Scielo RSS <![CDATA[Brazilian Archives of Biology and Technology]]> vol. 58 num. 2 lang. es <![CDATA[SciELO Logo]]> <![CDATA[Characteristics of Immobilized Urease on Grafted Alginate Bead Systems]]> This study evaluated the biological importance of immobilized urease enzyme over the free urease. The support material used for urease immobilization was alginate. Generally, the immobilization of urease in alginate gel showed a marked increase in Km and Vmax. However, the immobilized urease showed higher thermal stability than that of free enzyme. The rate of thermal inactivation of the immobilized enzyme decreased due to entrapment in gel matrix. Also, the activity of the immobilized urease was more stable in retention than that of the free enzyme during the storage in solution, although the activity of the immobilized enzyme was lower in comparison with the free enzyme. A stable immobilized system and long storage life are convenient for applications that would not be feasible with a soluble enzyme system. These results highlighted the technical and biochemical benefits of immobilized urease over the free enzyme. <![CDATA[Efficient Expression and Purification of Recombinant Human Enteropeptidase Light Chain in <em>Esherichia coli</em>]]> Human enterokinase (synonym: enteropeptidase, EC light chain (hEKL) gene was designed and artificially synthesized with built-in codon blas towards Escherichia coli codon preference. The synthetic hEKL gene was cloned into prokaryotic expression vector pMAL-s and transferred into the expression strain E. coli BL21 (DE3). Recombinant hEKL protein with a maltose binding protein (MBP) tag was expressed at high levels in soluble form, which yielded about 42% of the total cellular protein. The target protein was then purified to the homogeneity (&gt; 95%) by affinity chromatography. The peptide substrate GST-Melittin with enterokinase recognition site was completely cleaved by the purified MBP-hEKL at the molar ratio of 1:5000 (enzyme:substrate). Tricine SDS-PAGE analysis showed that the activity of MBP-hEKL was approximately seven times that of bovine enterokinase catalytic subunit (EKMaxTM, Invitrogen). From 1 L flask culture, 206 mg pure active MBP-hEKL was with specific activity of 1.4×104 U/mg. <![CDATA[Increasing Scopolamine content in Hairy Roots of Atropa belladonna using Bioreactor]]> The aim of this study was to use the, hairy root system for increasing the scopolamine content in Atropa belladonna. Agrobacterium rhizogenes ATCC15834 was utilized to produce hairy roots. The culture was carried out in a 1.5-l bioreactor using the inoculum size of 0.5 g fr. wt of 10-day-old hairy roots and various parameters, including agitation, aeration, conductivity and the consumption of sucrose were evaluated. Results revealed that the highest amount of scopolamine production (1.59 mg/g-1 dry wt) occurred in the bioreactor with aeration and agitation 1.25 vvm (volume per volume per minute) and 70 rpm, respectively. Study of conductivity and the consumption of sucrose showed that the highest amount of sucrose consumption and the highest amount of minerals consumption also was at 1.25 vvm and 70 rpm. Transgenic hairy root lines were confirmed by polymerase chain reaction (PCR). <![CDATA[Kinetics Study of Extracellular Detergent Stable Alkaline Protease from Rhizopus oryzae]]> In this study, extracellular alkaline protease was produced from Rhizopus oryzae in submerged fermentation using dairy waste (whey) as a substrate. Fermentation kinetics was studied and various parameters were optimized. The strain produced maximum protease at initial medium pH of 6.0 medium depth of 26 mm, inoculum size of 2% at incubation temperature of 35ºC for 168 h of fermentation. Alkaline protease was purified to homogeneity by ammonium sulphate fractionation followed by sephadex G-100 chromatography. The molecular mass of alkaline protease was 69 kDa determined by 10% SDS-PAGE. The optimum pH and temperature of alkaline protease was 9.0 and 40ºC, respectively. Metal profile of the enzyme showed that the enzyme was non-metallic in nature. The Km , Kcat , Vmax and Kcat/Km values of purified protease were 7.0 mg/mL, 3.8 x102 S-1, 54.30 µmol/min and 54.28 s-1 mg -1.mL respectively, using casein as substrate. The purified alkaline protease had stability with commercial detergents. <![CDATA[Partial Purification and Characterization of β-glucosidase from <em>Monascus sanguineus</em>]]> The aim of the present work was to study the production and characterization of β-glucosidase from Monascus sanguineus. Agro-waste residues were screened to obtain the maximum yield of enzyme. Jack fruit seed was the best substrate for enzyme production. Studies on the optimization of pH and temperature showed acidic pH favorable for enzymatic activity, whereas the optimum temperature was 60°C. Enzyme kinetics studies with different concentration of pNPG showed the