Scielo RSS <![CDATA[Electronic Journal of Biotechnology]]> vol. 18 num. 6 lang. pt <![CDATA[SciELO Logo]]> <![CDATA[<strong>Evidence of a role for prolactin as regulators of ovarian follicular development in goose</strong>]]> Background Prolactin (PRL) regulates development and reproduction, and its effects are mediated by the prolactin receptor (PRLR). In order to clarify the role of PRLR and PRL in the process of follicular development in the goose ovary, the level of PRLR mRNA expression in the ovary and follicles of the Sichuan white goose was determined, as well as the PRL concentration in ovarian follicles. Results The level of PRLR mRNA in the hierarchical follicles (HFs) initially increased, and subsequently decreased, whereas PRLR expression was initially low and later increased in postovulatory follicles (POFs). The level of PRLR mRNA expression was the highest in the F4 follicles, and lowest in the F1 follicles in all of the examined follicles. Compared with the level of PRLR mRNA expression in the small white follicles (SWFs), the level of PRLR mRNA was 2.86- and 1.44-fold higher in the F4 and small yellow follicles (SYFs), respectively (P < 0.05). The level of PRLR mRNA expression in the F4 follicles was highest (P < 0.05) in HFs. The highest PRL concentration in all of the examined samples was observed in SYFs and F1, with concentration of 6162 mLU/g and 6197 mLU/g, respectively. The PRL concentration in SYFs was significantly higher compared with SWFs (P < 0.05). Conclusions The change of PRL concentration was similar to the PRLR mRNA expression level in preovulatory follicles. These results suggest that the PRL mediated by the PRLR plays a stimulatory role in the SWF to SYF transition. <![CDATA[<strong>Characterization of the acetohydroxyacid synthase multigene family in the tetraploide plant </strong><em><b>Chenopodium quinoa</b></em>]]> Background Currently, the technology called Clearfield® is used in the development of crops resistant to herbicides that inhibit the enzyme acetohydroxy acid synthase (AHAS, EC AHAS is the first enzyme of the biosynthetic pathway that produces the branched-chain of the essential amino acids valine, leucine, and isoleucine. Therefore, multiple copies of the AHAS gene might be of interest for breeding programs targeting herbicide resistance. In this work, the characterization of the AHAS gene was accomplished for the Chenopodium quinoa Regalona-Baer cultivar. Cloning, sequencing, and Southern blotting were conducted to determine the number of gene copies. Results The presence of multiple copies of the AHAS gene as has been shown previously in several other species is described. Six copies of the AHAS gene were confirmed with Southern blot analyses. CqHAS1 and CqAHAS2 variants showed the highest homology with AHAS mRNA sequences found in the NR Database. A third copy, CqAHAS3, shared similar fragments with both CqAHAS1 and CqAHAS2, suggesting duplication through homeologous chromosomes pairing. Conclusions The presence of multiple copies of the gene AHAS shows that gene duplication is a common feature in polyploid species during evolution. In addition, to our knowledge, this is the first report of the interaction of sub-genomes in quinoa. <![CDATA[<strong>Active anti-acetylcholinesterase component of secondary metabolites produced by the endophytic fungi of </strong><em><b>Huperzia serrata</b></em>]]> Background An endophytic fungus lives within a healthy plant during certain stages of, or throughout, its life cycle. Endophytic fungi do not always cause plant disease, and they include fungi that yield different effects, including mutual benefit, and neutral and pathogenic effects. Endophytic fungi promote plant growth, improve the host plant's resistance to biotic and abiotic stresses, and can produce the same or similar biologically active substances as the host. Thus, endophytic fungal products have important implications in drug development. Result Among the numerous endophytic fungi, we identified two strains, L10Q37 and LQ2F02, that have anti-acetylcholinesterase activity, but the active compound was not huperzine A. The aim of this study was to investigate the anti-acetylcholinesterase activity of secondary metabolites isolated from the endophytic fungi of Huperzia serrata. Microbial cultivation and fermentation were used to obtain secondary metabolites. Active components were then extracted from the secondary metabolites, and their activities were tracked. Two compounds that were isolated from endophytic fungi of H. serrata were identified and had acetylcholine inhibitory activities. In conclusion, endophytic fungal strains were found in H. serrata that had the same anti-acetylcholinesterase activity. Conclusion We isolated 4 compounds from the endophytic fungus L10Q37, among them S1 and S3 are new compounds. 