Scielo RSS <![CDATA[Electronic Journal of Biotechnology]]> vol. 19 num. 6 lang. pt <![CDATA[SciELO Logo]]> <![CDATA[<strong>Identification and genetic diversity analysis of </strong><em><b>Memecylon </b></em><strong>species using ISSR, RAPD and Gene-Based DNA barcoding tools</strong>]]> Background: Memecylon species are commonly used in Indian ethnomedical practices. The accurate identification is vital to enhance the drug's efficacy and biosafety. In the present study, PCR based techniques like RAPD, ISSR and DNA barcoding regions, such as 5s, psbA-trnH, rpoC1, ndh and atpF-atpH, were used to authenticate and analyze the diversity of five Memecylon species collected from Western Ghats of India. Results: Phylogenetic analysis clearly distinguished Memecylon malabaricum from Memecylon wightii and Memecylon umbellatum from Memecylon edule and clades formed are in accordance with morphological keys. In the RAPD and ISSR analyses, 27 accessions representing five Memecylon species were distinctly separated into three different clades. M. malabaricum and M. wightii grouped together and M. umbellatum, M. edule and Memecylon talbotianum grouped in the same clade with high Jaccard dissimilarity coefficient and bootstrap support between each node, indicating that these grouped species are phylogenetically similar. Conclusion: Data from the present study reveals that chloroplast psbA-trnH region could be used as a potential candidate region for identifying Memecylon species, and ISSR marker system could be used for estimating genetic diversity since it has high percent polymorphism compared to RAPD marker. <![CDATA[<strong>Enhancement of botrallin and TMC-264 production in liquid culture of endophytic fungus </strong><em><b>Hyalodendriella </b></em><strong>sp. Ponipodef12 after treatments with metal ions</strong>]]> Background: Hyalodendriella sp. Ponipodef12, an endophytic fungus from a poplar hybrid, was a high producer of botrallin and TMC-264 with various bioactivities. In this study, the influences of eight metal ions (i.e.,Mn2+,Na+, Mg2+,Zn2+,Cu2+,Fe2+,Fe3+ and Al3+) on botrallin and TMC-264 production in liquid culture of the endophytic fungus Hyalodendriella sp. Ponipodef12 were investigated. Results: Three most effective metal ions (Zn2+,Cu2+ and Mg2+) along with their optimum concentrations were screened. The optimum addition time and concentrations of Zn2+,Cu2+ and Mg2+ were further obtained respectively for improving botrallin and TMC-264 production. The combination effects of Zn2+,Cu2+ and Mg2+ on the production of botrallin and TMC-264 by employing statistical method based on the central composite design (CCD) and response surface methodology (RSM) were evaluated, and two quadratic predictive models were developed for botrallin and TMC-264 production. The yields of botrallin and TMC-264, which were predicted as 144.12 mg/L and 36.04 mg/L respectively, were validated to be 146.51 mg/L and 36.63 mg/L accordingly with the optimum concentrations of Zn2+ at 0.81 mmol/L, Cu2+ at 0.20 mmol/L, and Mg2+ at 0.13 mmol/L in medium. Conclusion: The results indicated that the enhancement of botrallin and TMC-264 accumulation in liquid culture of the endophytic fungus Hyalodendriella sp. Ponipodef12 by the metal ions and their combination should be an effective strategy. <![CDATA[<strong>Xylitol production and furfural consumption by a wild type </strong><em><b>Geotrichum </b></em><strong>sp.</strong>]]> Background: Xylitol is a five carbons polyol with promising medical applications. It can be obtained from chemical D-xylose reduction or by microbial fermentation of Sugarcane Bagasse Hemicellulosic Hydrolysate. For this last process, some microbial inhibitors, as furfural, constitute severe bottleneck. In this case, the use of strains able to produce xylitol simultaneously to furfural neutralization is an interesting alternative. A wild-type strain of Geotrichum sp. was detected with this ability, and its performance in xylitol production and furfural consumption was evaluated. Furthermore, were analyzed its degradation products. Results: Geotrichum sp. produced xylitol from D-xylose fermentation with a yield of 0.44 g-g-1. Furfural was fully consumed in fermentation assay and when provided in the medium until concentration of 6 g-L-1. The furfural degradation product is not an identified molecule, presenting a molecular weight of 161 g-mol-1, an uncommon feature for the microbial metabolism