calculated value of Km approximately 0.89 mM with the non-linear regression and 0.98 mM with the linear regression techniques. The enzyme was predominantly inhibited by KCl (69.8%) and moderately inhibited by CaCl2 (14.8%). Studies on the sensitivity for glucose showed that after 100 mM concentration of glucose, inhibition in pNPG hydrolysis took place. The molecular weight of the protein was estimated as 116 and 66 kDa with SDS- PAGE and zymography was carried out to verify the specific activity. <![CDATA[Serological Identification of Virus in Watermelon Production Fields in the Tocantins State]]> Watermelon (Citrullus lanatus) cultivated in almost all tropical and subtropical regions of the world, has its largest output in China, and then, according to FAO data, Turkey, Iran and Brazil, being one of the main crops cultivated in State of Tocantins, Brazil. In this work was investigated the occurrence and distribution of the watermelon viruses, totaling 752 samples taken in a stratified experimental design in four representative regions of production: Gurupi (150), Lagoa da Confusao (232), Formoso do Araguaia (265) and Porto Nacional (105). The sampling and collecting the leaves of plants with the presence of symptoms were performed once a week during the entire cultivation cycle. As a result, were observed by Dot-ELISA method, different types of viruses, such as Papaya ringspot W (PRSV-W), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV) (potyvirus), Cucumber mosaic virus ( CMV) (Cucumovirus) and Zucchini lethal chlorosis virus (ZLCV) (Tospovirus). Of these, PRSV-W was predominant (22%), followed by WMV (15%), ZLCV (11%), CMV (5%) and ZYMV (4%). Mixed infections with PRSV-W + WMV and PRSV-W + ZLCV were also observed around 20% frequency (expressed with symptoms differently from a single infection). The results provide important support for the program management viruses. <![CDATA[Antimicrobial, Antioxidant and Cytotoxic Activity of Marine <em>Streptomyces parvulus</em> VITJS11 Crude Extract]]> The main aim of the study was to evaluate the bioactive properties of ethyl acetate crude extract of Streptomyces parvulus VITJS11 with a view to assess their therapeutic potential. The biological activity of ethyl acetate extract was tested against fungal and bacterial pathogens. The free radical scavenging potential of the crude extract was determined by DPPH assay. The chemo preventive properties of S. parvulus VITJS11 ethyl acetate extract was examined by MTT assay on HepG2 cells. The morphological, physiological and the biochemical properties of the strain S. parvulus VITJS11 was confirmed by conventional methods. Genotypic characterization was done using 16S r-DNA partial gene amplification and sequencing. The authenticity of the crude chemical constitutes were determined by the GC-MS. The ethyl acetate extract of VITJS11 showed maximum antifungal activity against three Aspergillus species and prominent antibacterial activity against two Gram positive and Gram negative bacteria at 20 mg/mL. The antioxidant potential of the crude extract exhibited strong reducing power activity at 5mg/ mL with 85% inhibition and the cytotoxic effect was found with IC50 of 500µg/ mL on HepG2 cell lines. The GC-MS analysis and the chromatogram patterns revealed 16 peaks, indicating the presence of bioactive constituents, which included several important organic compounds, namely 9-(2',2'-dimethylpropanoilhydrazono)-3,6-dichloro-2,7-Bis-[2-(diethylamino)-ethoxy]fluorine (23.1) Dotriacontylpentafluoropropionate,(25.0) Octadecanoic acid, (20.0); Trans-2-methyl-4-n-butylthiane, S, S-dioxide.(19.0). The results showed the benefit of ethyl acetate extract from S. parvulus VITJS11 in treating microbial infections and indicated their broad spectrum of activity with beneficial virtues for therapeutic use. <![CDATA[Aqueous Extract of Terminalia chebula Induces Apoptosis in Lung Cancer Cells Via a Mechanism Involving Mitochondria-mediated Pathways]]> The current study was designed to evaluate the aqueous extract of Terminalia chebula activity, and the main pathway was detected on lung cancer by extracts of T. chebula. Aqueous extract of T. chebula was separated using a zeolite, and five fractions of T. chebula extract were obtained and analyzed by ultraviolet (UV) and infrared (IR) spectroscopy. Antiproliferative activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods against human lung cancer (A549) and mouse lung cancer cell line LLC. T. chebula acts by regulating the Bcl-2 family protein-mediated mitochondrial pathway detected by western blot. Fraction 4 of the T. chebula extract showed much function and was thus studied further. Fraction 4 increased the activation of caspase-3, induced PARP cleavage, and promoted cytochrome c release into the cytoplasm. These data suggest that T. chebula acts by regulating the Bcl-2 family protein-mediated mitochondrial pathway and provide evidence that T. chebula deserves further investigation as a natural agent for treating and preventing cancer. <![CDATA[Evaluation of fractions and 5,7-dihydroxy-4',6-dimethoxy-flavone from <em>Clerodendrum phlomidis</em> Linn. F. against <em>Helicoverpa armigera</em> Hub.]]