micro flora includes preventing proliferation of pathogenic intestinal bacteria and facilitates digestion and absorption of nutrients. (C) 2011 Elsevier Ltd. All rights reserved.

Production of xylanase by Aspergilli using alternative carbon sources: application of the crude extract on cellulose pulp biobleaching

The ability of xylanolytic enzymes produced by Aspergillus fumigatus RP04 and Aspergillus niveus RP05 to promote the biobleaching of cellulose pulp was investigated. Both fungi grew for 4-5 days in liquid medium at 40A degrees C, under static conditions. Xylanase production was tested using different carbon sources, including some types of xylans. A. fumigatus produced high levels of xylanase on agricultural residues (corncob or wheat bran), whereas A. niveus produced more xylanase on birchwood xylan. The optimum temperature of the xylanases from A. fumigatus and A. niveus was around 60-70A degrees C. The enzymes were stable for 30 min at 60A degrees C, maintaining 95-98% of the initial activity. After 1 h at this temperature, the xylanase from A. niveus still retained 85% of initial activity, while the xylanase from A. fumigatus was only 40% active. The pH optimum of the xylanases was acidic (4.5-5.5). The pH stability for the xylanase from A. fumigatus was higher at pH 6.0-8.0, while the enzyme from A. niveus was more stable at pH 4.5-6.5. Crude enzymatic extracts were used to clarify cellulose pulp and the best result was obtained with the A. niveus preparation, showing kappa efficiency around 39.6% as compared to only 11.7% for that of A. fumigatus.

Application of a species-specific PCR-RFLP to identify Ancylostoma eggs directly from canine faeces

Apart from their veterinary importance, the hookworms Ancylostoma caninum, Ancylostoma braziliense and Ancylostoma caninum are also capable of causing zoonotic disease in humans. A highly sensitive and species-specific PCR-RFLP technique was utilised to detect and differentiate the various canine Ancylostoma spp directly from eggs in faeces. This technique was utilised to screen 101 canine faecal samples from parasite endemic tea growing communities in Assam, India, as part as an ongoing epidemiological investigation into canine parasitic zoonoses. The prevalence of hookworms in dogs was found to be 98% using a combination of PCR and conventional microscopy. Overall, 36% of dogs were found positive for single hookworm infections with A. caninum, 24% positive for single infections with A. braziliense and 38% had mixed infections with both A. caninum and A. braziliense. No dogs were found positive for A. ceylanicum in the community under study. The high prevalence of A. caninum and A. braziliense in dogs in this community may account for the high incidence of cutaneous larva migrans (CLM) observed among the human population residing at the tea estates. The PCR-RFLP technique described herein allows epidemiological screening of canine hookworms to be conducted rapidly, with ease and accuracy, and has the potential to be applied to a number of different clinical, pharmacological and epidemiological situations. (C) 2004 Elsevier B.V. All rights reserved.

Application of Ti/RuO(2)-Ta(2)O(5) electrodes in the electrooxidation of ethanol and derivants: Reactivity versus electrocatalytic efficiency

The influence of the preparation method on the performance of RuO(2)-Ta(2)O(5) electrodes was evaluated toward the ethanol oxidation reaction (EOR). Freshly prepared RuO(2)-Ta(2)O(5) thin films containing between 30 and 80 at.% Ru were prepared by two different methods: the modified Pechini-Adams method (DPP) and standard thermal decomposition (STD). Electrochemical investigation of the electrode containing RuO(2)-Ta(2)O(5) thin films was conducted as a function of electrode composition in a 0.5-mol dm(-3) H(2)SO(4) solution, in the presence and absence of ethanol and its derivants (acetaldehyde and acetic acid). At a low ethanol concentration (5 mmol dm(-3)), ethanol oxidation leads to high yields of acetic acid and CO(2). On the other hand, an increase in ethanol concentration (15-1000 mmol dm(-3)) favors acetaldehyde formation, so acetic acid and CO(2) production is hindered, in this case. Electrodes prepared by DPP provide higher current efficiency than STD electrodes for all the investigated ethanol concentrations. This may be explained by the increase in electrode area obtained with the DPP preparation method compared with STD. (c) 2008 Elsevier Ltd. All rights reserved.

Development of nanostructured bioanodes containing dendrimers and dehydrogenases enzymes for application in ethanol biofuel cells

This paper describes the use of the electrostatic layer-by-layer (LbL) technique for the preparation of bioanodes with potential application in ethanol/O(2) biofuel cells. More specifically, the LbL technique was employed for immobilization of dehydrogenase enzymes and polyamidoamine (PAMAM) dendrimers onto carbon paper support. Both mono (anchoring only the enzyme alcohol dehydrogenase, ADH) and bienzymatic (anchoring both ADH and aldehyde dehydrogenase, AldDH) systems were tested. The amount of ADH deposited onto the Toray (R) paper was 95 ng cm(-2) per bilayer. Kinetic studies revealed that the LbL technique enables better control of enzyme disposition on the bioanode, as compared with the results obtained with the bioanodes prepared by the passive adsorption technique. The power density values achieved for the mono-enzymatic system as a function of the enzyme load ranged from 0.02 to 0.063 mW cm(-2) for the bioanode containing 36 ADH bilayers. The bioanodes containing a gas diffusion layer (GDL) displayed enhanced performance, but their mechanical stability must be improved. The bienzymatic system generated a power density of 0.12 mW cm(-2). In conclusion, the LbL technique is a very attractive approach for enzyme immobilization onto carbon platform, since it enables strict control of enzyme disposition on the bioanode surface with very low enzyme consumption. (C) 2010 Elsevier B.V. All rights reserved.