of this product. Conclusion: This strain presents most remarkable potential in performing furfural consumption simultaneous to xylitol production. Subsequent efforts must be employed to establish bioprocess to simultaneous detoxification and xylitol production by Geotrichum sp. <![CDATA[<strong>Strategy of oxygen transfer coefficient control on the L-erythrulose fermentation by newly isolated </strong><em><b>Gluconobacter kondonii</b></em>]]> Background: The effect of diverse oxygen transfer coefficient on the L-erythrulose production from meso-erythritol by a newly isolated strain, Gluconobacter kondonii CGMCC8391 was investigated. In order to elucidate the effects of volumetric mass transfer coefficient (K La) on the fermentations, baffled and unbaffled flask cultures, and fed-batch cultures were developed in present work. Results: With the increase of the K La value in the fed-batch culture, L-erythrulose concentration, productivity and yield were significantly improved, while cell growth was not the best in the high K La. Thus, a two-stage oxygen supply control strategy was proposed, aimed at achieving high concentration and high productivity of L-erythrulose. During the first 12 h, Klawas controlled at 40.28 h-1 to obtain high value for cell growth, subsequently K La was controlled at 86.31 h-1 to allow for high L-erythrulose accumulation. Conclusions: Under optimal conditions, the L-erythrulose concentration, productivity, yield and DCW reached 207.9 ± 7.78 g/L, 6.50 g/L/h, 0.94 g/g, 2.68 ± 0.17 g/L, respectively. At the end of fermentation, the L-erythrulose concentration and productivity were higher than those in the previous similar reports. <![CDATA[<strong>Enzyme activity and thermostability of a non-specific nuclease from </strong><em><b>Yersinia enterocolitica </b></em><strong>subsp. </strong><em><b>palearctica </b></em><strong>by site-directed mutagenesis</strong>]]> Background: To identify the critical amino acid residues that contribute to the high enzyme activity and good thermostability of Yersinia enterocolitica subsp. palearctica (Y. NSN), 15 mutants of Y. NSN were obtained by site-directed mutagenesis in this study. And their enzyme activity and thermostability were assayed. Effect of several factors on the enzyme activity and thermostability of Y. NSN, was also investigated. Results: The results showed that the I203F and D264E mutants retained approximately 75% and 70% enzyme activity, respectively, compared to the wild-type enzyme. In addition to the I203F and D264E mutants, the mutant E202A had an obvious influence on the thermostability of Y. NSN. According to the analysis of enzyme activity and thermostability of Y. NSN, we found that Glu202, Ile203 and Asp264 might be the key residues for its high enzyme activity and good thermostability. Conclusions: Among all factors affecting enzyme activity and thermostability of Y. NSN, they failed to explain the experimental results well. One reason might be that the enzyme activity and thermostability of Y. NSN were affected not only by a single factor but also by the entire environment. <![CDATA[<strong>A fast and simple assay to quantify bacterial leukotoxin activity</strong>]]> Background: Mannheimia haemolytica is the primary bacterial pathogen in causing bovine respiratory disease with tremendous annual losses in the cattle industry. The leukotoxin from M. haemolytica is the predominant virulence factor. Several leukotoxin activity assays are available but not standardized regarding sample preparation and cell line. Furthermore, these assays suffer from a high standard error, a prolonged time consumption and often complex sample pretreatments, which is important from the bioprocess engineering point of view. Results: Within this study, an activity assay based on the continuous cell line BL3.1 combined with a commercial available adenosine triphosphate viability assay kit was established. The leukotoxin activity was found to be strongly dependent on the sample preparation. Furthermore, the interfering effect of lipopolysaccharides in the sample could be successfully suppressed by adding polymyxin B. We reached a maximum relative P95 value of 14%, which is more than seven times lower compared to current available assays as well as a time reduction up to 88%. Conclusion: Ultimately, the established leukotoxin activity assay is simple, fast and has a high reproducibility. Critical parameters regarding the sample