> Twelve fractions from chloroform extract of Clerodendrum phlomidis and 5,7-dihydroxy- 4',6-dimethoxy-flavone (pectolinaringenin) were evaluated against Helicoverpa armigera. Maximum antifeedant (89.41%), larvicidal (83.77%) and ovicidal (69.25%) activities were observed in fraction 5. The least LC50 value for antifeedant (178.09 ppm) and larvicidal (198.23 ppm) were observed in fraction 5. No adult emergence was recorded in fractions 4-6 at 1000 ppm. The oviposition deterrent activity was 100% in fraction 5 at all the concentrations. Pectolinaringenin recorded maximum antifeedant (74.68%) and larvicidal (81.11%) activities at 100 ppm; it completely prevented the adult emergence of H. armigera at 100 ppm. Maximum ovicidal activity at 100 ppm concentration was 67.95%. The oviposition deterrent activity was 100% in 100 and 50 ppm concentrations. C. phlomidis could be effectively used to develop a new formulation to control the economically important pests. <![CDATA[Identification of Primordial Germ Cells: Cytological, Histological and Immunohistochemical Aspects]]> Primordial germ cells (PGCs) constitute an embryonic cell type that migrate to gonadal precursors and form the gametes. In many animals, PGCs are set apart from somatic cells early during embryogenesis. These cells migrate to gonadal precursors and then constitute gonads so they are useful models for cell motility studies. They have a highlighted importance for development and reproduction studies. Primordial germ cells have morphological differences from the somatic cells. Structure of these cells can be detected with light and electron microscopy in early development stages. This review describes the morphological, histological, molecular and ultrastructural features of primordial germ cells in different animals and gives an overview for simplified identification. <![CDATA[Protective Effects of Sodium Selenite and Vitamin E on Mercuric Chloride-Induced Cardiotoxicity in Male Rats]]> This study was designed to investigate the protective effects of sodium selenite and/or vitamin E against mercuric chloride-induced cardiotoxicity. Male Wistar rats (n=48, 310±10 g) were administered mercuric chloride (1.0 mg/kg bw), sodium selenite (0.25 mg/kg bw), vitamin E (100 mg/kg bw), sodium selenite plus mercuric chloride, vitamin E plus mercuric chloride and sodium selenite plus vitamin E plus mercuric chloride daily via gavage for four weeks. Malondialdehyde (MDA) level, antioxidant enzyme activities [total superoxide dismutase (SOD), catalase (CAT), total glutathione peroxidase (GPx) and total glutathione-S-transferase (GST)], and histopathological changes in the heart tissue were evaluated. Results showed that mercuric chloride exposure resulted in an increase in the MDA level and a decrease in the SOD, CAT, GPx and GST activities, with respect to the control. Light microscopic investigations revealed that mercuric chloride induced histopathological changes in the heart tissue. A significant decrease in the MDA level and a significant increase in the SOD, CAT, GPx and GST activities were observed on the supplementation of sodium selenite and/or vitamin E to mercuric chloride-treated rats, which showed that, sodium selenite and/or vitamin E significantly reduced mercuric chloride induced cardiotoxicity, but not protected completely. <![CDATA[Quality Control of Biotechnological Inputs Detecting <em>Mycoplasma</em>]]> The aim of this work was to study the Polymerase Chain Reaction (PCR) as a tool of quality control of bovine sera and cellular cultures used in the biotechnological industry. A total of 46 samples of bovine sera derived from two slaughterhouses and 33 samples of BHK21 cells derived from two biotechnological industries were evaluated using the primers GPO-3 (sense) and MGSO (antisense). The PCR technique sensibility analysis showed that 280 bp were amplified for the quantities of 50 ng to 0.006 ng of Micoplasma DNA. The primers specificity was confirmed in the test using Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Candida albicans; except by the positive control, none of the samples showed amplification. The presence of Mycoplasma in bovine sera and in the cultures of BHK21 cells showed that 56.5 and 15.2%, respectively, were contaminated. Thus, it was possible to conclude that PCR was a fast and confident technique to detect mycoplasma and that it could be used to control the quality of immunobiological products and inputs, such as sera and cultures of BHK21 cells. <![CDATA[Chemotaxonomic Fingerprinting of Chilean Lichens Through Maldi and Electrospray Ionization Mass Spectrometry]]> The aim of this