Scielo RSS <![CDATA[Electronic Journal of Biotechnology]]> vol. 18 num. 4 lang. pt <![CDATA[SciELO Logo]]> <![CDATA[<strong>Utilization of genes encoding osmoprotectants in transgenic plants for enhanced abiotic stress tolerance</strong>]]> Global agriculture in the context of growing and expanding populations is under huge pressure to provide increased food, feed, and fiber. The recent phenomenon of climate change has further added fuel to the fire. It has been practically established now that the global temperature has been on the increase with associated fluctuations in annual rainfall regimes, and the resultant drought and flood events and increasing soil and water salinization. These challenges would be met with the introduction and utilization of new technologies coupled with conventional approaches. In recent years, transgenic technology has been proved very effective in terms of production of improved varieties of crop plants, resistant to biotic stresses. The abiotic stresses such as salt and drought are more complex traits, controlled by many genes. Transgenic plant development for these stresses has utilized many single genes. However, much emphasis has been placed on genes catalyzing the biosynthetic pathways of osmoprotectants. This review focuses on the current status of research on osmoprotectant genes and their role in abiotic stress tolerance in transgenic plants. <![CDATA[<strong>Association between biometric characteristics of tomato seeds and seedling growth and development</strong>]]> Background The size and weight of tomato seeds depend on genetics and can be modified by environment and management. In some species, a strong relation has been described between physical aspects of the seeds and the quality of the corresponding seedlings, but this cannot be considered a general rule. The objective of this research was to identify any association between the biometric characteristics of tomato seeds and the growth and development of their seedlings. Results A total of 18 lots of hybrid tomato seeds were used (from indeterminate plants with round fruits), belonging to six varieties from two reproduction seasons. Each lot was evaluated for seed size and weight, and seed quality, in terms of the germination test (5 and 14 d after sowing). The number of normal roots emerged with a length above 2 mm was also evaluated at d 3, 4 and 5 after sowing. The length of the seedlings and their total and partial dry weight were measured 5 d after sowing. The results indicate that there was no association between seed size and weight and subsequent seedling emergence, and only weak correlations were found between the dry weight of the radicle and cotyledon and seed size. Conclusion There is little association between the physical characteristics of the seeds and the subsequent seedlings, making it impossible to propose the use of seed weight or size as a compliment to quality evaluation tests. <![CDATA[<strong>User-friendly optimization approach of fed-batch fermentation conditions for the production of iturin A using artificial neural networks and support vector machine</strong>]]> Background In the field of microbial fermentation technology, how to optimize the fermentation conditions is of great crucial for practical applications. Here, we use artificial neural networks (ANNs) and support vector machine (SVM) to offer a series of effective optimization methods for the production of iturin A. The concentration levels of asparagine (Asn), glutamic acid (Glu) and proline (Pro) (mg/L) were set as independent variables, while the iturin A titer (U/mL) was set as dependent variable. General regression neural network (GRNN), multilayer feed-forward neural networks (MLFNs) and the SVM were developed. Comparisons were made among different ANNs and the SVM. Results The GRNN has the lowest RMS error (457.88) and the shortest training time (1 s), with a steady fluctuation during repeated experiments, whereas the MLFNs have comparatively higher RMS errors and longer training times, which have a significant fluctuation with the change of nodes. In terms of the SVM, it also has a relatively low RMS error (466.13), with a short training time (1 s). Conclusion According to the modeling results, the GRNN is considered as the most suitable ANN model for the design of the fed-batch fermentation conditions for the production of iturin A because of its high robustness and precision, and the SVM is also considered as a very suitable alternative model. Under the tolerance of 30%, the prediction accuracies of the GRNN and SVM are both 100% respectively in repeated