Scielo RSS <![CDATA[Electronic Journal of Biotechnology]]> vol. 19 num. 4 lang. pt <![CDATA[SciELO Logo]]> <![CDATA[<strong>Response surface methodology based optimization for degradation of align in </strong><em><b>Laminaria japónica </b></em><strong>feedstuff via fermentation by </strong><em><b>Bacillus </b></em><strong>in </strong><em><b>Apostichopus japónicas </b></em><strong>farming</strong>]]> Background: The alga Laminaria japonica is the most economically important brown seaweed cultured in China, which is used as food and aquatic animal feedstuff. However, the use of L. japonica as a feedstuff in Apostichopus japonicasfarming is not ideal because A. japonicas does not produce enough enzyme activity for degrading the large amount of algin present in L. japonica. In this study, semi solid fermentation of the L. japonica feedstuff employing a Bacillus strain as the microbe was used to as a mean to degrade the algin content in L. japonica feedstuff. Results: The Bacillus strain, Bacillus amyloliquefaciens WB1, was isolated by virtue of its ability to utilize sodium alginate as the sole carbon source. Eight factors affecting growth and algin-degrading capacity of WB1 were investigated. The results of Plackett-Burman design indicated that fermentation time, beef extract, and solvent to solid ratio were the significant parameters. Furthermore, the mutual interaction between the solvent to solid ratio and beef extract concentration was more significant than the other pairs of parameters on algin degradation. Optimal values obtained from Central-Composite Design were 113.94 h for fermentation time, 0.3% (w/v) beef extract and 44.87 (v/w) ratio of solvent to feedstuff. Under optimal conditions, 56.88% of the algin was degraded when a 50-fold scale-up fermentation was carried out, using a 5-L fermenter. Conclusions: This study provides an alternative and economical way to reduce the algin content in L. japonicathrough degradation by WB1, making it a promising potential source of feed for cultured L japonica. <![CDATA[<strong>A quantitative PCR approach for determining the ribosomal DNA copy number in the genome of </strong><em><b>Agave tequila </b></em><strong>Weber</strong>]]> Background: Agave tequilana has a great economic importance in Mexico in order to produce alcoholic beverages and bioenergy. However, in this species the structure and organization of the rDNAs in the genome are limited, and it represents an obstacle both in their genetic research and improvement as well. rDNA copy number variations per eukaryotic genome have been considered as a source of genetic rearrangements. In this study, the copy number of 18S and 5S rDNAs in the A. tequilana genome was estimated, and an absolute quantitative qPCR assay and genome size was used. In addition, an association between the rDNAs copy number and physical mapping was performed to confirm our results. Results: The analysis were successfully applied to determine copy number of 18S and 5S rDNAs in A. tequilana genome, showing high reproducibility with coefficient of variation (CV) values of 0.014-0.0129%, respectively. A variation of 51 times in the copy number the 18s regarding 5s rDNA was found, thus contributing to genome size of 1.47 and 8.38 x 10-3%, respectively. Similarly, data show a linear relationship (R [2] = 0.992) between rDNA copy number and the detected signals for each of the loci by FISH. The comparison of the rDNA copy number of agave showed differential relationship with other organisms and it may be due to evolutionary ecology.Conclusions: Results show that the proposed method a) can correctly detect the rDNA copy number, b) could be used as species-specific markers and c) might help in understanding the genetic diversity, genome organization and evolution of this species. <![CDATA[Statistical optimization of thermo-alkali stable xylanase production from <em>Bacillus tequilensis </em>strain ARMATI]]> Background: Xylanase from bacteria finds use in prebleaching process and bioconversion of lignocelluloses into feedstocks. The xylanolytic enzyme brings about the hydrolysis of complex biomolecules into simple monomer units. This study aims to optimize the cellulase-free xylanase production and cell biomass of Bacillus tequilensis strain ARMATI using response surface methodology (RSM). Results: Statistical screening of medium constituents and the physical factors affecting xylanase and biomass yield of the isolate were optimized by RSM using central composite design at N = 30, namely 30 experimental runs with 4 independent variables. The central composite design showed 3.7 