فهرست مطالب

Biotechnology - Volume:16 Issue: 3, Summer 2018

Iranian Journal of Biotechnology
Volume:16 Issue: 3, Summer 2018

  • تاریخ انتشار: 1397/06/04
  • تعداد عناوین: 9
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  • Tahereh Ghasemi-Kahrizsangi, Sayed-Amir Marashi *, Zhaleh Hosseini Pages 164-172
    Background
    A genome-scale metabolic network model (GEM) is a mathematical representation of an organism’s metabolism. Today, GEMs are popular tools for computationally simulating the biotechnological processes and for predicting biochemical properties of (engineered) strains.
    Objectives
    In the present study, we have evaluated the predictive power of two GEMs, namely iBsu1103 (for Bacillus subtilis 168) and iMZ1055 (for Bacillus megaterium WSH002).
    Materials And Methods
    For comparing the predictive power of Bacillus subtilis and Bacillus megaterium GEMs, experimental data were obtained from previous wet-lab studies included in PubMed. By using these data, we set the environmental, stoichiometric and thermodynamic constraints on the models, and FBA is performed to predict the biomass production rate, and the values of other fluxes. For simulating experimental conditions in this study, COBRA toolbox was used.
    Results
    By using the wealth of data in the literature, we evaluated the accuracy of in silico simulations of these GEMs. Our results suggest that there are some errors in these two models which make them unreliable for predicting the biochemical capabilities of these species. The inconsistencies between experimental and computational data are even greater where B. subtilis and B. megaterium do not have similar phenotypes.
    Conclusions
    Our analysis suggests that literature-based improvement of genome-scale metabolic network models of the two Bacillus species is essential if these models are to be successfully applied in biotechnology and metabolic engineering.
    Keywords: Biochemical capability, Bacillus Species, Computational biotechnology, Model validation, Systems biology
  • Adele Sadat Haghighat Hoseini, Mitra Mirzarezaee* Pages 173-184
    Background
    Prediction of the protein localization is among the most important issues in the bioinformatics that is used for the prediction of the proteins in the cells and organelles such as mitochondria. In this study, several machine learning algorithms are applied for the prediction of the intracellular protein locations. These algorithms use the features extracted from protein sequences. In contrast, protein interactions have been less investigated.
    Objectives
    As protein interactions usually occur in the same or adjacent places, using this feature to find the location would be efficient and impressive. This study did not aim at increasing the total accuracy of the conducted research. The study has focused on the features of the proteins’ interaction and their employment which lead to a higher accuracy.
    Materials And Methods
    In this study, we have examined the protein interaction network as one of the features for prediction of the protein localization and its effects on the prediction results. In this regards, we have gathered some of the most common features including Amino Acid Composition, Dipeptide Compositions, Pseudo Amino Acid Compositions (PseAAC), Position Specific Scoring Matrix (PSSM), Functional Domain, Gene Ontology information, and the Pair-wise sequence alignment. The results of the classification are compared to the ones using protein interactions. For achieving this goal different machine learning algorithms were tested.
    Results
    The best-obtained results of using single feature set obtained using SVM classifier for PseAAC feature. The accuracy of combining all features with PPI data, using the Decision Tree and Random Forest classifiers, was 82.49% and 83.35%, respectively. In another experiment, using just protein interaction data with the different cutting points resulted in obtaining an accuracy of 93.035% for the protein location prediction.
    Conclusion
    In total, it was shown that protein(s) interaction has a significant impact on the prediction of the mitochondrial proteins’ location. This feature can separately distinguish the locations well. Using this feature the accuracy of the results is raised up to 5%.
    Keywords: Machine learning, Mitochondria, Protein localization, Protein-Protein Interaction (PPI)
  • Ibrahim Torktaz, Jafar Hemmat *, Ali Asghar Karkhane, Garshasb Rigi, Amin Rostami, Jafar Khezri, Reza Behroozi Pages 185-192
    Background
    Considering natural thermal stability, Geobacillus stearothermophilus amylase and Cel5E from Clostridium thermocellum are good candidates for industrial applications. To be compatible with the industrial applications, this enzyme should be stable in the high temperatures, so any improvement in their thermal stability is valuable.