preparation were characterized and optimized making complex sample purification superfluous. <![CDATA[<strong>Antioxidant activity and protective role on protein glycation of synthetic aminocoumarins</strong>]]> Background: Synthesized aminocoumarins are heterocyclic compounds possessing potential for the treatment of insulin-dependent diabetes mellitus with unexplored anti-glycative action. Results: In this study 4-aminocoumarin derivatives (4-ACDs) were evaluated in vitro for antiglycation (AG) activities by using the human serum albumin (HSA)/glucose system, for 8 weeks of incubation. The glycation and conformational alteration of HSA in the presence of the tested compounds were evaluated by Congo red assay, fluorescence and circular dichroism spectroscopy. The antioxidant (AO) capacity were also tested by four different assays including: DPPH (2,2'-diphenyl-1-picrylhydrazyl radical), ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulphonate) diammonium salt), FRAP (ferric reducing antioxidant power) and β-carotene-linoleic acid assay. The tested compounds showed AG and AO effects. The intensity of the accomplished AO potential is related to the type of the used assay. Significant alterations in the secondary (monitored by CD spectropolarimetry) and tertiary structure (assessed by spectrofluorimetry) of HSA upon glycation were mitigated by the 4-ACDs, suggesting their suppressive role in the late stage (post-Amadori) of the HSA glycation. Conclusions: By the analogues, in vitro ascertained AO and AG properties of 4-ACD may be recognized as rationale for their protective role against oxidative changes of proteins, thereby precluding diabetic complications in humans. <![CDATA[<strong>Expression analysis of </strong><em><b>vasa </b></em><strong>in Asian paddle crab </strong><em><b>(Charybdis japónica) </b></em><strong>exposed to Bisphenol A</strong>]]> Background: Bisphenol A (BPA) is an endocrine-disrupting chemical (EDC) with a weak estrogen-like activity in fish that is found ubiquitously in aquatic environments. However, there has been little study about BPA on the endocrine disrupting effects of crab. In the present study, cDNA of vasa was cloned and characterized in the Charybdis japonica. Histological structures of testis and expression patterns of vasa gene in the testis of C. japonica after treatment with BPA were investigated. Results: The cDNA of vasa is composed of 3051 bp with a 2166 bp open reading frame encoding 721 AA. The deduced amino acid sequence contained eight conserved domains of the DEAD-box protein family. The tissue distribution showed that vasa mRNA was specifically expressed in ovary and testis. Histologically, the sperm cells were decreased in number and an acellular zone was seen in the testis. The transcript level of vasa gradually increased with a significant difference between the experimental and control groups. After BPA exposure with 0.50 and 1.00 mg/L for 1,3, 6 and 9 d, the expression levels of vasa increased. Conclusion: These findings suggest that BPA can increase the expression level of vasa mRNA and influence the development of the testis in C. japonica. <![CDATA[<strong>A novel pH-stable, endoglucanase (JqCel5A) isolated from a salt-lake microorganism, </strong><em><b>Jonesia quinghaiensis</b></em>]]> Background: Endoglucanase, one of three type cellulases, can randomly cleave internal p-1,4-linkages in cellulose polymers. Thus, it could be applied in agricultural and industrial processes. Results: A novel endoglucanase gene (JqCel5A) was cloned from Jonesia quinghaiensis and functionally expressed in Escherichia coli Rosetta (DE3). It contained 1722 bp and encoded a 573-residue polypeptide consisting of a catalytic domain of glycoside hydrolase family 5 (GH5) and a type 2 carbohydrate-binding module (CBM2), together with a predicted molecular mass of 61.79 kD. The purified JqCel5A displayed maximum activity at 55°C and pH 7.0, with 21.7 U/mg, 26.19 U/mg and 4.81 U/mg towards the substrate carboxymethyl cellulose, barley glucan and filter paper, respectively. Interestingly, JqCel5A exhibited high pH stability over a broad pH range of pH (3-11), and had good tolerance to a wide variety of deleterious chemicals including heavy metals and detergent. The catalytic mechanism of JqCel5A was also investigated by site mutagenesis and homology-modeling in this study. Conclusions: It was believed that these properties might make JqCel5A to be