Modares Journal of Biotechnology

Abstract Introduction: Amyloid beta (Aβ) is the major constituent of harmful plaques in the Alzheimer’s patients. Thus, study of Aβ and understanding its related molecular and cellular mechanisms is essential for diagnosis and therapeutic interventions. This study introduces a rapid, simple, and cost-effective technique for production and purification of this peptide, utilizing the expression of Aβ gene within bacterial system.
Materials and methods: Aβ gene was synthesized and transferred into the expression vector pET26b. After induction by Lactose and 24 hours of incubation for Aβ expression the cell sediment was analyzed for presence of recombinant peptide using SDS-PAGE and Western blot. Then the purification of recombinant peptide was carried using nickel chloride affinity chromatography. Characterization of purified Aβ was performed by evaluating cell cytotoxicity in 25 µM and 50 µM concentrations using MTT assay on Alzheimer cell line model SH-SY5Y.
Results: Colony PCR and sequencing results showed the correct insertion of Aβ coding fragment into the expression vector. Presence of bands with the expected size in the results of SDS PAGE and western blot had confirmed successful expression of his-tagged recombinant peptide. MTT assay results showed the purified peptide has respectively 30 and 50% cytotoxicity for 25 µM and 50 µM concentrations.
Discussion: Production of amyloid beta peptide in bacterial hosts seems to be favorable. Obtaining Aβ peptide in soluble phase is an important advantage of this study. Hence according to toxicity of the purified peptide, it can be utilized for cell line treatments and further researches on Alzheimer disease.

Abstract New technologies in determining the gender of eggs will greatly help to end the extermination of male chickens, which will save a lot in the poultry industry. These technologies are so valuable and important that many companies and research centers are willing to make large investments to progress in this field. Today, two invasive and non-invasive methods are used to determine the gender of the egg. Invasive diagnostic methods often lead to a decrease in the viability of samples, while non-invasive methods with high accuracy and viability of samples have created a great development capability among researchers. In other words, invasive diagnostic methods determine the gender of the embryos inside the egg with a high percentage, but it can endanger the continuation of the hatching process and jeopardize food safety. However, the use of non-invasive methods in line with industrial use has priority due to the fact that there is no danger to the chick embryo in the process of sex determination. In this review study, while examining the importance of gender determination during hatching for the poultry industry, an attempt has been made to examine and compare all the new technologies used to determine gender in the egg-laying and hatching stages and compare its advantages and disadvantages.

Abstract Microorganisms play an important role in formation of mines. In this research, the bacteria inhabiting in Aq-Darreh Takab gold mine were isolated and compared with agricultural soils. The isolates were characterized using 16S rDNA sequencing and the homology searches were performed using BlastN, EzTaxon, and RDP Classifier web tools. Resistance of the isolates was also investigated against arsenic and silver in the presence and absence of 3.5 ppm gold. Although the control soil showed a wide variety of bacterial diversity (43 isolates belonging to 13 genera), only 17 isolates belonging to 11 genera were isolated from mine soils including Acinetobacter, Agrobacterium, Comamonas, Deinococcus, Listeria, Microbacterium, Micrococcus, Pseudomonas, Rhizobium, Roseomonas and Staphylococcus. Among the isolates, A. radiobacter, D. ficus, M. antarcticus, M. luteus, R. radiobacter and R. selenitidurans were able to tolerate different amounts of arsenic and silver in the presence of gold, among which A. radiobacter and D. ficus showed the highest resistance in such a way that they grew in the presence of 50 ppm arsenic, 50 ppm silver, and 3.5 ppm gold. Our results showed that bacterial diversity in soils containing gold, silver and arsenic metals is less than agricultural soils. It was also found that the bacterial diversity in gold mines depends on the amount of gold and the amount and type of associated elements. Due to high resistance of two endogenous bacterial species to arsenic and silver, A. radiobacter and D. ficus, have also the potential for industrial purposes in environments contaminated with these metals.