HPLC-FLD methods to quantify chloroaluminum phthalocyanine in nanoparticles, plasma and tissue: application in pharmacokinetic and biodistribution studies

Analytical and bioanalytical methods of high-performance liquid chromatography with fluorescence detection (HPLC-FLD) were developed and validated for the determination of chloroaluminum phthalocyanine in different formulations of polymeric nanocapsules, plasma and livers of mice. Plasma and homogenized liver samples were extracted with ethyl acetate, and zinc phthalocyanine was used as internal standard. The results indicated that the methods were linear and selective for all matrices studied. Analysis of accuracy and precision showed adequate values, with variations lower than 10% in biological samples and lower than 2% in analytical samples. The recoveries were as high as 96% and 99% in the plasma and livers, respectively. The quantification limit of the analytical method was 1.12 ng/ml, and the limits of quantification of the bioanalytical method were 15 ng/ml and 75 ng/g for plasma and liver samples, respectively. The bioanalytical method developed was sensitive in the ranges of 15-100 ng/ml in plasma and 75-500 ng/g in liver samples and was applied to studies of biodistribution and pharmacokinetics of AlClPc. (C) 2011 Elsevier B.V. All rights reserved.

Lipid microspheres loaded with antigenic membrane proteins of the Leishmania amazonensis as a potential biotechnology application

Lipid microspheres (LM) are excellent drug delivery or vaccines adjuvant systems and are relatively stable. The aim of this work is to develop and characterize a system that is able to encapsulate and present antigenic membrane proteins from Leishmania amazonensis. Membrane proteins are important for vaccine`s formulation because these proteins come in contact with the host cell first, triggering the cell mediated immune response. This is a useful tool to avoid or inactivate the parasite invasion. The LM are constituted by soybean oil (SO), dipalmitoylphosphatidilcholine (DPPC), cholesterol and solubilized protein extract (SPE). The particles formed presented an average diameter of 200 run, low polydispersion and good stability for a period of 30 days, according to dynamic light scattering assays. Isopycnic density gradient centrifugation of LM-protein showed that proteins and lipids floated in the sucrose gradient (5-50%w/v) suggesting that the LM-protein preparation was homogeneous and that the proteins are interacting with the system. The results show that 85% of SPE proteins were encapsulated in the LM. Studies of cellular viability of murine peritoneal macrophages show that our system does not present cytotoxic effect for the macrophages and still stimulates their NO production (which makes its application as a vaccine adjuvant possible). LM-protein loaded with antigenic membrane proteins from L. amazonensis seems to be a promising vaccine system for immunization against leishmaniasis. (C) 2009 Elsevier Inc. All rights reserved.

Production of fibrolytic enzymes by Aspergillus japonicus C03 using agro-industrial residues with potential application as additives in animal feed

Solid-state fermentation obtained from different and low-cost carbon sources was evaluated to endocellulases and endoxylanases production by Aspergillus japonicus C03. Regarding the enzymatic production the highest levels were observed at 30 degrees C, using soy bran added to crushed corncob or wheat bran added to sugarcane bagasse, humidified with salt solutions, and incubated for 3 days (xylanase) or 6 days (cellulase) with 70% relative humidity. Peptone improved the xylanase and cellulase activities in 12 and 29%, respectively. The optimum temperature corresponded to 60 degrees C and 50-55 degrees C for xylanase and cellulase, respectively, both having 4.0 as optimum pH. Xylanase was fully stable up to 40 degrees C, which is close to the rumen temperature. The enzymes were stable in pH 4.0-7.0. Cu(++) and Mn(++) increased xylanase and cellulase activities by 10 and 64%, respectively. A. japonicus C03 xylanase was greatly stable in goat rumen fluid for 4 h during in vivo and in vitro experiments.

Late-season non-selective herbicide application reduces Lolium rigidum seed numbers, seed viability, and seedling fitness

Experiments were conducted to investigate the effect of Lolium rigidum (annual ryegrass) seed developmental stage and application rate of glyphosate and SpraySeed (paraquat 135 g/L+ diquat 115 g/L) on the number, germinability, and fitness of seeds produced. Glyphosate (450 g/L) was most effective when applied at a rate of 0.5-1 L/ha during heading and anthesis, reducing the number of filled seeds produced compared with unsprayed plants. Application post-anthesis, when seeds were at the milk to soft dough stage, was less effective. SpraySeed was most effective when applied post-anthesis, during the milk and early dough stages of seed development at a rate of 0.5-1L/ha, resulting in the production of few viable seeds. Although some filled seeds were produced, most of the seeds were dead. Application during anthesis or once the seeds reached soft dough stage was less effective. For both herbicides, those seeds that were capable of germinating were smaller and had slower radicle and coleoptile growth, resulting in slower early seedling growth and reduced biomass production within the first month of growth. Additionally, glyphosate application reduced the proportion of seeds exhibiting dormancy. The anticipated reduction in seed competitive ability and altered emergence timing resulting from late-season herbicide application, even when application timing is not optimal, could be exploited to reduce the likelihood of successful L. rigidum establishment in the following season.