6 compounds were isolated from LQ2F02, all 6 compounds are new compounds. After tested anti acetylcholinesterase activity, S5 has the best activity. Other compounds' anti acetylcholinesterase activity was not better compared with huperzine A. <![CDATA[<strong>Effect of edible quinoa protein-chitosan based films on refrigerated strawberry (</strong><em><b>Fragaria</b></em><strong> × </strong><em><b>ananassa</b></em><strong>) quality</strong>]]> Background Strawberries are non-climacteric fruits with a low respiration rate, but are subject to serious fungal deterioration during postharvest handling. The edible coatings based on chitosan (CH), quinoa protein-chitosan (Q/CH) and quinoa protein-chitosan-sunflower oil (Q/CH/SO) may provide a solution to this problem. Thus, in this work CH, Q/CH and Q/CH/SO were elaborated and applied to fresh strawberries, and its effect on the strawberries shelf life during storage for 15 d was evaluated by mold and yeast count, fungal decay, carbon dioxide rate, physicochemical properties, and sensory evaluation. Results On all analysis days, the strawberries coated with the film-forming CH, Q/CH and Q/CH/SO solutions presented a significant lower amount of mold and yeast growth than the uncoated strawberries. Coated strawberries with Q/CH/SO decreased the CO2 emission rate by 60% compared to the uncoated strawberries. The color of the strawberries was not influenced by the films. There was no significant difference between the different coating groups and the uncoated group in the physicochemical parameters. Sensory analysis showed that the coating application retained the total sensorial quality. Conclusions Fresh strawberries coated with CH, Q/CH/SO and Q/CH edible films had longer shelf lives than uncoated fruits. <![CDATA[<strong>Repetitive element palindromic PCR (rep-PCR) as a genetic tool to study interspecific diversity in Euphorbiaceae family</strong>]]> Background The classification of diversity in germplasm collections is important for plant breeding. The repetitive element palindromic-polymerase chain reaction (rep-PCR) technique was used to investigate inter-specific diversity within 17 species from the Euphorbiaceae family using REP and BOX primers. Results The agglomerative cluster analysis was used to evaluate the scoring data. BOX and REP gave amplification with polymorphism of 98.84% and 100% respectively. REP marker demarcated between the subgenus peltatae. Both markers confirmed Jatropha tanjorensis as a natural hybrid between Jatropha gossypifolia and Jatropha curcas. Five random sequences from the rep-PCR gels were chosen, cloned and sequenced. The blast results demonstrated that the amplified products were from the mitochondrial genomes. Conclusion The rep-PCR molecular tool can be used to characterize diversity in plants as they are suitable for distinguishing eukaryotic genomes effectively. <![CDATA[<strong>Copper-induced adaptation, oxidative stress and its tolerance in </strong><em><b>Aspergillus niger</b></em><strong> UCP1261</strong>]]> Background The effects of exposure to copper, during growth, on the production of biomass, total protein, catalase, glutathione-S transferase, glutathione peroxidase, peroxidase, polyphosphate, acid and alkaline phosphatases, ultrastructure and the ability to remove this metal from Aspergillus niger, obtained from caatinga soil, were evaluated. Results All parameters tested were influenced by the concentration of metal in the culture medium. The presence of metal induced high levels of antioxidant enzymes, including lipid peroxidation, thereby revealing the appearance of an oxidative stress response. The variation in polyphosphate levels indicates the participation of the polymer in response to stress induced by copper. The activities of the phosphatases were positively influenced by growing them in the presence of copper. Ultrastructure changes in the cell surface, electron density, thickness, and septation were visualized by exposing cells to increasingly larger concentrations of metal. The isolate was able to remove the agent from the growth medium, while maintaining its physiological functions. The metal removed from the cultures exposed to 0.5 mM, 1 mM and 2 mM copper exhibited percentages of removal equivalent to 75.78%, 66.04% and 33.51%. Conclusions The results indicate that the isolate was able to grow in high concentrations of copper, activates mechanisms for adaptation and tolerance in the presence of metal, and is highly efficient at removing the agent. Such data are fundamental if a better understanding is to be reached of the cellular and molecular abilities of native isolates, which can be used to develop bioprocesses in environmental and industrial areas. <![CDATA[<strong>Biodegradation of deproteinized potato wastewater and glycerol during cultivation of </strong><em><b>Rhodotorula glutinis</b></em><strong> yeast</strong>]]> Background Deproteinized