fold and 1.5 fold increased xylanase production and biomass yield of the isolate respectively compared to 'one factor at a time approach',inthe presence of the basal medium containing birchwood xylan (1.5% w/v) and yeast extract (1% w/v), incubated at 40°C for 24 h. Analysis of variance (ANOVA) revealed high coefficient of determination (R2)of0.9978 and 0.9906 for the respective responses at significant level (p < 0.05). The crude xylanase obtained from the isolate showed stability at high temperature (60°C) and alkaline condition (pH 9) up to 4 h of incubation. Conclusions: The cellulase-free xylanase showed an alkali-tolerant and thermo-stable property with potentially applicable nature at industrial scale. This statistical approach established a major contribution in enzyme production from the isolate by optimizing independent factors and represents a first reference on the enhanced production of thermo-alkali stable cellulase-free xylanase from B. tequilensis. <![CDATA[<strong>Asn336 is involved in the substrate affinity of glycine oxidase from </strong><em><b>Bacillus cereus</b></em>]]> Background: Glycine oxidase (GO), a type of D-amino acid oxidase, is of biotechnological interest for its potential in several fields. In our previous study, we have characterized a new glycine oxidase (BceGO) from Bacillus cereus HYC-7. Here, a variant of N336K with increased the affinity against all the tested substrate was obtained by screening a random mutant library of BceGO. It is observed that the residue N336 is invariable between its homogeneous enzymes. This work was aimed to explore the role of the residue N336 in glycine oxidase by site-directed mutagenesis, kinetic assay, structure modeling and substrate docking. Results: The results showed that the affinity of N336H, N336K and N336R increased gradually toward all the substrates, with increase in positive charge on side chain, while N336A and N336G have not shown a little significant effect on substrate affinity. The structure modeling studies indicated that the residue Asn336 is located in a random coil between |J-18 and a-10. Also, far-UV CD spectra-analysis showed that the mutations at Asn336 do not affect the secondary structure of enzyme. Conclusion: Asn336 site was located in a conserved GHYRNG loop which adjoining to substrate and the isoalloxazine ring of FAD, and involved in the substrate affinity of glycine oxidase. This might provide new insight into the structure-function relationship of GO, and valuable clue to redesign its substrate specificity for some biotechnological application. <![CDATA[<strong>The exopolysaccharides biosynthesis by </strong><em><b>Candida </b></em><strong>yeast depends on carbon sources</strong>]]> Background: The exopolysaccharides (EPS) produced by yeast exhibit physico-chemical and rheological properties, which are useful in the production of food and in the cosmetic and pharmaceutical industries as well. The effect was investigated of selected carbon sources on the biosynthesis of EPS by Candida famata and Candida guilliermondii strains originally isolated from kefirs. Results: The biomass yields were dependent on carbon source (sucrose, maltose, lactose, glycerol, sorbitol) and ranged from 4.13 to 7.15 g/L. The highest biomass yield was reported for C. guilliermondii after cultivation on maltose. The maximum specific productivity of EPS during cultivation on maltose was 0.505 and 0.321 for C. guilliermondii and C. famata, respectively. The highest EPS yield was found for C. guilliermondii strain. The EPS produced under these conditions contained 65.4% and 61.5% carbohydrates, respectively. The specific growth rate (µ) of C. famata in medium containing EPS as a sole carbon source was 0.0068 h-1 and 0.0138 h-1 for C. guilliermondii strain. Conclusions: The most preferred carbon source in the synthesis of EPS for both Candida strains was maltose, wherein C. guilliermondii strain showed the higher yield of EPS biosynthesis. The carbon source affected the chemical composition of the resulting EPS and the contribution of carbohydrate in the precipitated preparation of polymers was higher during supplementation of maltose as compared to sucrose. It was also found that the EPS can be a source of carbon for the producing strains. <![CDATA[<strong>Expression of Hemagglutinin-Neuraminidase and fusion epitopes of Newcastle Disease Virus in transgenic tobacco</strong>]]> Background: Newcastle disease is an important avian infectious disease that brings about vast economic damage for poultry industry. Transgenic plants represent a cost-effective system for the