Scielo RSS <![CDATA[Electronic Journal of Biotechnology]]> vol. 10 num. 4 lang. es <![CDATA[SciELO Logo]]> <![CDATA[Amplification <strong>of genome-integrated BeYDV replicons</strong> <strong>by transient expression</strong> <strong>of Rep in </strong><em><b>Arabidopsis thaliana</b></em>]]> Bean yellow dwarf geminivirus (BeYDV) has been used as a potential vector to improve foreign gene expression, specifically, to achieve higher yields of vaccine proteins in plants. Previously, we have shown that when the BeYDV replication initiator protein Rep was provided in trans, replication and gene expression of GUS were enhanced enormously from a BeYDV expression vector in a transient assay system. In this paper, transgenic lines of Arabidopsis (cv. Columbia) were generated harboring the BeYDV cis-acting elements required for replication. Constructs encoding BeYDV Rep or intronless Rep open reading frames (ORFs) were transiently introduced into transgenic plants via Agrobacterium-mediated infiltration in order to examine the relative levels of replication and expression of the genome-integrated GUS reporter gene. This study shows that expression of Rep protein was regulated in trans from a separate cassette which enabled the rescue, replication and enhancement of the genome-integrated GUS gene in transgenic Arabidopsis. We conclude that Rep expression can be effectively controlled in Arabidopsis plants, and that regulation of Rep expression can result in the amplification of a genome-integrated foreign gene by circumventing the negative effects of gene silencing. <![CDATA[<b>Assessment of genetic structure in Mexican charolais herds using microsatellite markers</b>]]> Knowledge of livestock genetic diversity is an essential step to respond to commercial demands and reach production objectives in different environments and production systems. The evaluation of animal genetic diversity is achieved by using molecular markers. Microsatellites are the most used markers for studies of this type. Eleven microsatellites were used to evaluate the genetic variation from three populations of Charolais cattle located in northeast Mexico. The studied populations exhibited a high allelic variability with a mean heterozygosity of 0.5. A moderate genetic differentiation between the Charolais populations (F ST = 0.079; P < 0.001) was observed. This suggests subdivisions in Charolais breed established in Mexico, due to genetic material origin, reproductive and selective management and local isolation. <![CDATA[<b>Bioconversion of (+)- and (-)-alpha-pinene to (+)- and (-)-verbenone by plant cell cultures of <i>Psychotria brachyceras</i> and <i>Rauvolfia sellowii</i></b>]]> This work describes the bioconversion of (-)- and (+)-alpha-pinene (2,6,6-trimethyl-bicyclo[3.1.1]hept-2-ene), targeted at the production of (-)- and (+)-verbenone (4,6,6-trimethyl-bicyclo (3.1.1) hept-3-en-2-one), respectively, using Psychotria brachyceras and Rauvolfia sellowii cell suspension cultures. P. brachyceras showed selectivity to (-)-alpha-pinene with 80.9% conversion (relative integrated area gas chromatography-mass spectrometry (GC-MS)) of (-)-verbenone in 10-day-incubation, whereas R. sellowii was able to convert both pinene enantiomers (37.6% conversion of (-)-verbenone in 7-day-incubation and 32.2% conversion of (+)-verbenone in 10-day-incubation). In both systems trans-verbenol was formed as main product and then slowly biocatalyzed to verbenone. Verbenone were also present among the autoxidation products during control experiments, but in much lower amounts and accompanied by several by-products, highlighting the usefulness of the biotransformation process. <![CDATA[<b>Comparative esterification of phenylpropanoids versus hydrophenylpropanoids acids catalyzed by lipase in organic solvent media</b>]]> The esterification of phenylpropanoid and hydrophenylpropanoid acids, catalyzed by candida antarctica lipase B (CAL-B), with several alcohols has demonstrated that the substitution pattern on the aromatic ring has a very significant influence on the reactivity of the carboxyl group due, mainly, to electronic effects, when compared to the unsaturated acids with the hydrogenated acids. It is also clear that in the saturated acids there still remain some unclear effects related to the aromatic substituents. <![CDATA[<b>Comparison on the removal of hydrogen sulfide in biotrickling filters inoculated with <i>Thiobacillus thioparus </i>and <i>Acidithiobacillus thiooxidans</i></b>]]> Emissions of hydrogen sulfide (H2S) by industrial activities is frequent cause of corrosion and unpleasant odours. Treatment of gaseous emissions contaminated with H2S by biotrickling filters inoculated with single cultures of sulfur oxidizer bacteria exhibit several advantages over physicochemical methods, such as shorter adaptation times and higher removal ability. Biofilms of Thiobacillus thioparus and Acidithiobacillus thiooxidans have proved to exhibit high removal capacities, yet no comparative studies between