Modares Journal of Biotechnology

Abstract Although biosurfactants have great advantages over chemical surfactants, their wider industrial applications have been constrained by their relatively high production cost. Using renewable, sustainable and cheap substrates such as different industrial by-products and wastes maybe decrease biosurfactant production costs. Since in different countries, there are a variety of by-products and wastes so use of these substrates rely on their types and concentrations in countries. In addition to hydrocarbon compounds, molasses has been considered as a dominant by-product in Iran. In this study, among 16 crude oil degrading isolates, strain Pseudomonas aeruginosa ZN was selected as an efficient biosurfactant producer by screening methods for detection of biosurfactant producing bacteria. For investigation of molasses concentrations effect on bacterial growth and biosurfactant production, a wide range of molasses concentrations from 2-12% (v/v) were used. This strain was able to grow and produce biosurfactant in all range of molasses concentrations while the best concentrations were 4-6%. Also, at the optimum molasses concentration, reduction of surface tension from 70 to 32-34 mN/m was observed. The concnetrations more than these values decreased the growth and production process. Acid precipitation and solvent extract (ethyl acetate: hexane) methods were carried out for recovery of biosurfactant from the culture broth, then results of spraying on developed TLC and staining fermentation broth without bacterial cells showed the two produced biosurfactants were glycolipid.

Abstract
Inhibitor of apoptosis (IAP) are a family of proteins that block cell death through caspase activity. Survivin is smallest IAPs family member that overexpresses in different cancer types but not in normal tissue except embryonic tissue. Survivin may be used as a new marker to stratify cancer patients for more optimal treatment modalities. The aim of the current study was to investigate survivin DNA cloning into pET-28a and its expression in E.coli.
The sequence of survivin gene was amplified by PCR using specific primers and pcDNA-survivin temple. PCR product and pET-28a plasmid were digested by HindIII/NheI restriction enzymes and survivin was ligated into the digested vector. Then, the ligation product was transformed into the E.coli DH5a competent cells and screened by antibiotic selection marker (kanamycin). Positive colonies were selected by colony PCR and screened by double digestion of isolated plasmid. One positive colony was sequenced and confirmed. The recombinant plasmid was transformed into the expression strain of E.coli (BL21) by chemical method. The expression of survivin was induced in the different conditions and expression level investigated by SDS-PAGE.
The size of PCR product in agarose gel showed the correctness of amplification. The digested pET-28a plasmid also indicated the correctness of enzymatic reaction. The sequence of the cloned fragment revealed a 100% similarity to the human survivin. In expressing, adding IPTG increased the expression of survivin protein in all conditions, especially 37 ᵒC from 2 h after induction. At all conditions, most of survivin accumulated in the bacteria as inclusion body.

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 Glucoamylase, is an important economic enzyme due to its ability to hydrolyze starch and β-D-glucose polymers. Understanding of factors affecting the thermal stability of the glucoamylase enzyme is critical in the production of isoenzymes with high heat or cold stability. In this study, the effect of temperature on the structure and properties of each of the isoenzymes of the mesophilic, thermophilic and psychrophilic glucoamylase were studied. For this purpose, molecular dynamics simulation was used to assess these factors and structural differences. 240 nanosecond of MD simulation was done for three isoenzymes of glucoamylase in four temperatures at 300, 350, 400 and 450 K. The variations of each of these parameters were compared for three isoenzymes, and it was found that among the computable factors in molecular dynamics simulation, electrostatic energy of protein with water, van der Waals energy between proteins and water, free energy solubility (∆G_solvation), instability parameter, nonpolar solvent accessible surface, and total solvent accessible surface can be used to predict thermal stability of a protein during increase of temperature.

Abstract Atherosclerosis is a chronic vascular disease and remains the leading cause of death and morbidity worldwide. Endothelial dysfunction is an important factor in the progression of atherosclerotic disease. Increased expression of cell adhesion index genes and decreased cell-binding proteins lead to abnormal endothelial function. These molecular changes are one of the most important indicators of endothelial cell dysfunction and the progression of atherosclerosis. CXCR3 is a G-protein-coupled chemokine receptor expressed by endothelial cells. The role of the receptor CXCR3 and its ligands in endothelial cells and heart disease is not yet fully understood. In this study, we evaluated the effect of CXCR3 downregulation on the expression level of adhesion (I-CAM-1, V-CAM-1), tight junction (TJP1), related to endothelial dysfunction.
In order to reduce the expression of the CXCR3 gene, the RNA-cleaving DNAzyme was used against the mRNA of the CXCR3 gene. DNAzyme was transfused into HUVEC cells by TurboFect^TM. After confirmation of decreased CXCR3 gene expression, RNA extraction and cDNA synthesis were performed and then the expression of markers was evaluated by RT-qPCR technique.
Our result was showed the expression level of I-CAM-1 and V-CAM-1 were showed significant up-regulation in transfected cells compared with control cells, while the TJP1 gene was not showed significant change. It seems that reducing the CXCR3 gene expression could induce endothelial dysfunction through the change of adhesion markers genes expression. Therefore, this receptor can be considered as a potential molecular target for a better understanding of the mechanism of atherosclerosis.