Abstract The process of diphtheria toxoid production was designed by using SuperPro Designer and the effect of the applied changes in process on the yield and costs of the manufacturing was investigated. First, giving the information of the real process of the toxoid production, a bioreactor with improved operational conditions and a disc stack centrifuge instead of the filter press, which is applied for the bacterial debris separation, were utilized. Such alterations followed the addition of a pump between the bioreactor and centrifuge. The results indicated that improvement of the bioreactor operational conditions can lead to the 25% increase in the toxin production, i.e., the increase of toxoid production from 7,000,000 doses to 8,750,000 doses. The toxin waste through filter press (14%) may be remarkably reduced by using the centrifuge, which in turn resulted in the 44% enhancement in the toxoid production. Such alterations can result in the 16% reduction in the separation operation time, 29% reduction in water consumption and 32% increase in the energy consumption. Overall, the simulation results showed that the costs of the new equipment suggested to be used in the improved process can be recoverable through running two batches.

Abstract In recent years, biocatalysts have widespread application in industry because they can do chemical reactions with the lowest energy and highest efficiency. Bacterial enzymes are more useful in this field due to simple cloning and expression process in the manipulated host. By considering specific role of endoglucanase enzymes in cellulose hydrolyzing reactions, these types of enzymes are more applicable in related industries. The produced glucose through enzymatic hydrolysis could be used in different industries such as biofuel and ethanol production and in the food industry as sweetener. Therefore, cloning and production of Endoglucanase in manipulated hosts has been developed in recent years. This study was performed to isolate, screen and identify native endoglucanase -producing strains from soil around the roots of the maple tree. Isolated strains were identified using 16S rRNA gene sequencing. After identifying of the bacteria (Enterobacter hormaechei), Endoglucanase enzyme gene was amplified using degenerate primers at first and then by specific primers with restriction enzymes sequences. DNA fragment and plasmid vector were treated by specific restriction enzymes and then ligated to each other. Then recombinant plasmid transferred to the E. coli BL-21 as expression host and kinetic properties of recombinant enzyme were evaluated. Expression of the target protein was done by stimulating the Lac operon by using 1 mM of IPTG and the kinetic features of the recombinant enzyme such as Vmax and Km evaluated as 45 µmol/min and 1.4 mg/ml respectively. The optimum conditions for enzyme activity tend to be 37°C at a pH of 7.

Abstract In the production of fuel briquettes, different additives are used in order to improve the technical parameters. In the present study, two types of lignocellulosic binders including nanocellulose and lignin have been used. Due to the different chemical structure and difference in calorific value of each of these two materials (lignin and nanocellulose) and the difference in their mechanism of action on improving the thermal properties of fuel briquettes, in order to evaluate the final product of thermal analysis using calorific value and TGA and DTA diagrams were used. The results show the positive effects of the use of cellulosic binders in improving the thermal behavior of biofuel briquettes. Thermal analysis showed that 9% nanocellulose and 9% lignin treatments with 19.85 MJ / Kg and 25.75 MJ / Kg had the highest calorific value compared to the control sample, respectively. The diagram obtained from thermal analysis (TGA) of the control sample and the samples treated with lignin and nanocellulose show that the treated samples have lower weight loss rate, higher burning rate and higher burning temperature.

Abstract In this study, the wheat germ was fermented with industrial bakery yeast powder to produce FWGE with high 2,6-DMBQ content in a Bench-scale bioreactor by scale-up approach. The 2,6-DMBQ content of FWGE was increased by optimizing the three initial variables of pH, fermentation temperature, and agitation rate at two levels using the Taguchi method. The 2,6-DMBQ content of the samples was determined at 14, 16, and 18 hours of the fermentation process. Then, the results were analyzed by Qualitek software. The effect of centrifugation speed on turbidity and the yeast's number in the final supernatant was then investigated. Finally, the supernatant was dried by spray dryer with an inlet temperature of 120 °C and outlet temperature of 70°C, and the amount of active 2,6-DMBQ, pH, moisture, and ash was determined. Under optimal conditions: initial pH of 6, fermentation temperature of 32 °C, and agitation rate of 80 rpm, maximum 1.527 mg of 2,6-DMBQ per gram of FWGE obtained. The separation results showed that the centrifugation rate doesn't have a significant effect on the final turbidity and the number of yeasts left, and thus 3000 g was selected as the optimal speed. However, because of the high content of yeast in the supernatant, filtration was required after centrifugation. Due to the high speed of sample drying, the low moisture of the final product, and high efficiency on an industrial scale, the samples were dried using a spray dryer. Finally, the moisture, protein, ash, and pH of the final product were measured.