work was to study a fast, new, sensitive, and simple method for the chemotaxonomic classification of Chilean lichens (Teloschistes chrysophthalmus, Ramalina farinacea, Usnea pusilla, Ramalina chilensis and Stereocaulon ramulosum) using MALDI-TOF-MS and UPLC-ESI(-)-MS data. Lichens soluble proteins fingerprints were acquired by MALDI-TOF-MS and they were analyzed by chemometric (PCA). Lichens organic extracts fingerprints were obtained by UPLC-ESI(-)-MS. MALDI-TOF-MS associated with chemometric analysis was used to detect new m/z patterns of soluble proteins that were compared with Protein Data Bank of UnitPro. These data also permitted the satisfactory distinction among the families and species. UPLC-ESI(-)-MS fingerprints analyses of the organic extracts showed the presence of five major lichen compounds (atranorin, parietin, teloschistin, ramalinolic and usnic acids). In contrast to other techniques, MALDI-TOF-MS associated with chemometric analysis and UPLC-ESI(-)-MS provided a new, fast and sensitive method for chemotaxonomic characterization of lichens. <![CDATA[Haematological Parameters of the Hybrid Surubim (<em>Pseudoplatystoma reticulatum x P. corruscans</em>) farmed in Brazil]]> This study evaluated the haematological parameters of the hybrid surubim (Pseudoplatystoma corruscans x P. reticulatum) farmed in intensive and semi intensive system. A total of 240 fish were examined, 120 from fish farm and 120 from a semi-intensive for comparison between the hot and cold season. The water quality was weekly measured to evaluate the influence of environmental conditions on the haematology. Fish from intensive system showed hematocrit and white blood cell count (WBC) higher in hot season while the total plasmatic protein, hemoglobin concentration, mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular volume (MCV) were higher in cold season. In fish from semi intensive system, the red blood cells count (RBC) and total thrombocyte number was higher in hot season while the MCHC and MCV were higher in cold season. There was no alterations in the numbers of lymphocytes, neutrophils, basophils, eosinophils and PAS - positive granular leukocyte (PAS-LG). The results showed the influence of seasonality on the hematological parameters of the hybrid surubim kept under normal farming conditions. Regarding water quality, hot season was directly related to increased water temperature and decreased dissolved oxygen while the opposite was found in cold season. <![CDATA[Physicochemical and antioxidant properties of kiwifruit as a function of cultivar and fruit harvested month]]> The present study was carried out to find the effect of fruit harvesting stage (October, November and December) on the physicochemical and antioxidant properties in five kiwi cultivars (Abbot, Bruno, Allison, Hayward, Monty). Results showed that soluble solid content (SSC) and pH increased while ascorbic acid (Vit C), titrated acidity (TAD) and SSC/TAD decreased in all the cultivars with delay in harvesting. Total polyphenols (TP) were decreased while total flavonoids (TF) increased in all tested cultivars with delay in harvesting. The highest concentration of TP (2.02 mg gallic acid equivalent/g fresh weight) and TF (51.12 mg catechin equivalent/100g FW) were found in cultivar 'Allison' in the month of October and December, respectively. Antioxidant activities (AA) were genotype depended and no trend was observed with month of harvesting. Principal component analysis (PCA) showed strong correlation between Vit C, TP and antioxidant activities. Two major clusters were computed using agglomerative hierarchical clustering (AHC). All the studied important traits may be used in the breeding programmes to increase the variability for different physiochemical and antioxidative characteristics and to make suitable selections that could be acceptable to consumers. <![CDATA[Influence of Cooling on the Glycolysis Rate and Development of PSE (<em>Pale, Soft, Exudative</em>) Meat]]> The aim of this work was to evaluate pH values fall rate in chicken breast meat under commercial refrigeration processing conditions and the development of PSE (pale, soft, exudative) meat. Broiler breast samples from the Cobb breed, both genders, at 47 days of age (n = 100) were taken from refrigerated carcasses (RS) immersed in water and ice in a tank chilled at 0°C (±2). pH and temperature (T) values were recorded at several periods throughout refrigeration in comparison to samples left at room T as control (CS). The ultimate pH (pHu) value of 5.86 for RS carcasses were only reached at 11°C after 8.35 h post mortem (PM) while, for CS samples, pHu value was 5.94 at 22°C after 4.08 h PM. Thus, under commercial refrigeration conditions, the glycolysis rate was retarded by over 4.0 h PM and the breast meat color was affected. At 24.02 h PM, PSE meat incidence was 30% while for CS, meat remained dark and PSE meat