experiments. <![CDATA[<strong>Fermentation optimization of maltose-binding protein fused to neutrophil-activating protein from </strong><em><b>Escherichia coli</b></em><strong> TB1</strong>]]> Background The fermentation conditions of recombinant maltose-binding protein fused to neutrophil-activating protein (rMBP-NAP) of Helicobacter pylori were optimized from Escherichia coli TB1 with varying medium, inoculum age and size, time, inducer, pH and temperature in batch fermentation. Results It was revealed that the optimal conditions for the production of rMBP-NAP in shake flask were as follows: M9 medium (with 3% yeast extract powder added), inoculum age of 19 h, inoculum size of 6%, initial pH of 6.6, temperature of 37°C, and 0.7 mmoL/L IPTG inducted 21 h in a 50 mL/250 mL shake flask. The recombinant protein yield was increased from 59 to 592 mg/L after optimization. Fermentation process conducted in a 10 L fermenter with similar conditions could get 30 g/L wet cell and 1.738 g/L soluble protein with the rMBP-NAP expression level of 11.9%. Conclusion The results improve the expression level of rMBP-NAP, and it is expected that these optimized conditions can be well applied for large scale production of rMBP-NAP. <![CDATA[<strong>Effects of aroma quality on the biotransformation of natural 2-phenylethanol produced using ascorbic acid</strong>]]> Background Natural 2-phenylethanol (2-PE) is an important flavoring that emits the aroma of roses. During biotransformation, the aroma quality of natural 2-PE is affected by its main by-products, which include butanol, isobutyric acid, butyric acid, and isovaleric acid. Thus, controlling undesirable by-product formation can reduce the effect of odor on 2-PE aroma quality. Results 2-PE was produced through biotransformation using l-phenylalanine as a substrate and glucose as a carbon source. Ascorbic acid was added to the system to improve the redox reaction and suppress the generation of by-products. Principal component analysis of the aroma quality of 2-PE was performed using an electronic nose. Similarity analysis revealed that the effects of four by-products on 2-PE aroma quality may be ranked in the following order: isovaleric acid > butyric acid > isobutyric acid > butanol. The sample that exhibited the best similarity to the standard 2-PE sample (99.19%) was the sample to which ascorbic acid had been added during glucose metabolism. Conclusions 2-PE produced through the addition of ascorbic acid exhibited the closest aroma similarity to the standard 2-PE sample. <![CDATA[<strong>Antiproliferative evaluation of tall-oil docosanol and tetracosanol over CHO-K1 and human melanoma cells</strong>]]> Background Polycosanols derived from plant species have traditionally been used in medicine as antiproliferative agents for treating various viruses (primarily the herpes simplex virus). However, few studies have studied their effects on hyperproliferative cell lines. In this work, the antiproliferative capacity of polycosanols from tall-oil pitch, obtained from black liquor soaps in the kraft pulping process of cellulose (specifically from Pinus radiata, Pinus taede, and Eucalyptus globulus), was evaluated on CHO-K1 and CRL-1974 human melanoma cell lines. Results The proliferative capacities and cell viabilities were measured for 72 and 140 h, respectively. Treatment with docosanol produced differential effects on the CHO-K1 and human melanoma cells and significantly affected their proliferation rates, but not their cell viabilities. Tetracosanol produced a significant negative effect on the proliferation of human melanoma cells, and this effect was less than that caused by docosanol. However, it had no effect on the proliferation of CHO-K1 cells and did not induce any significant effect on the viability of the studied cell lines. Conclusion Docosanol and tetracosanol induced antiproliferative effects on the studied cell lines and exhibited significantly greater effects on the oncogenic cell lines. Prior to this study, the capacity of these polycosanols has never been investigated. Future studies will be necessary to determine their mechanisms of action on these cell systems. <![CDATA[<strong>Phenolic compound production and biological activities from </strong><em><b>in vitro</b></em><strong> regenerated plants of gherkin (</strong><em><b>Cucumis anguria</b></em><strong> L.)