Abstract Among the sources of oils (vegetable, animal, microorganism), microbial oil has attracted the attention of many researchers. Oily microorganisms are able to accumulate 20 to 80% of lipids in each dry biomass. Among various microorganisms (bacterias, microalgae, fungal species including yeasts), some yeasts are considered to be the superior source of oil production. Yarrowia lipolytica is an excellent example of oily microorganisms with high fat production efficiency. By using cheap, native and available pulp as a production medium, the cost of oil produced by yeasts can be reduced. The microbial oil produced is used for medicinal, food and cosmetic purposes. In this study, the pleomorphism of Yarrowia lipolytica (ATCC 18942) was examined microscopically in different culture media. After culturing the yeast in media containing olive, sesame and sunflower pulp, in semi-open culture conditions, the fatty acids produced were analyzed using GC-MS and FTIR techniques. After reviewing the results, the medium containing olive pulp was selected and the microbial lipid produced in this medium was extracted. Then dry weight of biomass and microbial fat were measured. The results showed that the fatty acids extracted from the medium containing olive oli cake included oleic acid, palmitic acid, linoleic acid and stearic acid, which had the best production of fatty acids among the pulp. The content of microbial fat and dry weight were 4.07 and 7.83 g/l, respectively, and microbial fat production efficiency was 51.97%.

Abstract Investigation of factors affecting endothelial cell proliferation is an essential part of angiogenesis studies. Given the importance of inhibiting angiogenesis in the treatment of cancers, and due to the side effects and high cost of anti-angiogenic drugs such as Avastin, the use of physical agents to aid in treatment and reduce the need for high doses of the drug is noteworthy. Magnetic fields are of interest due to their long-distance and non-invasive effects, and many studies have been conducted on their effects on biological phenomena, including angiogenesis, with inconsistent results. In the present study, the effect of a 2 mT alternating magnetic field with a frequency of 200 Hz and Austin on the proliferation of human umbilical vein endothelial cells (HUVEC) was investigated. Cells were treated for 48 hours under a mixture of 50 μg/ml solution of vascular endothelial growth factor (VEGEF) and Avastin at concentrations (zero (drug control), 50, 100, 200 and 400 μg/ml) as well as field treatment groups for They were exposed to magnetic fields for 3, 6, 12, 24 and 48 hours. Then, cell proliferation was assessed using Alamar Blue colorimetric test. Data were analyzed by three-way analysis of variance. According to the findings, the exposure times of 12, 24 and 48 hours showed a significant reduction in cell proliferation compared to the control group, but this difference was not significant in the 3 and 6 hour treatments. Also, the degree of interaction of these factors with each other on HUVEC proliferation was investigated.

Abstract In this study, cellulase enzyme production by Trichoderma reesei on three lignocellulosic substrates (corn bran, sawdust and wheat bran) and percentage of different combinations of sawdust and wheat bran by solid-state fermentation method for 6 days in scale checked out. Then, under optimal substrate component proportions obtained from Erlenmeyer-scale, the effect of aeration at three levels of 0.5, 1 and 1.5 liters per hour of initial dry substrate (l/(h.gds)) on the production of this enzyme in 0.5-Liter packed-bed bioreactor was studied. The initial substrate moisture and pH were 70 %(w/w) and 5 respectively, and the heating temperature was set at Erlenmeyer-scale and bioreactor at 30 and 28 °C, respectively. Cellulase enzyme production was evaluated based on the activity of endoglucanase and exoglucanase enzymes. The highest amount of endoglucanase and exoglucanase activity at substrate combination of 75% wheat bran and 25% sawdust in Erlenmeyer-scale at day 6 and 3 were obtained 13 and 6.4 U/gds, respectively, and in bioreactor at aeration of 1.5 (l/(h.gds)) at day 3 were attained 36 and 10 U/gds, respectively.

