Cloning, expression, and characterization of a D-psicose 3-epimerase from Clostridium cellulolyticum H10

The noncharacterized protein ACL75304 encoded by the gene Ccel_0941 from Clostridium cellulolyticum H10 (ATCC 35319), previously proposed as the xylose isomerase domain protein TIM barrel, was cloned and expressed in Escherichia coli . The expressed enzyme was purified by nickel-affinity chromatography with electrophoretic homogeneity and then characterized as d-psicose 3-epimerase. The enzyme was strictly metal-dependent and showed a maximal activity in the presence of Co(2+). The optimum pH and temperature for enzyme activity were 55 °C and pH 8.0. The half-lives for the enzyme at 60 °C were 6.8 h and 10 min when incubated with and without Co(2+), respectively, suggesting that this enzyme was extremely thermostable in the presence of Co(2+) but readily inactivated without metal ion. The Michaelis-Menten constant (K(m)), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) values of the enzyme for substrate d-psicose were estimated to be 17.4 mM, 3243.4 min(-1), and 186.4 mM min(-1), respectively. The enzyme carried out the epimerization of d-fructose to d-psicose with a conversion yield of 32% under optimal conditions, suggesting that the enzyme is a potential d-psicose producer.     

Stability of omega-3 fatty acids in fortified surimi seafoods during chilled storage

Physical, chemical, and sensory properties of cooked surimi seafood gels (crab analogue) fortified with omega-3 fatty acids (FA) were monitored during chilled storage. Three sources of stabilized omega-3 FA (fish oil concentrate, menhaden oil, and a purified marine oil) were each incorporated into gels to an omega-3 FA content of 1.5 or 2.5%, w/w. Omega-3 FA stability, development of off-flavors, and changes in color and texture were monitored during chilled storage for 2 months. Gels with fish oil concentrate developed fishy flavor and aroma within 30 days and were eliminated from the study. Gels containing menhaden oil and purified marine oil exhibited little change in sensory properties or oxidation products throughout 2 months of storage. Relative polyene index values (ratio of polyunsaturated to saturated fatty acids) indicated that the omega-3 FA were stable at both levels of addition. Omega-3 fortified gels were whiter than control gels, and gel texture was modified when menhaden and purified oils were added but not significantly affected by the level of omega-3 addition.     

Enhancement of lutein production in Chlorella sorokiniana (Chorophyta) by improvement of culture conditions and random mutagenesis

Chlorella sorokiniana has been selected for lutein production, after a screening of thirteen species of microalgae, since it showed both a high content in this carotenoid and a high growth rate. The effects of several nutritional and environmental factors on cell growth and lutein accumulation have been studied. Maximal specific growth rate and lutein content were attained at 690 μmol photons m(-2) s(-1), 28 °C, 2 mM NaCl, 40 mM nitrate and under mixotrophic conditions. In general, optimal conditions for the growth of this strain also lead to maximal lutein productivity. High lutein yielding mutants of C. sorokiniana have been obtained by random mutagenesis, using N-methyl-N'-nitro-nitrosoguanidine (MNNG) as a mutagen and selecting mutants by their resistance to the inhibitors of the carotenogenic pathway nicotine and norflurazon. Among the mutants resistant to the herbicides, those exhibiting both high content in lutein and high growth rate were chosen. Several mutants exhibited higher contents in this carotenoid than the wild type, showing, in addition, either a similar or higher growth rate than the latter strain. The mutant MR-16 exhibited a 2.0-fold higher volumetric lutein content than that of the wild type, attaining values of 42.0 mg L(-1) and mutants DMR-5 and DMR-8 attained a lutein cellular content of 7.0 mg g(-1) dry weight. The high lutein yield exhibited by C. sorokiniana makes this microalga an excellent candidate for the production of this commercially interesting pigment.     

Molecular detection of Coxiella burnetii and Neospora caninum in equine aborted foetuses and neonates

Abortion, stillbirth and neonatal death are major causes of equine mortality and cause severe economic loss to the equine industry. The present study was based on a complete necropsy protocol associated with classical microbiological examinations and molecular biology on 407 cases of abortion, stillbirths and neonate death. Based on this retrospective survey, "less common" abortive infectious agents were characterised by molecular tools in nine independent cases of abortion or neonate mortality. Among others, Chlamydophila abortus (1 case), Coxiella burnetii (6 cases) and Neospora caninum (3 cases) were detected by real-time PCR; one of these samples being co-infected by N. caninum and C. burnetii. DNA detection of this latter bacterium is reported here for the first time in equine abortion samples. C. burnetii should, along with other common pathogens, probably be taken into account in equine abortion.     