</strong>]]> Background The effect of polyamines (PAs) along with cytokinins (TDZ and BAP) and auxin (IBA) was induced by the multiple shoot regeneration from leaf explants of gherkin (Cucumis anguria L.). The polyphenolic content, antioxidant and antibacterial potential were studied from in vitro regenerated and in vivo plants. Results Murashige and Skoog (MS) medium supplemented with 3% sucrose containing a combination of 3.0 µM TDZ, 1.0 µM IBA and 75 µM spermidine induced maximum number of shoots (45 shoots per explant) was achieved. Regenerated shoots elongated in shoot elongation medium containing 1.5 µM GA3 and 50 µM spermine. The well-developed shoots were transferred to root induction medium containing 1.0 µM IBA and 50 µM putrescine. Rooted plants were hardened and successfully established in soil with a 95% survival rate. Twenty-five phenolic compounds were identified by ultra-performance liquid chromatography (UPLC) analysis The individual polyphenolic compounds, total phenolic and flavonoid contents, antioxidant and antibacterial potential were significantly higher with in vitro regenerated plants than in vivo plants. Conclusions Plant growth regulators (PGRs) and PAs had a significant effect on in vitro plant regeneration and also a biochemical accumulation of flavonols, hydroxybenzoic and hydroxycinnamic acid derivatives in C. anguria. Due to these metabolic variations, the antioxidant and antibacterial activities were increased with in vitro regenerated plants than in vivo plants. This is the first report describing the production of phenolic compounds and biological activities from in vitro and in vivo regenerated plants of C. anguria. <![CDATA[<strong>Expression and purification of soluble single-chain Fv against human fibroblast growth factor receptor 3 fused with Sumo tag in </strong><em><b>Escherichia coli</b></em>]]> Background Overexpression or mutated activation of Fibroblast growth factor receptor 3 (FGFR3) is involved in the pathogenesis of many tumors. More and more studies focus on the potential usage of therapeutic antibodies against FGFR3. Results In this study, a novel single-chain Fv (ScFv) against FGFR3 was prepared and characterized. To achieve the soluble expression, ScFv was fused with Sumo (Small ubiquitin-related modifier) by polymerase chain reaction (PCR), and cloned into pET-20b. The recombinant bacteria were induced by 0.5 mM Isopropyl-ß-d-thiogalactopyranoside (IPTG) for 16 h at 20°C, and the supernatant liquid of Sumo-ScFv was harvested and purified by Ni-NTA chromatography. After being cleaved by the Sumo protease, the recombinant ScFv was released from the fusion protein, and further purified by Ni-NTA chromatography. The purity of ScFv was shown to be higher than 95% and their yield reached 4 mg per liter of bacterial culture. In vitro data showed that ScFv can significantly attenuate FGF9-induced phosphorylation of FGFR3. Conclusion We provide a novel method to produce soluble expression and bioactive functions of ScFv in Escherichia coli. <![CDATA[<strong>Purification and characterization of xylanases from </strong><em><b>Trichoderma inhamatum</b></em>]]> Background Two xylanases, Xyl I and Xyl II, were purified from the crude extracellular extract of a Trichoderma inhamatum strain cultivated in liquid medium with oat spelts xylan. Results The molecular masses of the purified enzymes estimated by SDS-PAGE and gel filtration were, respectively, 19 and 14 kDa for Xyl I and 21 and 14.6 kDa for Xyl II. The enzymes are glycoproteins with optimum activity at 50°C in pH 5.0-5.5 for Xyl I and 5.5 for Xyl II. The xylanases were very stable at 40°C and in the pH ranges from 4.5-6.5 for Xyl I and 4.0-8.0 for Xyl II. The ion Hg2+ and the detergent SDS strongly reduced the activity while 1,4-dithiothreitol stimulated both enzymes. The xylanases showed specificity for xylan, Km and Vmax of 14.5, 1.6 mg·mL-1 and 2680.2 and 462.2 U·mg of protein-1 (Xyl I) and 10.7, 4.0 mg·mL-1 and 4553.7 and 1972.7 U·mg of protein-1 (Xyl II) on oat spelts and birchwood xylan, respectively. The hydrolysis of oat spelts xylan released xylobiose, xylotriose, xylotetrose and larger xylooligosaccharides. Conclusions The enzymes present potential for application in industrial processes that require activity in acid conditions, wide-ranging pH stability, such as for animal feed, or juice and wine industries. <![CDATA[<strong>Production of </strong><strong>β</strong><strong>-glucosidase on solid-state fermentation by </strong><em><b>Lichtheimia ramosa</b></em><strong> in agroindustrial residues</strong>: <strong>Characterization and catalytic properties of the enzymatic extract</strong>]]> Background β-Glucosidases catalyze the hydrolysis of cellobiose and cellodextrins, releasing glucose as the main product. This enzyme is used in the food, pharmaceutical, and biofuel industries. The aim of this work is to improve the β-glucosidase