production of therapeutic proteins and are widely used for the production of poultry vaccines. In an attempt to develop a recombinant vaccine, a plant expression binary vector pBI121, containing the genes encoding Hemagglutinin-Neuraminidase (HN) and Fusion (F) epitopes of Newcastle Disease Virus (NDV) under the control of CaMV35S promoter and NOS terminator was constructed and introduced into the tobacco ( Nicotiana tabacum) plant by Agrobacterium-mediated transformation. Results: Putative transgenic plants were screened in a selection medium containing 50 mg/L kanamycin and 30 mg/L meropenem. Integration of the foreign gene in plant genome was confirmed by PCR. Expression of foreign gene was analyzed at transcription level by RT-PCR and at translation level by means of dot blotting and ELISA. All analyses confirmed the expression of recombinant protein. Conclusion: Developments in genetic engineering have led to plant-based systems for recombinant vaccine production. In this research, tobacco plant was used to express F and HN epitopes of NDV. Our results indicate that for the production of recombinant vaccine, it is a novel strategy to use concatenated epitopes without their genetic fusion onto larger scaffold structure such as viral coat protein. <![CDATA[<strong>Expression, purification and thermal stability evaluation of an engineered amaranth protein expressed in </strong><em><b>Escherichia coli</b></em>]]> Background: The acidic subunit of amarantin (AAC)-the predominant amaranth seed storage protein-has functional potential and its third variable region (VR) has been modified with antihypertensive peptides to improve this potential. Here, we modified the C-terminal in the fourth VR of AAC by inserting four VY antihypertensive peptides. This modified protein (AACM.4) was expressed in Escherichia coli. In addition, we also recombinantly expressed other derivatives of the amarantin protein. These include: unmodified amarantin acidic subunit (AAC); amarantin acidic subunit modified at the third VR with four VY peptides (AACM.3); and amarantin acidic subunit doubly modified, in the third VR with four VY peptides and in the fourth VR with the RIPP peptide (AACM.3.4). Results: E. coli BL21-CodonPlus (DE3)-RIL was the most favorable strain for the expression of proteins. After 6 h of induction, it showed the best recombinant protein titer. The AAC and AACM.4 were obtained at higher titers (0.56 g/L) while proteins modified in the third VR showed lower titers: 0.44 g/L and 0.33 g/L for AACM.3 and AACM.3.4, respectively. As these AAC variants were mostly expressed in an insoluble form, we applied a refolding protocol. This made it possible to obtain all proteins in soluble form. Modification of the VR 4 improves the thermal stability of amarantin acidic subunit; AAC manifested melting temperature (Tm) at 34°C and AACM.4 at 37.2°C. The AACM.3 and AACM.3.4 did not show transition curves. Conclusions: Modifications to the third VR affect the thermal stability of amarantin acidic subunit. <![CDATA[<strong>Evaluation of fitness in F<sub>2</sub> generations of Africa Biofortified Sorghum event 188 and weedy </strong><em><b>Sorghum bicolor </b></em><strong>ssp. </strong><em><b>drummondii</b></em>]]> Background: Introgression of transgenes from crops to their wild species may enhance the adaptive advantage and therefore the invasiveness of and weedy forms. The study evaluated the effect of Africa Biofortified Sorghum (ABS) genes from ABS event 188 on the vegetative and reproductive features of the F2 populations derived from crosses with Sorghum bicolor subsp. drummondii. Results: F1 populations were obtained from reciprocal crosses involving ABS event 188 and its null segregant with inbred weedy parents from S. bicolor subsp. drummondii. Four F2 populations and four parental populations were raised in RCBD with 4 replications in a confined field plot for two seasons. Vegetative and reproductive traits were evaluated. The vigour shown in the F2 populations from the reciprocal crosses involving ABS event 188 and S. bicolor subsp. drummondii was similar to that in the crosses involving the null segregant and S. bicolor subsp. drummondii. Differences in vegetative and reproductive parameters were observed between the parental controls and the F2 populations. Examination of the above and below ground vegetative biomass showed lack of novel weedy related features like rhizomes. Conclusions: Therefore, release of crops with ABS 188 transgenes into cropping systems is not likely to pose a risk of conferring additional adaptive advantage in the introgressing