potato wastewater and glycerol are two by-products which are difficult to dispose. The objective of this study was to determine the ability of Rhodotorula glutinis to use glycerol and nitrogen compounds present in deproteinized potato wastewater and to evaluate the ability of simultaneous biodegradation of potato wastewater and glycerol via microbiological methods. Results It has been found that R. glutinis used glycerol and potato wastewater as a source of carbon and nitrogen, respectively. The highest degree of glycerol content (70.6%) reduction was found after cultivation of the investigated strain using a medium with 5% glycerol. In this medium, a significant reduction in the total protein content, estimated at 61%, was observed. The process of 72 h cultivation of yeast in a medium containing potato wastewater and 5% glycerol reduced the chemical oxygen demand (COD) more than 77%. Supplementation of media with high doses of glycerol (i.e. 20 and 25%) led to decreased metabolic activity in the yeast strain tested. Conclusion It has been found that there is a possibility of simultaneous biodegradation of potato wastewater and glycerol during the cultivation of R. glutinis. <![CDATA[<strong>Phytoconstituents and antioxidant properties among commercial tea (</strong><em><b>Camellia sinensis</b></em><strong> L.) clones of Iran</strong>]]> Background Tea (Camellia sinensis), a well-known beverage is consumed frequently worldwide due to its high antioxidant properties. The present study determines the amount of phytochemicals and antioxidant activities among 12 high yielding tea clones cultivated in Iran. Results Among the 12 clones studied, tea clone Iran 100 had the highest total phenolic content and total flavonoid content with values of 8.44 ± 1.03 mg gallic acid equivalents per gram dry weight and 4.50 ± 0.16 mg rutin equivalents per gram dry weight respectively. High performance Liquid Chromatography (HPLC) analysis of phenolics and flavonoids in 12 clones revealed the presence of (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, (-)-epigallocatechin-gallate, (-)-epicatechingallate, gallic acid and caffeine. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay showed the existence of variation in the antioxidant activity ranging from 22.67 to 65.36%. The highest antioxidant activity with IC50 value of 218.24 µg/mL was observed in the leaf extract of the clone Iran 100, while the lowest was found in the clone Iran 482 with IC50 value of 234.44 µg/mL. The antioxidant activity had a positive correlation with total phenolic content, total flavonoid content, (-)-epigallocatechin-gallate, (-)-epicatechingallate and caffeine (0.59 = r = 0.97, P < 0.05). Conclusion From the study it can be concluded that the clone Iran 100 has a superior quality compared to any other clones studied due to occurrence of more phenolic compounds and a greater antioxidant activity. Hence, we recommend the use of tea clone Iran 100 for commercial planting. <![CDATA[<strong>Use of AFLP markers to estimate molecular diversity of </strong><em><b>Phakopsora pachyrhizi</b></em>]]> Background Asian soybean rust (SBR) caused by Phakopsora pachyrhizi Syd. & Syd., is one of the main diseases affecting soybean and has been reported as one of the most economically important fungal pathogens worldwide. Knowledge of the genetic diversity of this fungus should be considered when developing resistance breeding strategies. We aimed to analyze the genetic diversity of P. pachyrhizi combining simple sampling with a powerful and reproducible molecular technique. Results We employed Amplified Fragment Length Polymorphism (AFLP) technique for the amplification of P. pachyrhizi DNA extracted from naturally SBR-infected plants from 23 production fields. From a total of 1919 markers obtained, 77% were polymorphic. The high percentage of polymorphism and the Nei's genetic diversity coefficient (0.22) indicated high pathogen diversity. Analysis of molecular variance showed higher genetic variation within countries than among them. Temporal analysis showed a higher genetic variation within a year than between years. Cluster, phylogenetic and principal co-ordinate analysis showed that samples group by year of collection and then by country sampled. Conclusions The study proposed combining a simple collection of urediniospore with a subsequent analysis by AFLP was useful to examine the molecular polymorphism of samples of P. pachyrhizi collected and might have a significant contribution to the knowledge of its genetic diversity. Also, AFLP analysis is an important and potent molecular tool for the study of genetic diversity and could be useful to carry out wider genetic diversity studies. <![CDATA[<strong>Methanogenic toxicity evaluation of chlortetracycline hydrochloride</strong>]]> Background Anaerobic digestion is a technology applied successfully