production by the fungus Lichtheimia ramosa by solid-state fermentation (SSF) using various agroindustrial residues and to evaluate the catalytic properties of this enzyme. Results A high production of β-glucosidase, about 274 U/g of dry substrate (or 27.4 U/mL), was obtained by cultivating the fungus on wheat bran with 65% of initial substrate moisture, at 96 h of incubation at 35°C. The enzymatic extract also exhibited carboxymethylcellulase (CMCase), xylanase, and β-xylosidase activities. The optimal activity of β-glucosidase was observed at pH 5.5 and 65°C and was stable over a pH range of 3.5-10.5. The enzyme maintained its activity (about 98% residual activity) after 1 h at 55°C. The enzyme was subject to reversible competitive inhibition with glucose and showed high catalytic activity in solutions containing up to 10% of ethanol. Conclusions β-Glucosidase characteristics associated with its ability to hydrolyze cellobiose, underscore the utility of this enzyme in diverse industrial processes. <![CDATA[<strong>Analysis of a suppressive subtractive hybridization library of </strong><em><b>Alternaria alternata</b></em><strong> resistant to 2-propenyl isothiocyanate</strong>]]> Background Isothiocyanates (ITCs) are natural products obtained from plants of the Brassicas family. They represent an environmentally friendly alternative for the control of phytopathogenic fungi. However, as it has been observed with synthetic fungicides, the possibility of inducing ITC-resistant strains is a major concern. It is, therefore, essential to understanding the molecular mechanisms of fungal resistance to ITCs. We analyzed a subtractive library containing 180 clones of an Alternaria alternata strain resistant to 2-propenyl ITC (2-pITC). After their sequencing, 141 expressed sequence tags (ESTs) were identified using the BlastX algorithm. The sequence assembly was carried out using CAP3 software; the functional annotation and metabolic pathways identification were performed using the Blast2GO program. Results The bioinformatics analysis revealed 124 reads with similarities to proteins involved in transcriptional control, defense and stress pathways, cell wall integrity maintenance, detoxification, organization and cytoskeleton destabilization; exocytosis, transport, DNA damage control, ribosome maintenance, and RNA processing. In addition, transcripts corresponding to enzymes as oxidoreductases, transferases, hydrolases, lyases, and ligases, were detected. Degradation pathways for styrene, aminobenzoate, and toluene were induced, as well as the biosynthesis of phenylpropanoid and several types of N-glycan. Conclusions The fungal response showed that natural compounds could induce tolerance/resistance mechanisms in organisms in the same manner as synthetic chemical products. The response of A. alternata to the toxicity of 2-pITC is a sophisticated phenomenon including the induction of signaling cascades targeting a broad set of cellular processes. Whole-transcriptome approaches are needed to elucidate completely the fungal response to 2-pITC. <![CDATA[<strong>Ethanol induction of laccase depends on nitrogen conditions of </strong><em><b>Pycnoporus sanguineus</b></em>]]> Background Ethanol has been pointed out as a laccase inducer. However, there are controversial reports about its efficiency with some fungi. In this study, we hypothesized that ethanol laccase induced in Pycnoporus sanguineus depends on nitrogen nutriment conditions. To prove this, we assessed laccase production in submerged cultures of P. sanguineus, with different nitrogen concentrations and with, or without ethanol added in a factorial designed experiment. Results In order to analyze the effects of factors on the response variables, a factorial ANOVA, and response-surface models were performed. It was found that the nitrogen source was the main factor that affected laccase production in P. sanguineus. The treatments with yeast extract (2 g/L) and ethanol (3 g/L) induced the highest laccase activity (31.01 ± 4.9 U/L), while the treatments with urea reached the lowest activity (less than 1.6 U/L). Ethanol had positive and synergic effects on laccase production, in accordance with the surface response model, as long as simple nitrogen sources (urea) were not available. Conclusions We suggest that laccase in P. sanguineus is regulated by a catabolic nitrogen repression mechanism; laccase activity is strongly inhibited by urea used as nitrogen source and it decreases when the amount of urea increases; contrarily, a synergic positive effect was observed between yeast extract and ethanol on laccase production.