was not detected. Results show retardation in the glycolysis rate and PSE meat development was promoted by the refrigeration treatment when compared with samples stored at processing room temperature. <![CDATA[Torularhodin and Torulene: Bioproduction, Properties and Prospective Applications in Food and Cosmetics - a Review]]> Torularhodin and torulene are two widespread microbial carotenoids with relatively few studies, as compared to other nutraceutical carotenoids such as β-carotene, lycopene and astaxanthin. Several genera of microorganisms produce it in high concentration (up to 0.1% of the cell dry weight), probably as a protection against photooxidation and free radicals. These pigments, which differ by a terminal carboxylic group, have provitamin-A activity and, being red, have potential use as food and cosmetic color additives. Several factors affect the biosynthesis of these substances, including: the composition of culture media, light irradiation, which may enhance the carotenoid production up to 25% of the non-irradiated cultures, and temperature, which changes the carotenoid balance towards more of the acidic carotenoid (torularhodin) or the hydrocarbon (torulene). The biomass may be directly extracted using non polar solvents such as hexane or a hexane-acetone mixture, without need of cell disruption. Extensive purification is not needed for using the pigments as food or cosmetic additives, but it is still necessary to evaluate the bioactivity of the pigments in humans. <![CDATA[Properties of films obtained from biopolymers of different origins for skin lesions therapy]]> In this study, the effects of the origin of xanthan used, in combination with chitosan, to prepare films for the treatment of skin lesions were evaluated. The characteristics of the films obtained with xanthan commercially available for the food industry sector and xanthan originated from a fermentation process conducted in a pilot plant were compared. Results showed that the source did not strongly interfere in many of the properties of the films, such as the mechanical properties, cytotoxicity to L929 cells, absorption of simulated body fluid and culture medium, stability in water and saline solution. Hence, even though the properties of biopolymers of different sources might vary, the films prepared with two distinct types of xanthan gum could be considered as potentially safe and similar in terms of relevant characteristics considering the aimed application. <![CDATA[Resistance of oxidative stress in biofilm and planktonic cells]]> This work studied the susceptibility of biofilm produced by E. coli to oxidative stress, and compared the components of free radicals defences: level of glutathione, catalase and dismutase activities in planktonic and biofilm located cells. Results showed the diversity of responses to oxidative stress in bacterial cells in log or stationary phases in both planktonic and biofilm forms. The bacteria were exposed to free-radical donors (H2O2, tBOOH, menadione, SIN-1 or peroxynitrite) in a wide range of final concentrations, from 0.5 to 10mM. Different level of toxicity of individual donors, independence of cell type (planktonic forms or biofilm) and phases of growth were observed. The highest oxidative stress resistance was observed for the cells in logarithmic phase of growth treated with H2O2, both in planktonic and biofilm forms, whereas for the cells in stationary phase, the highest resistance was observed for menadione. These results showed higher efficiency of agents based on superoxide anion donors in combating bacteria colonizing abiotic surfaces stainless steel (AISI 316L). <![CDATA[The Mutagenic Potential Caused by the Emissions from Combustion of Crude Glycerin and Diesel Fuel]]> This study evaluated the use of crude glycerin as an alternative of energy generation to replace the traditional fuels. The Tradescantia stamen hair mutation assay (Trad-SH) was applied to study the mutagenic effects caused by the emissions generated in the direct combustion of diesel oil and glycerin in a flame tube furnace. Tradescantia inflorescences were exposed to gaseous emissions from the combustion tests in a fumigation chamber for 30-40 min. The analysis of variance and the Tukey test were applied to compare the differences between six test groups (intoxicated with emissions from glycerin and diesel oil combustion) and a control group. Only one glycerin group showed statistical differences (0.05), possibly due to the complexity of the burning process and impurities, besides the acrolein present in its emissions. The high heating value (HHV) of crude glycerin (25.5 MJ/kg) was lower than diesel oil (45.19 MJ/kg), but it was comparable to other fuels. Although the use of glycerin as a biofuel could be an important aspect to be considered, the results showed that the glycerin had a substantial mutagenic potential similar to that of diesel oil.