    Objectives
    Using in silico approach and identifying point mutations in the structure amylase of G. stearothermophilus and Cel5E from C. termocellum we tried to increase thermal stability of the enzymes along with their catalytic activity to reach a new industrial amylase with higher thermostability and an improved function.
    Materials And Methods
    In this study we predicted the 3D structure of the enzymes, then simulated the molecular docking study using MolDock, PLANTS, and Lamarkian genetic algorithm as scoring functions for the docking and in silico engineering of the protein aiming to increase the thermal stability and catalytic activity.
    Results
    A series of thermal stability increasing point mutations were exerted around the active site of the enzyme, then by docking procedure, the binding affinity was measured and finally a list of mutations which theoretically improved the increased thermal stability as well as catalytic activity were proposed.
    Conclusions
    Based on the in silico results obtained the modified enzymes seems to be suitable candidates for considering in both laboratory and industrial scales.
    Keywords: Amylase, Catalytic activity, Cel5E, Geobacillus stearothermophilus, Industry, Thermal stability
  • Masumeh Shaeyan, Hassan Tirandaz, Somayeh Ghanbarpour, Nasim Seyedipour, Mahmoud Shavandi, Seyed Mohammad Mehdi Dastgheib * Pages 193-199
    Background
    Petroleum drilling and exploration operations generate a significant amount of oily wastes. Comparing with physical and chemical remediation methods, bioremediation is cost-effective, highly efficient, and environment-friendly technology.
    Objectives
    The aim of this study was to evaluate biological treatment efficiency for an oil-contaminated soil prepared from an abandoned drilling mud pit located in the Khangiran region , Khorasan Razavi Provinvce, Iran.
    Materials And Methods
    Following chemical analysis of the contamination, isolation and phylogenetic analysis of the predominant hydrocarbon-utilizing bacteria were performed in order to elucidate microbial diversity. Afterwards, the full factorial design was used to optimize the concentration of nitrogen (0, 100, 250, and 500 ppm) and phosphate (0, 10, 25, and 50 ppm) in the soil. The prepared microcosms were incubated at 30 °C for a 60-day period. The total heterotrophic bacterial content was enumerated during bioremediation process. The total petroleum hydrocarbon concentration was analyzed after 60 days to evaluate biodegradation extent.
    Results
    The mud pit was mainly contaminated with the weathered diesel compounds ranging from C12 to C30. Investigation of the indigenous microflora showed that a variety of hydrocarbon-utilizing bacteria were present in the untreated soil dominated by Actinobacteria including the genera Gordonia, Nocardia, Nocardioides, and Rhodococcus. The full-factorial soil microcosms’ nutrient optimization indicated that nitrogen amendment could be the most effective at 250 ppm, while excess nitrogen would lead to a diminished microbial activity. The main effect statistical analysis demonstrated that addition of phosphate did not cause a significant improvement in the remediation process. In the optimal nutrient conditions, the total microbial count increased from 1.76 × 107 CFU.g-1 to 1.24 × 109 CFU.g-1 and led into 75.4 % w/w biodegradation in two months, while in the control microcosm without nutrient amendment only 36.6 % of the depletion was observed.
    Conclusions
    These results indicate that biostimulation could be a promising candidate for a successful field-scale bioremediation process in the studied drilling mud pit.
    Keywords: Bioremediation, Diesel, Mud pit, Microflora, Nutrient optimization, Oil-based drilling
  • Vasamsetty Shanthi *, Marthoor Roymon Pages 200-212
    Background
    Plant biomass and agricultural waste products disposal is a serious problem in agriculture based countries. These wastes, usually rich in xylan can be satisfactorily converted to industrially important and useful products by efficient biotechnological application of potent xylanase producing bacteria which generally have high temperature and pH optima.
    Objective
    The aim was to isolate and identify xylanolytic bacterial isolates from Bhilai-Durg region of Chhattisgarh, India which was otherwise unexplored for isolation of thermoalkalophilic xylanase producing bacteria. Partial scale up of process development was performed.
    Materials And Methods
    Xyalanse producing bacteria were isolated from probable samples following three stages of screening procedures. The potent isolates were identified and various parameters affecting xylanase production were optimized using the conventional one-factor-at-a-time approach.