them have been reported. This article reports the efficiency of biotrickling filters inoculated with T. thioparus and A. thiooxidans under similar conditions excepting the pH, that was the optimal for the bacterial growth, for the removal of H2S. The support was selected by determining the respirometric coefficients of the biomass. The maximum removal capacity of the biofilter inoculated with T. thioparus, operating within the range of pH (5.5-7.0) was 14 gS m-3 h-1, lower the value obtained for the biotrickling filter inoculated with A. thiooxidans; 370 gS m-3 h-1. Therefore, it is concluded that acid biotrickling filter inoculated with A. thiooxidans constitute the best strategy to remove H2S, with the advantage that the system not require an exhaustive pH control of the liquid media. <![CDATA[<b>Electrogeneration of hydrogen peroxide applied to the peroxide-mediated oxidation of (<i>R</i>)-limonene in organic media</b>]]> Horse radish peroxidase (HRP) from Armoracia rusticana catalyses the oxidation of (R)-limonene into the oxidized derivatives carveol and carvone. This study compares the direct addition (DA) of hydrogen peroxide with its continuous electrogeneration (EG) during the enzymatic oxidation of (R)-limonene. Reaction mixtures containing HRP, (R)-limonene as substrate, and hydrogen peroxide, added directly or electrogenerated, in 100 mM sodium-potassium phosphate buffer pH 7.0, at 25ºC were studied. Two electrochemical systems for the hydrogen peroxide electrogeneration were evaluated, both containing as auxiliary electrode (AE) a platinum wire and saturated calomel electrode (SCE) as reference. Reticulated vitreous carbon foam (RVCF) and an electrolytic copper web (CW) were evaluated as working electrodes (WE). Results were compared in terms of hydrogen peroxide electrogeneration, (R)-limonene residual concentration or conversion and product selectivity. Best results in terms of maximum H2O2 concentration (1.2 mM) were obtained using the CW electrode at -620 mV SCE, and continuous aeration. Use of the EG system under optimized conditions, which included the use of acetone (30% v/v) as a cosolvent in a 3 hrs enzymatic reaction, lead to a 45% conversion of (R)-limonene into carveol and carvone (2:1). In comparison to the results obtained with DA, the use of EG also improved the half-life of the enzyme. <![CDATA[<b>Equilibrium sorption isotherm studies of Cd(II), Pb(II) and Zn(II) ions detoxification from waste water using unmodified and EDTA-modified maize husk</b>]]> The mobilization of heavy metals in the environment due to industrial activities is of serious concern due to the toxicity of these metals in humans and other forms of life. The equilibrium adsorption isotherms of Cd(II), Pb(II) and Zn(II) ions, detoxification from waste water using unmodified and EDTA-modified maize husk have been studied. Maize husk was found to be an excellent adsorbent for the removal of these metal ions. The amount of these metal ions adsorbed increased as the initial concentration increased. Also, EDTA-modification enhanced the adsorption capacity of maize husk due to the chelating ability of ethylenediamine tetra acetic acid (EDTA). Among the three adsorption isotherms tested, Dubinin-Radushkevich isotherm gave the best fit with R² value ranging from 0.7646 to 0.9988 and an average value of 0.9321. This is followed by Freundlich and then Langmiur isotherms. The sorption process was found to be mostly a physiosorption process as seen from the apparent energy of adsorption which ranged from 1.03 KJ/mol to 12.91 KJ/mol. Therefore, this study demonstrates that maize husk which is an environmental pollutant could be used to adsorb heavy metals and achieve environmental cleanliness. <![CDATA[<b>Experimental and bioinformatic approaches for analyzing and visualizing cyanobacterial nitrogen and hydrogen metabolism</b>]]> Many cyanobacteria are capable of utilizing light energy for nitrogen fixation. As a by-product of this nitrogenase mediated catalysis, hydrogen gas is produced. Several approaches to increase hydrogen production from cyanobacteria exist. Usually, these approaches are non-targeted. Here we exemplify how DNA-microarray based gene-expression analysis and bioinformatic visualization techniques can be used to analyze nitrogen and hydrogen metabolism from the filamentous, heterocyst forming cyanobacterium Nostoc PCC 7120. We analyzed the expression of 1249 genes from major metabolic categories under nitrogen fixing and non-nitrogen fixing growth. Of the selected genes, 494 show a more than 2-fold expression difference in the two conditions analyzed. Under nitrogen-fixing conditions 465 genes, mainly involved in energy metabolism, photosynthesis, respiration and nitrogen-fixation, were found to be stronger expressed, whereas only 29 genes showed a stronger expression under non-nitrogen fixing conditions. To help understanding probe hybridization, all expression data were correlated with potential target secondary structures and probe