to converting organic matter into biogas. However, the presence of inhibitory compounds such as antibiotics can adversely affect methane production. The aim of this study is to evaluate the toxic effect of chlortetracycline hydrochloride (CLOR) on the methanogenic bacteria. In order to study the methanogenic toxicity of CLOR, different concentrations of CLOR (10, 50, 100, 200 mg L- 1) were evaluated by methanogenic toxicity assays using three feedings. Results Maximum methane production was obtained for the assays with 10 mg CLOR L- 1, the values obtained were 277 ± 4.07; 193 ± 11.31 and 166 ± 7.07 mL for the first, second and third feedings, respectively. The average values for acetic, propionic and butyric acid at start of the experiments were 2104 ± 139; 632 ± 7.6; 544 ± 26 mg L- 1, respectively. The VFA values obtained finally of the experiment were dependent on the evaluated antibiotic concentrations, indicating that the efficiency of methanogenesis is directly affected by the CLOR concentration. Conclusions CLOR is an effective methanogenic bacteria inhibitor. Moreover, the results show that CLOR has a bactericidal effect on methanogenic activity given that methane production did not recover during the third feeding. This study shows that the 50% inhibitory concentration (IC50) for methanogenic bacteria in 10 mg L- 1. <![CDATA[<strong>Molecular characterization of a novel thermostable laccase PPLCC2 from the brown rot fungus </strong><em><b>Postia placenta</b></em><strong> MAD-698-R</strong>]]> Background Laccase has been considered important for the degradation of lignocellulose by wood rot fungi. The properties and functions of laccase in white rot fungi have been investigated extensively, but those from brown rot fungi remain largely unknown. In this paper, a laccase isoform Pplcc2 from the brown rot fungus Postia placenta MAD-698-R was expressed heterologously in Pichia pastoris GS115, purified and the properties of the enzyme were determined. Results The molecular weight of the protein was determined to be 67 kDa using SDS-PAGE. It cannot oxidize syringaldazine (SGZ), but it can oxidize 2,2'-azino-di-(3-ethylbenzothialozin-6-Sulfonic acid) (ABTS) and 2,6-dimethoxyphenol (DMP). Specific activity for ABTS was 1960 ± 19 Unit/mg. The catalytic constant (k cat) was 1213 ± 18.3 s-1 for ABTS and 293.2 ± 21.9 s-1 for DMP. Km was 22.08 µM for ABTS and 11.62 µM for DMP. The optimal pH for the oxidation of ABTS and DMP was 3.5 and 5.0 respectively. The optimal temperature for the oxidation of ABTS and DMP was 60°C. Conclusions This is the first identified thermo activated and thermostable laccase in brown rot fungi. This investigation will contribute to understanding the roles played by laccases in brown rot fungi. <![CDATA[<strong>Amplification of </strong><em><b>tlh</b></em><strong> gene in other Vibrionaceae specie by specie-specific multiplex PCR of </strong><em><b>Vibrio parahaemolyticus</b></em>]]> Background The surveillance of Vibrio parahaemolyticus in the Chilean coast has been mainly performed by multiplex PCR amplification of three different hemolysin genes, which are specie-specific virulence factors. These genes are also employed in the determination of V. parahaemolyticus pathogenic load in seafood and for characterization of pathogenic strains associated to diarrhea cases in human. During environmental surveillance that we performed every summer, we occasionally observed a thermolabile hemolysin (tlh) PCR product of a slightly smaller size than expected, which was coincident with low loads of V. parahaemolyticus in the environment. In order to understand this observation, we probed the specificity of tlh primers for the detection of V. parahaemolyticus at different bacterial loads and DNA concentrations. Results Primers used for the detection of V. parahaemolyticus specific tlh amplified a slightly smaller tlh gene, which is found in Vibrio alginolyticus and other related strains. These amplicons were observed when V. parahaemolyticus was absent or in undetectable loads in the environment. Conclusions Surveillance of V. parahaemolyticus using tlh primers can be imprecise because amplification of a V. parahaemolyticus specific marker in V. alginolyticus and other related strains occurs. This situation complicates potentially the estimation of bacterial load in seafood, because do not ensure the correct identification of V. parahaemolyticus when his load is low. Additionally, it could complicate the tracking of outbreaks of V. parahaemolyticus infections, considering the genetic markers used would not be specie-specific. <![CDATA[<strong>Characterization of a multi-tolerant tannin acyl hydrolase II from </strong><em><b>Aspergillus carbonarius</b></em><strong> produced under solid-state fermentation</strong>]]> Background Tannases are enzymes with biotechnological potential produced mainly by