Abstract The microalgal strain Aurantiochytrium sp. shy contains considerable amounts of poly-unsaturated fatty acids (PUFAs), chiefly docosahexaenoic acid (DHA) with potential pharmaceutical and health-related attributes. Effects of various concentrations of glucose, meat extract, monosodium glutamate and sea salt on the algal biomass and DHA production have been investigated in this study. Maximum algal biomass (7.1 g/l) was obtained when the culture medium contained 60 g/l of glucose, 6 g/l of meat extract, 6 g/l monosodium glutamate and sea salt at 25ppt. Lipid contents of the alga exceeded 30% of its dry cell weight, with palmitic acid and DHA as the most abundant components. When the effect of a single additive was concerned, meat extract was significantly effective, while interaction between meat extract and glucose was the most effective in comparison with other interactions (P < 0.0001). According to the results, glucose can assure more algal and fatty acids production when adequate amounts of meat extract exist in the culture medium. Optimal results attained when the ratios of glucose to meat extract and C/N concentrations were 10 and 5, respectively. Due to its remarkable growth rate and the capability to produce substantial quantities of biomass and fatty acids, Aurantiochytrium sp.shy was found to be a major source of the beneficial ingredients, whose productivity can magnify if its culture conditions is optimized using favorable blend of growth-promoting materials.

Abstract Fibrinogen is a major component of the coagulation cascade following tissue damage and rapidly forms an insoluble fibrin scaffold. Fibrin is a filamentous biopolymer that naturally forms from fibrinogen polymerization during blood clotting. After tissue damage and coagulation cascade initiation, soluble fibrinogen polymerization by thrombin enzymebegins and forms an insoluble fibrin network and blood clots with platelets. This fibrin network is crucial for the development of homeostasis after tissue damage. This biopolymer also plays a key role in the wound healing as a temporary scaffoldand due to its unique structural properties and physiological function; it is used in reconstructive medicine. Fibrin is able to absorb extracellular matrix proteins (ECM) such as fibronectin and growth factors. The main types of fibrin scaffolds like platelet-rich fibrin (PRF) and platelet-rich plasma (PRP) are being used as autologous biomaterials in reconstructive medicine, wound healing, orthopedics and skin reconstruction and cosmetic sciences. Fibrin derivatives and degradation products also play an important role in the process of wound healing by stimulating cell infiltration and tissue regeneration and they are being widely used in developing new products as a biological material for over a century.

Abstract Biosurfactants are metabolites produced by microorganisms which have potential capabilities in various industries due to abundant beneficial properties. In spite of great advantages, commercial utilization of biosurfactants especially in food industry and pharmaceuticals are limited for the reasons of technical and commercial such as low yield, high production cost, and the type of producing strain. Majority of biosurfactant producer microorganisms have ever evaluated, are pathogenic strains which are not acceptable in industrial and environmental utilization particularly in health and cosmetics, pharmaceuticals and food industries. However, the present study aims to investigate high production of cell-bound biosurfactant by lactic acid bacterium Lactobacillus plantarum through optimization of the main carbon source of specific culture medium. Therefore, three culture media with different amount of glucose were evaluated for biomass and biosurfactant (by surface tension reduction of phosphate buffered saline) production in shake flasks and bioreactor (controlled temperature, pH and agitating speed). The results from both shake flasks fermentation and bioreactor showed the maximum biomass concentration of 3.9 and 4.17 g/L, the minimum surface tension of 41.17 and 40.48 mN/m and subsequently the maximum biosurfactant production in culture medium including 30 g/L of glucose, respectively. Furthermore, fourier transform infrared spectroscopy analysis indicated the biosurfactants are structurally a mixture of protein, polysaccharide and possibly phosphate group, possessing glycoprotein structure.