Application of a molecularly imprinted polymer for the extraction of kukoamine a from potato peels

A molecularly imprinted polymer (MIP) for the purification of N(1),N(12)-bis(dihydrocaffeoyl)spermine (kukoamine A) was computationally designed and tested. The properties of the polymer were characterized. The protocol of the solid phase extraction (SPE) of kukoamine A from potato peels was optimized. A HPLC-MS method for the quantification of kukoamine A was developed and used for all optimization studies. The capacity of the MIP in relation to kukoamine A from the potato peels extract was estimated at 54 mg/g of the polymer. The kukoamine A purified from potato extract using MIP was exceptionally pure (≈ 90%). Although the corresponding blank polymer was less selective than the MIP for the extraction of kukoamine A from the potato extract, it was shown that the blank polymer could be effectively used for the purification of the crude synthetic kukoamine (polymer capacity = 80 mg of kukoamine A/g of the adsorbent, kukoamine A purity ≈ 86%). Therefore, selective adsorbents could be computationally designed for other plant products, allowing their purification in quantities that would be sufficient for more detailed studies and potential practical applications.     

An outbreak of Francisella tularensis in captive prairie dogs: an immunohistochemical analysis.

An immunohistochemical assay was developed and tested for detection of Francisella tularensis lipopolysaccaride antigen in tissues of captive prairie dogs (Cynomys ludovicianus). Tissues from 59 cases of F. tularensis were examined by this technique, which was corroborated by direct fluorescent antibody assay and direct isolation of the organism. In infected prairie dogs, studies indicated multiple, severe, necroprurulent foci occurring in the liver, lung, spleen, terminal ileum, and mandibular lymph node. Immunohistochemical analysis of the same formalin-fixed tissues indicated the presence of F. tularensis antigen in neutrophils and macrophages of these lesions and occurring extracellularly in areas of necrosis. This report demonstrates that immunohistochemical analysis is a rapid procedure that can be used to determine the pathogenesis of F. tularensis in rodent populations.     

Pre-parturition staphylococcal mastitis in primiparous replacement goats: persistence over lactation and sources of infection

This investigation reported for the first time the occurrence of intramammary infections caused by Staphylococcus in primiparous replacement goats before parturition and the persistence of clinical Staphylococcus aureus infection during the lactation period. Subclinical infections, mainly caused by coagulase negative staphylococci (CoNS), did not persist during lactation. Genotyping analysis indicated that environment seems to play a moderate role as source of intramammary infections to goats before parturition, but causative agents of mastitis in lactating animals are not genotypically related to environmental staphylococci. The occurrence and persistence of intramammary infections in replacement goats demonstrate the need to consider those animals as potential sources of infections in dairy goat herds.     

Manure-Based Organic Soil Amendments Provide an Adequate Nutritional Source for Corn Production in Chile When Combined with Urea Applications

ABSTRACT

The use of manure-based organic soil amendments is common in countries with food animal production. A two-season field study was carried out in central Chile to evaluate the effect of different poultry litter (PL) and pig compost (PC) rates (7.5, 10.0, 12.5, and 15.0 Mg ha^?1for each amendment) combined with different nitrogen (N) rates (0, 100, 200, and 300 kg ha^?1) on dry matter (DM), grain production, and macronutrient concentration in corn (Zea mays L.) produced during the 2008–2009 (PL) and 2009–2010 (PC) seasons. The soil was clay loam. Organic amendments were applied before sowing, whereas inorganic N (urea) was applied at the 6-leaf stage (V6). When PL was combined with N, DM production ranged between 21.44 and 31.01 Mg ha^?1 and grain yield was between 12.76 and 17.86 Mg ha^?1. When PC was combined with N, DM production values were between 21.07 and 26.18 Mg ha^?1 and grain yield was between 14.01 and 17.14 Mg ha^?1. Results indicate that applying a combination of PL and N improved DM production, but applying a combination of PC and N did not affect this parameter. Grain production was only affected by increasing N rates. Applying both PL and N improved plant macronutrient concentration, while only applying PC increased N concentration. These results could be considered as the best recommended practices for using PL and PC.