Abstract According to the formation and evolution of life along with static magnetic fields,the permanent exposure has given adaptive ability to beings. Therapeutic magnetism is one of the branches of complementary medicine which uses the low intensity and non-harmful magnetic fields to the body. By studying in infertile couples (20% male factor), the only cause of infertility and in 50% of cases it is considered as an intermediate factor. One of the influential factors in infertility in men is sperm. In the present study, normal specimens in the magnetic field under the intensities of 1,6 and 12 millitesla and at 1,3 and 5 h intervals.
Sperm movement rate was evaluated by CASA, as well as sperm viability, by eosin staining of necrosin and morphology by staining Papanicula. The results of this step on normal sperm showed a significant reduction in the sperm movement ,which that was not affected by the field.
Morphological studies also show that sperm motility is not affected by magnetic field.. the survival rate of sperm was affected by the magnetic field was significantly reduced, and the sperm morphology remained unchanged

Abstract Hyper-glycosylation is an approach to introduce new N-glycosylation consensus sequence(s) (َAsn-Xxx-Ser/Thr three-peptide) into a protein primary amino acid sequences by site-directed mutagenesis which is followed by the attachment of a new glycan to the Asn residue located within the three-peptide sequence. Hyper-glycosylation has attracted lots of interest especially in the protein therapeutics industry. The attached glycan may improve the pharmacokinetic properties of the hyper-glycosylated priteins and increase their half-life in the bloodstream. In the current study, a new N-glycosylation site was introduced into N-terminal Gla domain of hFIX. Arg³⁷ position of mature hFIX was targeted to be converted into Asn residue by site-directed mutagenesis using overlap extension PCR. Recombinant expression plasmids for native and mutant hFIX were constructed. The expression of the recombinant wild-type and mutant hFIX was analyzed in mammalian HEK293 cells using gradient SDS-PAGE and western blotting analysis. The results indicated in higher molecular weight for R37N mutant in compared with the native protein. The glycan attachment to R37N mutant was further confirmed by PNGase digestion and western blotting.

Abstract In the present study, the structure of three common anticancer drugs including 6-thioguanine (6-TG), hydroxyurea (NH) and busulfan were optimized using quantum computational and obtained minimum energy for them. Also, optimization structure of gold nanoparticle was investigated by density functional theory (DFT). Finally, the binding energy of Au nanoparticle was calculated with the optimized structures of drugs. All different sites of drugs that can be interacted with nanoparticle were considered and the most stable structure was chosen for further study. These calculations were performed using FHI-aims which is a software package based on DFT. The bond length and the best interaction energy were reported in this work. To better investigation of the location of the interaction, the type of orbitals involved in the interaction and their shapes are shown. Gap energy analysis showed that the lowest energy was related to the complex of gold nanoparticle with 6-thioguanine, which confirms the chemical stability of this drug with nanoparticle. Investigations showed that the binding energy of gold nanoparticle with drugs is busulfan > hydroxyurea> 6-thioguanine so busulfan has more affinity to bind with gold nanoparticle.
Au nanoparticle as an anticancer drug deliver was studied with the molecular docking calculations. The human albumin serum (HSA) binding with three anticancer drugs was docked individually with Hex 8 software and their active sites of interaction were shown as well as. Finally, the binding energies and types of interactions such as electrostatic, van der Waals and hydrogen bonds between HSA and Au@ drugs were presented, clearly.

Abstract Human alpha1-antitrypsin is a glycoprotein comprised of 394 amino acids and 52 kDa molecular weight, which is mostly synthesized and secreted by hepatocytes, diffuses to interstitial lung tissues, and has an essential function to protected tissues against neutrophil elastase. One of the significant challenges in dealing with alpha-1-antitrypsin is the structural instability of the folded form of protein and, consequently, the accumulation of polymers in lung tissue. This makes patients vulnerable to chronic obstructive pulmonary disease, severe asthma, and emphysema. Intravenous augmentation therapy of alpha 1-antitrypsin is one of the most prevalent therapies. Moreover, patients who are candidates for that have respiratory symptoms, and the use of bronchodilator (Salbutamol) is the first recommended standard treatment. In this study, protein purification was performed by using high-performance affinity chromatography and, its purity was confirmed by gel electrophoresis. The effect of different concentrations of salbutamol on heat-induced polymerization at 60 ° C was investigated by non-denaturing polyacrylamide gel electrophoresis, dynamic light scattering (DLS), and circular dichroism (CD) techniques. Protein activity was measured by trypsin inhibitor capacity (TIC) assay. The results indicated that salbutamol decreases the rate of polymerization by reducing the flexibility of the reactive center loop, thus decelerate protein activity reduction. Therefore, salbutamol can be an appropriate supplementary for alpha 1-antitrypsin and a proper option for the treatment of protein polymerization associated diseases.

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.