populations. The interaction of ABS genes in weedy backgrounds will also not have an effect towards enhancing the weedy features in these populations. <![CDATA[<strong>Enhancing production of lipase MAS1 from marine </strong><em><b>Streptomyces </b></em><strong>sp. strain in </strong><em><b>Pichia pastoris </b></em><strong>by chaperones co-expression</strong>]]> Background: A thermostable lipase MAS1 from marine Streptomyces sp. strain was considered as a potential biocatalyst for industrial application, but its production level was relatively low. Here, the effect of chaperones co-expression on the secretory expression of lipase MAS1 in Pichia pastoris was investigated. Result: Co-expression of protein disulfide isomerase (PDI), HAC1 and immunoglobulin binding protein could increase the expression level of lipase MAS1, whereas co-expression of Vitreoscilla hemoglobin showed a negative effect to the lipase MAS1 production. Among them, PDI co-expression increased lipase MAS1 expression level by 1.7-fold compared to the control strain harboring only the MAS1 gene. Furthermore, optimizing production of lipase MAS1 with Pichia pastoris strain X-33/MAS1-PDI in a 30-L bioreactor were conducted. Lower induction temperature was found to have a benefit effect for lipase MAS1 production. Lipase activity at 24 and 22°C showed 1.7 and 2.1-fold to that at 30°C, respectively. Among the induction pH tested, the highest lipase activity was obtained at pH 6.0 with activity of 440 U/mL after 144 h fermentation. Conclusion: Our work showed a good example for improving the production of recombinant enzymes in Pichia pastoris via chaperon co-expression and fermentation condition optimization. <![CDATA[<b>Effect of <em>Brachionus rubens </em>on the growth characteristics of various species of microalgae</b>]]> Background: Cultivation of algae for conversion to biofuels has gained global interest. Outdoor raceway cultivation is preferred because of its lower capital and operating costs. A major disadvantage of outdoor cultivation is susceptibility of algal crops to attack by predatory rotifers. In order to quantify the impact of rotifer attack on different species of algae, we evaluated the growth of eleven microalgal species over a 21-d period after being infected by the predatory rotifer Brachionus rubens. Results: Of the eleven species, Chlorella sorokiniana was the most susceptible with rapid decline in algal growth concomitant with increase in rotifer population growth (3.82/d). In contrast, Synechococcus elongatus andScenedesmus dimorphus were both resistant to the rotifer and suppressed rotifer growth (-0.06/d). An index of algal species susceptibility to be consumed by the rotifer was generated with C. sorokiniana as the baseline (index = 1.000) indicating most susceptible among species tested. Other species' susceptibilities are indicated in parenthesis as follows: Monoraphidium spp. (0.997), Chlamydomonas globosa (0.827), Botryococcus braunii(0.740), Chlorella minutissima (0.570), Chlamydomonas augustae (0.530), Chlamydomonas yellowstonensis (0.500), Scenedesmus bijuga (0.420), and Haematococcus pluvialis (0.360). Two species, namely, S. dimorphus andS. elongatus were unique in that they exhibited an ability to suppress the growth of the rotifer as indicated by the decline in rotifer populations in their presence. Conclusions: Variations in susceptibility of algal species to rotifer predation could be a result of their individual morphology, cell walls structure, or the biochemical composition of individual species. <![CDATA[<strong>Establishment of a HEK293T cell line able to site-specifically integrate and stably express GDNF by rAAV-2 vector</strong>]]> Background: Using recombinant adeno-associated virus 2 (rAAV-2), we attempted to establish a HEK293T cell line that is able to site-specifically integrate and stably express glial cell line-derived neurotrophic factor (GDNF). Results:Recombinant vector with enhanced green fluorescent protein (EGFP) and GDNF (pTR-P5-EGFP-IRES-GDNF), as well as that carrying Rep genes and SV40 promoters (pSVAV2) were constructed and packed. HEK293T cells were co-infected with rAAV-2/EGFP-GDNF and rAAV-2/SVAV2 virus separately at 1 x 10(4),1 x 10(5),and 1x10(6) of multiplicity of infection (MOI). The efficiency of transduction was detected using flow cytometry. Additionally, the infected HEK293T cells were separately validated by touchdown polymerase chain reaction (PCR) and Western-blot. After 72 h of transduction, the rate of EGFP positive cell was 22%, 45% and 49% at the MOIs of 1 x 10(4),1 x 10(5) and 1 x 