    Results
    Two potent indigenous bacterial isolates belonged to genus Bacillus and designated as Bacillus sp. MCC2728 and Bacillus sp. MCC2727 were isolated from forest soils with the ability to degrade xylan. Significant differences were observed in their morphology and phenotypic characters amongst themselves and with its closest type strains implying the novelty of the two isolates. After optimization, maximum xylanase levels were obtained at pH 9.0, 55 °C for Bacillus sp. MCC2728 and 50 °C for Bacillus sp. MCC2727, 5% inoculum, agitation speed (150 rpm). Yeast extract and peptone are best nitrogen sources and wheat bran, the best carbon source. The GenBank/EMBL/DDBJ accession numbers of strains Bacillus sp. MCC2728 and Bacillus sp. MCC2727 are KP742971 and KT444621 respectively. Wheat bran, Yeast extract and peptone proved to be the best carbon and nitrogen sources respectively and xylose as an additive was found to be contributing to maximize the xylanase yields.
    Conclusion
    Two potent thermoalkalophilic novel bacterial isolates were successfully isolated with xylan degrading ability which may be used as promising xylanase producing candidates for various industrial purposes using agricultural based waste residues.
    Keywords: Bacillus sp, Optimization, Phylogenetic analysis, Thermoalkalophilic bacteria, Xylanase
  • In Vitro Cytotoxic Activity of a Lactococcus lactis Antimicrobial Peptide Against Breast Cancer Cells
    Abasaleh Avand, Vajihe Akbari *, Shahin Shafizadegan Pages 213-220
    Background
    Nisin, an effective natural food preservative, is an antimicrobial peptide produced by Lactococcus lactis. Although it has been mainly studied and developed as a potential alternative for antibiotics, other pharmacological effects of the nisin including cytotoxic and anti-tumor activity have been attracted many attentions.
    Objectives
    Here, we aimed to evaluate in vitro cytotoxic activity of the nisin against breast cancer cells.
    Materials And Methods
    The effect of temperature, pH, and chemical composition of the medium on the yield of nisin production was evaluated. As well, the anti-proliferative effect of nisin against a breast cancer cell line (i. e., MCF-7) and a non-cancerous cell line (i.e, HUVEC) was determined using MTT assay. Furthermore, the potential of the synergistic effect of the nisin on the doxorubicin cytotoxicity was evaluated.
    Results
    The optimum culture condition for the nisin production by L. lactis was found to be MRS medium (pH 6.3) supplemented with the tryptone and incubation at 30 °C. MTT assay results indicate that nisin exhibits a high and selective cytotoxicity against MCF-7 cell line with IC50 value of 5 μM. Furthermore, a combination of nisin and doxorubicin at sub-inhibitory concentrations were more cytotoxic compared to either of drugs alone.
    Conclusion
    It could be suggested that nisin, either alone or in combination with other chemotherapeutic agents, could be a potential therapeutic for the breast cancer cells.
    Keywords: Antimicrobial peptide, Breast Cancer, Cytotoxicity, Nisin, Lactococcus lactis
  • Jafar Khezri, Farid Heidari *, Mahdi Shams Ara Pages 221-226
    Background
    Transgenic mice are being considered as invaluable tool in biological sciences towards comprehension of the cause of the genetic diseases. Manipulated embryonic stem (ES) cells are used to produce loss-of-function mutant mice. Microinjection of manipulated ES cells into blastocoel cavity, and morula fusion are the two main techniques in producing transgenic mice. So far, no reports have dealt with the comparison of these two methodologies provide.
    Objective
    The object of this study was to determine advantages and disadvantages of knockout mouse creation protocols.
    Materials And Methods
    Both blastocyst microinjection and morula aggregation were implemented to produce chimeric mice and the advantages and disadvantages of each technique were evaluated. For this, embryonic stem cells were transfected with a GFP-expression vector. In blastocyst microinjection technique, first transfected ES cell were cultured and appropriate colonies were selected. The cells were microinjected to blastocoel cavity of the expanded blastocyst. In morula aggregation technique, the transfected ES cell colonies were sandwiched between two naked morulas. After 16 h incubation in a 5% CO2 at 37 °C the morulas and infected ES cell were aggregated to produce a new morula. All the injected blastocyst and aggregated morulas were transferred to uterus of foster mice. The new born mice were analyzed for chimera confirmation.