microorganisms as filamentous fungi. In this context, the production and characterization of a multi-tolerant tannase from Aspergillus carbonarius is described. Results The filamentous fungus A. carbonarius produced high levels of tannase when cultivated under solid-state fermentation using green tea leaves as substrate/carbon source and tap water at a 1:1 ratio as the moisture agent for 72 h at 30°C. Two tannase activity peaks were obtained during the purification step using DEAE-Cellulose. The second peak (peak II) was purified 11-fold with 14% recovery from a Sepharose CL-6B chromatographic column. The tannase from peak II (tannase II) was characterized as a heterodimeric glycoprotein of 134.89 kDa, estimated through gel filtration, with subunits of 65 kDa and 100 kDa, estimated through SDS-PAGE, and 48% carbohydrate content. The optimal temperature and pH for tannase II activity was 60°C and 5.0, respectively. The enzyme was fully stable at temperatures ranging from 20-60°C for 120 min, and the half-life (T1/2) at 75°C was 62 min. The activation energy was 28.93 kJ/mol. After incubation at pH 5.0 for 60 min, 75% of the enzyme activity was maintained. However, enzyme activity was increased in the presence of AgNO3 and it was tolerant to solvents and detergents. Tannase II exhibited a better affinity for methyl gallate (Km = 1.42 mM) rather than for tannic acid (Km = 2.2 mM). Conclusion A. carbonarius tannase presented interesting properties as, for example, multi-tolerance, which highlight its potential for future application. <![CDATA[<strong>Application of bacterial and yeast biosurfactants for enhanced removal and biodegradation of motor oil from contaminated sand</strong>]]> Background This study investigated the potential application of two biosurfactants for enhanced removal capability and biodegradation of motor oil contaminated sand under laboratory conditions. The biosurfactants were produced by the yeast Candida sphaerica and by the bacterium Bacillus sp. cultivated in low-cost substrates. The ability of removing motor oil from soil by the two biosurfactants was identified and compared with that of the synthetic surfactants Tween 80 and Triton X-100. Results Both crude and isolated biosurfactants showed excellent effectiveness on motor oil removal from contaminated sand under kinetic conditions (70-90%), while the synthetic surfactants removed between 55 and 80% of the oil. A contact time of 5-10 min under agitation seemed to be enough for oil removal with the biosurfactants and synthetic surfactants tested. The crude and the isolated biosurfactant from C. sphaerica were able to remove high percentages of motor oil from packed columns (around 90%) when compared to the biosurfactant from Bacillus sp. (40%). For the degradation experiments conducted in motor oil contaminated sand enriched with sugar cane molasses, however, oil degradation reached almost 100% after 90 d in the presence of Bacillus sp. cells, while the percentage of oil degradation did not exceed 50% in the presence of C. sphaerica. The presence of the biosurfactants increased the degradation rate in 10-20%, especially during the first 45 d, indicating that biosurfactants acted as efficient enhancers for hydrocarbon biodegradation. Conclusions The results indicated the biosurfactants enhancing capability on both removal and rate of motor oil biodegradation in soil systems. <![CDATA[<strong>Topological analysis of carbon flux during multi-stress adaptation in </strong><em><b>Halomonas</b></em><strong> sp. AAD12</strong>]]> Background Osmolytes with their effective stabilizing properties are accumulated as protectants not only against salinity but also against denaturing harsh environmental stresses such as freezing, drying, high temperatures, oxygen radicals and radiation. The present work seeks to understand how Halomonas sp. AAD12 cells redirect carbon flux specifically to replenish reactions for biomass and osmolyte synthesis under changing salinity and temperature. To accomplish this goal, a combined FBA-PCA approach has been utilized. Results Experimental data were collected to supply model constraints for FBA and for the verification of the model predictions, which were satisfactory. With restrictions on the various combinations of selected anaplerotic paths (reactions catalyzed by phosphoenolpyruvate carboxylase, pyruvate carboxylase or glyoxylate shunt), two major phenotypes were found. Moreover, under high salt concentrations, when the glucose uptake rate was over 1.1 mmoL DCW- 1 h- 1, an overflow metabolism that led to the synthesis of ethanol caused a slight change in both phenotypes. Conclusions The operation of the glyoxylate shunt as the major anaplerotic pathway and the degradation of 6-phosphogluconate through the Entner-Doudoroff Pathway were the major factors in causing a distinction between the observed phenotypes.