Abstract The combustion of fossil fuels containing sulfur results in the release of sulfur dioxide into the atmosphere and environmental pollution. Hence, the researchers focused on the biological desulfurization method. Dibenzothiophene is used as the model molecule to study the ability of the desulfurization of microorganisms. The most suitable sources of carbon, nitrogen and sulfur concentration optimized by response surface method to obtain the highest cell growth and biological desulfurization activity. The performance of iron nanoparticles on the growth and biodesulfurization activity of thermophilic bacterium Bacillus thermoamylovorans strain EAMYO was investigated. Characterization of starch-modified iron nanoparticles was performed by TEM, SEM. The images of TEM and SEM of starch / Iron nanoparticles showed that the Fe₃O₄ and Fe0 nanoparticles were 20 and 30 nm, respectively. The investigating the growth of microorganism in the presence of iron nanoparticles showed that these nanoparticles not only did not have a toxic effect on microorganism growth, but also increased the growth of microorganism in 96 h (OD 660 = 1.864, 1.896 respectively in the presence of nanoparticles Fe₀ and Fe₃O₄), while the highest rate of growth in the absence of nanoparticles in 96 h (OD660 = 1.51). Also, the activity of desulfurization in the presence of starch/Fe₀ nanoparticles and starch/Fe₃O₄ / starch increased by 26.52% and 10.75%, respectively, compared to the cells without the coating of iron nanoparticles.

Abstract In this study, the effect of red and blue illumination on continuous culture of two different species of microalgae and cynicocytosis was studied. Comparison of blue and red lights in the cyanobacteria, Synechocystis sp. PCC6803, showed that this specie grows very fast under red light illumination, but it has very slow growth rate under blue light exposure. In spite of huge difference in growth rate, the lipid content and the fatty acid composition of Synechocystis was approximately the same for red and blue light illumination. For microalgae, Chlorella Sorokiniana, the blue light resulted to slightly higher growth rate than the red light. The C18:3 unsaturated fatty acid content was significantly higher for red light illumination compare to blue light illumination. Overall, considering the lower energy requirement for illumination of red, this light is more efficient than blue light for cultivation of Chlorella Sorokiniana.

Abstract The use of enzymes in organic solvents represents an important area of industrial and biotechnological development. However, organic solvents often cause protein denaturation, thereby reducing the activity and stability of enzymes. Use of stabilizing additives, protein engineering and chemical modification of enzymes are common strategies to overcome this problem. In this study, a cysteine protease from the latex of Ficus johannis was purified and the activity and stability of the protease were investigated in the presence of different organic solvents. The effect of trehalose, sorbitol, and sucrose on the enzyme activity was also studied in the presence of organic solvents. The results showed that the enzyme activity was elevated in the presence of low concentrations of organic solvents increased, while it was decreased with increasing concentration of organic solvents. However, the enzyme still retained 60% of its activity at 30% organic solvent concentration. The enzyme was considerably stable in the presence of organic solvents, maintaining almost 90% of its stability in the presence of 50% of all solvents. As stabilizing additives, sugars enhanced the catalytic activity and stability of the enzyme, and trehalose was the most effective sugar. The easy purification procedure and considerable activity and stability of the protease in the presence of organic solvents could suggest this enzyme as a good candidate for peptide synthesis industry.

Abstract With the approval of the law for supporting knowledge-based firms in 2010, a new wave in Iran's science, technology and innovation system began with a focus on the knowledge based economy and innovation-based. Currently, there are more than 4,000 knowledge-based firms in Iran that nearly 5% of them are active in biotechnology. The aim of the present study is to design an empirical model of the relationship between financial and tax incentives of this law on some of the performance indicators of biotechnology knowledge-based firms. For this purpose, after analyzing the content of related documents and designing the study model, for evaluating the direct and interacting effects between policy tools, identifying the important empirical factors and their level, "2³ factorial design" was used. Study target community includes 113 manufacturing knowledge-based firms in the field of biotechnology. The findings of this study on input additionality indicators show the positive effect of the threefold interaction of factors on the R&D expenditure and the positive effects of commercialization financing and technology financing and their interactions on R&D employee. In the present study, there was no relationship between the effectiveness of policy tools on output additionality indicators.