GC-content. For the first time the expression of high light-induced stress proteins (HLIP-family) is shown to be linked to the nitrogen availability. <![CDATA[<b>Heterologous expression, purification and refolding of an anti-listerial peptide produced by <i>Pediococcus acidilactici</i> K7</b>]]> The fusion protein, 6XHis-Xpress-PedA was constructed and expressed in Escherichia coli BL21 (DE3). The presence of a 12.8 kDa recombinant protein, localized in inclusion bodies (IBs) at high concentration, was confirmed by SDS-PAGE analysis and by western blotting using anti-His antibody. The rec-pediocin was purified by Nickel-nitrilotriacetic acid beads and refolded using 5 mM of β-mercaptoethanol along with 1 M glycine. Results indicated that the refolded rec-pediocin had an early elution profile in the RP-HPLC when compared to the unfolded protein and it exhibited biological activity against Listeria monocytogenes V7 which was approximately 25 times less active compared to native counterpart. The final yield of purified rec-pediocin was 3 mg/l of the culture and is estimated to be 8-10 times higher than the purification by conventional methods. <![CDATA[<b>Inter simple sequence repeats separate efficiently hemp from marijuana (<i>Cannabis sativa</i> L.)</b>]]> Cannabis sativa L. is a multiple-use plant that provides raw material for the production of seed oil, natural fiber for textiles, automotive and pulp industries. It has also been used in insulating boards, ropes, varnishes, animal feed, and as medicinal agents. Cannabis has potential to be used for phytoremediation: however, its cultivation is strictly controlled due to its psychoactive nature and usage in producing drugs such as marijuana, and hashish. In this study, psychoactive type Cannabis samples, which were seized from 23 different locations of Turkey, and nine hemp type Cannabis accessions, as well as an unknown accession were used. Our interest was to identify the genetic relatedness of the seized samples and to separate drug and hemp type plants. Inter Simple Sequence Repeats (ISSRs) were employed for analysis based on single plant material (SET1) and bulked samples of them (SET2). Data was analysed via cluster analysis and principal coordinate analysis (PCoA). PCoA analyses, by using SET1 and SET2, were able to efficiently discriminate the seized samples from the fiber type accessions. However, separation of the plants was not clear via unweighted pair-group method using arithmetic average (UPGMA) dendogram in SET1, while they were clearly separated in SET2. Hemp type accessions showed high levels of variation compared to drug type Cannabis both in SET1 and SET2. <![CDATA[<b>Micropropagation of <i>Lilium ledebourii</i> (Baker) Boiss as affected by plant growth regulator, sucrose concentration, harvesting season and cold treatments</b>]]> A protocol for the micropropagation in different harvesting time of Lilium ledebourii (Baker) Boiss, an endangered rare species endemic to Iran has been developed. In vitro scale culture of this species, using bulbs from three harvesting seasons (spring, summer and winter), was attempted. Among the various treatments tested, the Murashige and Skoog (MS) medium supplemented with 0.1 mg l-1 naphthaleneacetic acid (NAA) + 0.1 mg l-1 benzyladenin (BA) and 6% sucrose in all harvesting seasons proved to be superior to others. The best results for fresh weight of bulblets, rooting parameters and the survival rate after transplantation to greenhouse were obtained from early winter-harvested bulbs. Summer-harvested bulbs had the highest number of bulblets per explant. The bulblets at the end of the culture period were given cold treatment at 4ºC for 2-8 weeks at a 2-weeks interval and then transplanted to a potting mixture of sand, leaf mold and peat moss (1:1:1 v/v). The best emergence rate (90%) was achieved at 8 weeks cold treatment for winter harvested bulbs. <![CDATA[<b>Multiple model approach to modelling of <i>Escherichia coli</i> fed-batch cultivation extracellular production of bacterial phytase</b>]]> The paper presents the implementation of multiple model approach to modelling of Escherichia coli BL21(DE3)pPhyt109 fed-batch cultivation processes for an extracellular production of bacterial phytase. Due to the complex metabolic pathways of microorganisms, the accurate modelling of bioprocesses is rather difficult. Multiple model approach is an alternative concept which helps in modelling and control of complex processes. The main idea is the development of a model based on simple submodels for the purposes of further high quality process control. The presented simulations of E. coli fed-batch cultivation show how the process could be divided into different functional states and how the model parameters could be obtained easily using genetic algorithms. The obtained results and model verification demonstrate the effectiveness of the applied concept of multiple model approach and of the proposed identification scheme. <![CDATA[New strains obtained after