Application of biotechnology in breeding lentil for resistance to biotic and abiotic stress

Lentil is a self-pollinating diploid (2n = 14 chromosomes) annual cool season legume crop that is produced throughout the world and is highly valued as a high protein food. Several abiotic stresses are important to lentil yields world wide and include drought, heat, salt susceptibility and iron deficiency. The biotic stresses are numerous and include: susceptibility to Ascochyta blight, caused by Ascochyta lentis; Anthracnose, caused by Colletotrichum truncatum; Fusarium wilt, caused by Fusarium oxysporum; Sclerotinia white mold, caused by Sclerotinia sclerotiorum; rust, caused by Uromyces fabae; and numerous aphid transmitted viruses. Lentil is also highly susceptible to several species of Orabanche prevalent in the Mediterranean region, for which there does not appear to be much resistance in the germplasm. Plant breeders and geneticists have addressed these stresses by identifying resistant/tolerant germplasm, determining the genetics involved and the genetic map positions of the resistant genes. To this end progress has been made in mapping the lentil genome and several genetic maps are available that eventually will lead to the development of a consensus map for lentil. Marker density has been limited in the published genetic maps and there is a distinct lack of co-dominant markers that would facilitate comparisons of the available genetic maps and efficient identification of markers closely linked to genes of interest. Molecular breeding of lentil for disease resistance genes using marker assisted selection, particularly for resistance to Ascochyta blight and Anthracnose, is underway in Australia and Canada and promising results have been obtained. Comparative genomics and synteny analyses with closely related legumes promises to further advance the knowledge of the lentil genome and provide lentil breeders with additional genes and selectable markers for use in marker assisted selection. Genomic tools such as macro and micro arrays, reverse genetics and genetic transformation are emerging technologies that may eventually be available for use in lentil crop improvement.

Mode of action of back-line application of spinosad, a non-systemic parasiticide, on sheep

Spinosad, applied as a jetting solution or dip is an efficacious, non-systemic treatment for the control of Bovicola ovis in sheep. This paper describes the effect of back-line treatment width and group housing of animals on the efficacy of spinosad for the control of lice. A 0.4 mg/kg liveweight dose was found to be the suboptimal dose of spinosad for the control of body lice in a dose titration study and was used to investigate application and housing effects in a second study. Lousy Merino sheep were treated with either a narrow 3-cm application of spinosad or with a wider 25-cm swathe. After treatment they were either kept alone or in groups of 6 sheep per pen. Lice were counted at day 0 and every 14 days to 70 days after treatment before estimation of the percentage of lice control and analysis of treatment effects. A much higher percentage of lice control was achieved with 0.4 mg/kg in the second study than in the first, possibly because of differences in formulation used. The wider application width gave significantly higher (P < 0.05) control of lice than the narrow application when sheep were either housed alone or in groups up to day 42 post-treatment. Greater control of lice was seen in group-housed sheep compared with sheep housed individually (P < 0.05) up to day 70. Using broader application widths combined with holding the animals together after treatment with pour-on formulations may optimise the delivery and efficacy of ectoparasiticides for livestock.

An investigation of the anti-pronation effect of two taping methods after application and exercise

This study investigated the effect of two anti-pronation taping techniques on vertical navicular height, an indicator of foot pronation, after its application and 20 min of exercise. The taping techniques were: the low dye (LD) and low dye with the addition of calcaneal slings and reverse sixes (LDCR). A repeated measures study was used. It found that LDCR was superior to LD and control immediately after application and exercise. LD was better than control immediately after application but not after exercise. These findings provide practical directions to clinicians regularly using anti-pronation taping techniques.

Plant transformation: Problems and strategies for practical application

Plant transformation is now a core research tool in plant biology and a practical tool for cultivar improvement. There are verified methods for stable introduction of novel genes into the nuclear genomes of over 120 diverse plant species. This review examines the criteria to verify plant transformation; the biological and practical requirements for transformation systems; the integration of tissue culture, gene transfer, selection, and transgene expression strategies to achieve transformation in recalcitrant species; and other constraints to plant transformation including regulatory environment, public perceptions, intellectual property, and economics. Because the costs of screening populations showing diverse genetic changes can far exceed the costs of transformation, it is important to distinguish absolute and useful transformation efficiencies. The major technical challenge facing plant transformation biology is the development of methods and constructs to produce a high proportion of plants showing predictable transgene expression without collateral genetic damage. This will require answers to a series of biological and technical questions, some of which are defined.