    Results
    Five chimeric mice (21.75%) from morula aggregation and eight chimeric mice (63%) from blastocyst microinjection were born. The results indicated that both techniques can be used to generate chimeric mouse, however the success rate was higher in blastocyst microinjection.
    Conclusion
    Morula fusion stands out where the required instrumentations are in place. Furthermore, the quality of ES cells plays a prominent role in the success rate. When the cell quality is low the blastocoel microinjection is recommended. The microinjection technique is more effective than morula aggregation.
    Keywords: Blastocoel microinjection, Embryonic stem cell, Loss-of-function mice mutants, Morula fusion
  • Ajeet Singh *, Vipin Permar, A. Basavaraj, Singh Tomar Bhoopal, Shelly Praveen Pages 227-234
    Background
    Groundnut bud necrosis virus (GBNV) (Tospovirus genus, Bunyaviridae family) infects the major crops of solanaceae, leguminosae and cucurbitaceae in India. Temperature is an important factor which influences the plant growth and development under diseased conditions.
    Objective
    In the present study, we evaluated the effect of four different temperatures on the symptoms expression and viral RNA accumulation in the GBNV inoculated cowpea plants.
    Material and
    Methods
    For the evaluation of viral symptoms and RNA accumulation, we used 2-3 leaf stage cowpea plants grown in the controlled conditions. GBNV was mechanically inoculated by sap method to the cowpea plants and inoculated plants were incubated at four different temperatures (30, 25, 20 and 15 °C).
    Results
    The first visible symptom of GBNV infection at the inoculated site was observed in the form of chlorotic spots which were converted into the necrotic spots as the infections succeeded. Some yellow mosaic symptoms were also observed at the systemic site during viral infection cycle. Plants incubated at higher (30 and 25 °C) temperatures showed a severe necrosis and a higher viral RNA accumulation at the inoculated site and facilitated the viral spread at the systemic site. However, viral RNA accumulation was less at the systemic site than the inoculated site. In contrast, symptoms’ expression and viral RNA accumulation were decreased at the inoculated site at low (20 and 15 °C) temperatures, no viral symptoms were observed at the systemic site (15 °C); in addition to viral RNA accumulation suppression at this site. GBNV infection at the inoculated site induced the higher accumulation of H2O2 followed by the induction of cell death at higher temperatures (30 and 25 °C) than the lower (20 and 15 °C) temperatures.
    Conclusion
    This study suggests that viral RNA accumulation parallels with the H2O2 production and induction of cell death by GBNV infection in cowpea plants is temperature dependent.
    Keywords: Chlorosis, H2O2, Hypersensitive response (HR), Programmed cell death (PCD), Necrosis
  • Zongmin Liao, Haijuan Liu, Jian Liu, Mingsheng Cai, Tao Chen, Qing Hong, Xiaodong Luo, Xiaomin Li, Xue Ding, Haoxian Shen, Daixiong Chen* Pages 235-240
    Background
    Recently, the incidence of allergic diseases has been on the rise; Dust mite is the major indoor allergen which needs a special consideration.
    Objectives
    This study was carried out to identify and investigate the molecular properties of a new allergen named Hsp60 and to afford a foundation for future research of the allergic diseases caused by Dermatophagoides farinae.
    Materials And Methods
    Using polymerase chain reaction (PCR) with degenerate primer, the cDNA of Dermatophagoides farinae Hsp60 was amplified and sequenced. Next, the cDNA fragment was cloned into the prokaryotic expression vector pET-32a for the expression of the Hsp60. Then, it was further characterized by Elisa and Western Blot analysis.
    Results
    The partial cDNA sequence of the Dermatophagoides farinae Hsp60 was determined, and the recombinant Hsp60 was successfully expressed in Escherichia coli BL21. ELISA and Western blot analysis showed that the recombinant protein could be specifically recognized by SIgE from sera of the Dermatophagoides farina-allergic patients.
    Conclusions
    Our group has, for the first time, demonstrated the fact that there is an Hsp60 family of Dermatophagoides farinae and analyzed the allergenicity of the Hsp60 with immunological method. These results provide a foundation for further allergological research of the Dermatophagoides farinae Hsp60.
    Keywords: Dermatophagoides farinae, Expression, Hsp60