UV treatment and protoplast fusion of native <i>Trichoderma harzianum</i>: <b>their biocontrol activity on <i>Pyrenochaeta lycopersici</i> </b>]]> The obtainment of 30 new strains from native Trichoderma harzianum after UV light irradiation (UV-A and UV-C), and of 82 strains resulted from protoplast fusion were accomplished. The new strains, initially selected for their growing rate under low temperature and high pH conditions, as well as for their innocuousness on tomato plants, were tested for in vitro inhibition of Pyrenochaeta lycopersici in dual cultures and due to secretion of volatile and diffusible metabolites. All the UV-A and UV-C selected candidate mutants were innocuous to tomato plants, but none of them showed improvement in their biocontrol activity on P. lycopersici. Th12A20.1 increased 1.3 and 1.9 fold the total fresh weight of Fortaleza tomato plants when compared to its parental strains Th12 and Th11, respectively. The selected candidate mutants obtained through protoplast fusion were also innocuous to tomato plants, but only ThF1-2 and ThF4-4 inhibited 1.3 fold (in dual cultures) and 5 fold (due to secretion of volatile metabolites) the growth of P. lycopersici, respectively, in relation to the mean inhibitory effect of both parents. Therefore, these candidate mutants could be included in experiments under field conditions. <![CDATA[<b>Optimization of medium composition for transglutaminase production by a Brazilian soil<i> Streptomyces </i>sp.</b>]]> Finding a new microbial source of transglutaminase (MTGase) and the study of the medium composition for MTGase production were the goals of this work. A total of 200 actinomycete-like strains were isolated from Brazilian soil samples and two of them named T10b and P20 were selected based on their ability to produce 0.15 U.mL-1 and 0.25 U.mL-1 of MTGase, respectively. Strain P20 was chosen to continue the study and was identified as Streptomyces sp. In order to optimize the MTGase activity, modifications of the usual media composition described for enzyme production were tested. The strategy adopted was: (1) screening experiment for the best carbon and nitrogen sources; (2) fractional factorial design (FFD) to elucidate the key ingredients in the media (the results indicated that the soybean flour, peptone, KH2PO4 and MgSO4.7H2O had a significant effect on MTGase) production and (3) central composite design (CCD) to optimize the concentration of the key components. The experimental results were fitted to a second-order polynomial model at the 95% level of significance (P < 0.05). Under the proposed optimized conditions, the model predicted a MTGase activity of 1.37 U.mL-1, very closely matching the experimental activity of 1.4 U.mL-1. <![CDATA[<b>Phage-resistance of <i>Salmonella</i> <i>enterica</i> serovar Enteritidis and pathogenesis in <i>Caenorhabditis</i> <i>elegans</i> is mediated by the lipopolysaccharide</b>]]> Phage therapy has been used in the past as an alternative therapy against bacterial pathogens. However, phage-resistant bacterial strains can emerge. Some studies show that these phage-resistant strains are avirulent. In this study, we report that phage-resistant strains of Salmonella enterica serovar Enteritidis (hereafter S. Enteritidis) were avirulent in the Caenorhabditis elegans animal model. We isolated phage-resistant strains of S. Enteritidis ATCC 13076 by using three lytic phages (f2αSE, f3αSE and f18αSE). In these mutants, we explored different virulence factors like lipopolysaccharide (LPS), virulence plasmid (Pla), motility and type I fimbriae, all of which may have effects on virulence and could furthermore be related to phage resistance. The phage-resistant strains of S. Enteritidis showed loss of O-Polysaccharide (O-PS) and auto-agglutination, present a rough phenotype and consequently they are avirulent in the C. elegans animal model. We speculate that the O-PS is necessary for phage attachment to the S. Enteritidis cell surface. <![CDATA[<b>Methyl jasmonate induced overproduction of eleutherosides in somatic embryos of <i>Eleutherococcus senticosus</i> cultured in bioreactors</b>]]> This study was concentrated on the production of eleutherosides and chlorogenic acid in embryogenic suspension cultures of Eleutherococcus senticosus by exposing them to different concentrations (50-400 µM) of methyl jasmonate (MJ) during the culture period. In the bioreactor cultures, eleutheroside content increased significantly by elicitation of MJ, however, the fresh weight, dry weight and growth ratio of embryos was strongly inhibited by increasing MJ concentrations. The highest total eleutheroside (7.3 fold increment) and chlorogenic acid (3.9 fold increment) yield was obtained with 200 µM MJ treatment. There was 1.4, 3.4 and 14.9 fold increase in the eleutheroside B, E, and E1 production respectively with such elicitation treatment. These results suggest that MJ elicitation is beneficial for eleutheroside accumulation in the embryogenic cell suspension cultures.