10(6), respectively. On the 3rd, 6th and 9th day of cell passage, there was no significant difference in the cell viability and proliferation rate between transduction and control groups. Importantly, touchdown PCR showed that there was a specific PCR amplified product band in the lane of infected cells. Furthermore, GDNF expression was detected in the infected cells after 15 and 180 d of cultivation. Conclusions: A HEK293T cell line able to site-specifically integrate and stably express GDNF was established. <![CDATA[<strong>Production, purification and characterization of recombinant human antithrombin III by </strong><em><b>Saccharomyces cerevisiae</b></em>]]> Background: Antithrombin III (ATIII) is a protein that inhibits abnormal blood clots (or coagulation) by breaking down thrombin and factor Xa. ATIII helps to keep a healthy balance between hemorrhage and coagulation. The present work demonstrated the production, purification and characterization of recombinant human antithrombin (rhAT) from yeast Saccharomyces cerevisiae BY4741 was demonstrated. After expression of rhAT by S. cerevisiae, the biomass and rhAT concentration were analyzed through fed-batch fermentation process. Results: In fed-batch fermentation, the biomass (maximum cell dry weight of 11.2 g/L) and rhAT concentration (312 mg/L) of the expressed rhAT were achieved at 84 h of cultivation time. The maximum cell lysis efficiency (99.89%) was found at 8 s sonication pulse and 7 mL lysis buffer volume. The rhAT protein solution was concentrated and partially purified using cross-flow filtration with the recovery yield and purity of 95 and 94%, respectively. The concentrated solution was further purified by the single step ion exchange chromatography with the recovery yield and purity of 55 and >98%, respectively. The purified rhAT was characterized by various analytical techniques, such as RP-HPLC, FT-IR, CD, SDS-PAGE, western blotting, and Liquid chromatography mass spectrometry (LC-MS) analysis. The biological activity of rhAT was analyzed as heparin cofactor to meet the therapeutic grade applications. Conclusions: The simple, cost-effective and economically viable nature of the process used in the present study for the production of rhAT will be highly beneficial for the healthcare sector. This may also be used to produce other value-added therapeutic recombinant proteins expressed in S. cerevisiae, with greater effectiveness and ease. <![CDATA[<strong>Interaction of </strong><em><b>Acidithiobacillus ferrooxidans, Rhizobium phaseoli </b></em><strong>and </strong><em><b>Rhodotorula </b></em><strong>sp. in bioleaching process based on Lotka-Volterra model</strong>]]> Background: Nowadays, leaching-ore bacteria, especially Acidithiobacillus ferrooxidans is widely used to retrieve heavy metals, many researches reflected that extra adding microorganism could promote bioleaching efficiency by different mechanisms, but few of them discussed the interaction between microorganisms and based on growth model. This study aimed to provide theoretical support for the collaborative bioleaching of multiple microorganisms by using the Lotka-Volterra (L-V) model. Results: This study investigated the interaction of Acidithiobacillus ferrooxidans, Rhizobium phaseoli,and Rhodotorula sp. Results showed that the individual growth of the three microorganisms fit the logistic curves. The environmental capacities of A. ferrooxidans, R. phaseoli, and Rhodotorula sp. were 1.88 x 109, 3.26 x 108, and 2.66 x 108 cells/mL, respectively. Co-bioleaching showed mutualism between A. ferrooxidans and R. phaseoli with mutualism coefficients of a =1.19and /3 = 0.31, respectively. The relationship between A. ferrooxidans and Rhodotorula sp. could be considered as commensalism. The commensalism coefficient y of the effect of Rhodotorula sp. on A. ferrooxidans was 2.45. The concentrations of A. ferrooxidans and R. phaseoli were 3.59 x 109 and 1.44 x 109 cells/mL in group E, respectively, as predicted by the model. The concentrations of A. ferrooxidans and Rhodotorula sp. were 2.38 x 109 and 2.66 x 108 cells/mL, respectively. The experimental peak values of the concentrations in microorganism groups E and F were detected on different days, but were quite close to the predicted values. Conclusion: The relationship among microorganisms during leaching could be described appropriately by Lotka-Volterra model between the initial and peak values. The relationship of A. ferrooxidans and R. phaseoli could be considered as mutualism, whereas, the relationship of A. ferrooxidans and R. phaseoli could be considered as commensalism.