potentially used in the suitable industrial catalytic condition, which has a broad pH fluctuation and/or chemical disturbance. <![CDATA[<strong>Characterization and comparability of biosimilars</strong>: <strong>A filgrastim case of study and regulatory perspectives for Latin America</strong>]]> Background: Developing countries have an estimate of ten times more approved biosimilars than developed countries. This disparity demands the need of an objective regulation that incorporates health policies according to the technological and economical capabilities of each country. One of the challenges lies on the establishment of comparability principles based on a physicochemical and biological characterization that should determine the extent of additional non-clinical and clinical studies. This is particularly relevant for licensed biosimilars in developing countries, which have an extensive clinical experience since their approval as generics' in some cases more than a decade. To exemplify the current status of biosimilars in Mexico' a characterization exercise was conducted on licensed filgrastim biosimilars using pharmacopeial and extended characterization methodologies. Results: Most of the evaluated products complied with the pharmacopeial criteria and showed comparability in their Critical Quality Attributes (CQAs) towards the reference product. These results were expected in accordance with their equivalent performance during their licensing as generics. Accordingly' a rational approval and registration renewal scheme for biosimilars is proposed, that considers the proper identification of CQAs and its thoroughly evaluation using selected techniques. Conclusions: This approach provides support to diminish uncertainty of exhibiting different pharmacological profiles and narrows or even avoids the necessity of comparative clinical studies. Ultimately, this proposal is intended to improve the accessibility to high quality biosimilars in Latin America and other developing countries. <![CDATA[<strong>The use of chitosan in protecting wooden artifacts from damage by mold fungi</strong>]]> Background: Many buildings in Egypt e.g. museums, mosques and churches, do not possess controlled environments for minimizing the risks of damage of wooden artifacts due to the growth of fungi. Fungal damage usually appears as change in wood color, appearance of stains, and sometimes deformation of wooden surfaces. In this study we focused on the effect that some fungi exert on the properties of wooden artifacts and evaluated the effectiveness of different concentrations of chitosan on their protection against damage by mold fungi. Results: Samples were collected from different monuments and environments, and fungi growing on them were isolated and identified. The isolated Penicillium chrysogenum, Aspergillus flavus and /Aspergillus niger strains were used for the infestation of new pitch pine samples. The results revealed that the lightness of samples infected with any of the tested fungi decreased with increasing incubation times. XRD analysis showed that the crystallinity of incubated samples treated individually with the different concentrations of chitosan was lower than the crystallinity of infected samples. The crystallinity index measured by the first and the second method decreased after the first and second months but increased after the third and fourth months. This may due to the reducing of amorphous part by enzymes or acids produced by fungi in wooden samples. Conclusions: The growth of fungi on the treated wood samples decreased with increasing the concentration of chitosan. Hence, it was demonstrated that chitosan prevented fungal growth, and its use could be recommended for the protection of archeological wooden artifacts. <![CDATA[<strong>Efficient expression and characterization of a cold-active endo-1, 4-</strong><strong>β</strong><strong>-glucanase from </strong><em><b>Citrobacter farmeri </b></em><strong>by co-expression of </strong><em><b>Myxococcus xanthus </b></em><strong>protein S</strong>]]> Background: Cold-active endo-1, 4-β-glucanase (EglC) can decrease energy costs and prevent product denaturation in biotechnological processes. However, the nature EglC from C. farmeri A1 showed very low activity (800 U/L). In an attempt to increase its expression level, C. farmeri EglC was expressed in Escherichia coli as an N-terminal fusion to protein S (ProS) from Myxococcus xanthus. Results: A novel expression vector, pET(ProS-EglC), was successfully constructed for the expression of C. farmeri EglC in E. coli. SDS-PAGE showed that the recombinant protein (ProS-EglC) was approximately 60 kDa. The activity of ProS-EglC was 12,400 U/L, which was considerably higher than that of the nature EglC (800 U/L). ProS-EglC was active at pH 6.5-pH 8.0, with optimum activity at pH 7.0. The recombinant protein was stable at pH 3.5-pH 6.5 for 30 min. The optimal temperature for activity of ProS-EglC was 30°C-40°C. It showed greater than 50% of maximum activity even at 5°C, indicating that the ProS-EglC is a cold-active enzyme. Its activity was increased by Co2+ and Fe2+, but decreased by Cd2+, Zn2+, Li+, methanol, Triton-X-100, acetonitrile, Tween 80, and SDS. Conclusions: The ProS-EglC is promising in application of various biotechnological processes because of its cold-active characterizations. This study also suggests a useful strategy for the expression of foreign proteins in E. coli using a ProS tag. <![CDATA[<strong>Over-expression of </strong><em><b>Mycobacterium neoaurum </b></em><strong>3-ketosteroid-∆1-dehydrogenase in </strong><em><b>Corynebacterium crenatum </b></em><strong>for efficient bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione</strong>]]> Background: 3-Ketosteroid-∆¹-dehydrogenase (KSDD), a flavoprotein enzyme, catalyzes the bioconversion of 4-androstene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD). To date, there has been no report about characterization of KSDD from Mycobacterium neoaurum strains, which were usually employed to produce AD or ADD by fermentation. Results: In this work, Corynebacterium crenatum was chosen asa new host for heterologous expression of KSDD from M. neoaurum JC-12 after codon optimization of the KSDD gene. SDS-PAGE and western blotting results indicated that the recombinant C. crenatum harboring the optimized ksdd (ksdd n) gene showed significantly improved ability to express KSDD. The expression level of KSDD was about 1.6-fold increased C. crenatum after codon optimization. After purification of the protein, we first characterized KSDD from M. neoaurum JC-12, and the results showed that the optimum temperature and pH for KSDD activity were 30°C and pH 7.0, respectively. The Km and Vmax values of purified KSDD were 8.91 µM and 6.43 mM/min. In this work, C. crenatum as a novel whole-cell catalyst was also employed and validated for bioconversion of AD to ADD. The highest transformation rate of AD to ADD by recombinant C. crenatum was about 83.87% after 10 h reaction time, which was more efficient than M. neoaurum JC-12 (only 3.56% at 10 h). Conclusions: In this work, basing on the codon optimization, overexpression, purification and characterization of KSDD, we constructed a novel system, the recombinant C. crenatum SYPA 5-5 expressing KSDD, to accumulate ADDfromADefficiently. This work provided new insights into strengthening sterol catabolism by overexpressing the key enzyme KSDD, for efficient ADD production. <![CDATA[<strong>Introgression of </strong><em><b>LTP2 </b></em><strong>gene through marker assisted backcross in barley </strong><em><b>(Hordeum vulgare </b></em><strong>L.)</strong>]]> Background: Marker-assisted introgression currently represents the most widely spread application of DNA markers as an aid to selection in plant breeding. New barley germplasm should be supplemented by genes that facilitate growth and development under stressful conditions. The homology search against known genes is a fundamental approach to identify genes among the generated sequences. This procedure can be utilized for SNP search in genes of predicted function of interest and associated gene ontology (GO). Results: Backcross breeding enhanced by marker selection may become a powerful method to transfer one or a few genes controlling a specific trait. In the study, the integrated approach of combining phenotypic selection with marker assisted backcross breeding for introgression of LTP2 gene, in the background of semi-dwarf spring barley cultivar, was employed. This study discusses the efficiency of molecular marker application in backcrossing targeted on the selected gene. Conclusions: BC6 lines developed in this study can serve as a unique and adequate plant material to dissect the role of LTP2 gene. Due to its role in lipid transfer, the LTP2 may be crucial in lipidome modification in response to abiotic stress.