National Institute of Genetic Engineering and Biotechnology of IranIranian Journal of Biotechnology1728-30439420111001Overexpression of full-length core protein of hepatitis C virus by Escherichia coli cultivated in stirred tank fermentor2452527128ENJafar HemmatNational Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, I.R. Iran. Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST), P.O. Box
15815-3538, Tehran, I.R. Iran.Bagher YakhchaliNational Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, I.R. Iran.0000-0003-4031-877xKhosro KhajehDepartment of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares
University, P.O. Box 14115-154, Tehran, I.R. Iran.Ali Akbar Moosavi-MovahediInstitute of Biochemistry and Biophysics, University of Tehran, P.O. Box 13145-1384, Tehran, I.R. IranAli Asghar KarkhaneNational Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, I.R. Iran.Journal Article20111001The mature core protein of the Hepatitis C virus (HCVC173) carrying pelB as a signal peptide (PelB::core) was overexpressed in Escherichia coli as 18% and 23.3% of the host’s total protein, in flask and fermentor cultivation, respectively. A final specific yield of 25 ± 1 mg HCVC173/g dry cell weight and an overall<br />productivity of 51±1 mg HCVC173/l/h were obtained in the stirred-tank fermentor. The recombinant<br />PelB::core protein was overexpressed as the inclusion body (IB) form, higher than the expected level when<br />compared to the HCVC173, which was also showed by the analysis of secondary structure of mRNAs and<br />calculation of the Codon Adaptation Index of the gene. The results showed that the combined effects of protein fusion and the signal sequence significantly enhanced the production of recombinant mature<br />HCVC173 in E. coli. Therefore, the fusion form of the mature HCV core protein and the conditions defined in<br />this study provide an alternative strategy for HCVC173 production in high cell density culture of E. coli.<br /><br />https://www.ijbiotech.com/article_7128_45c6632f59efadc1fa8df487b69e343e.pdfNational Institute of Genetic Engineering and Biotechnology of IranIranian Journal of Biotechnology1728-30439420111001Optimizing refolding condition for recombinant tissue plasminogen activator2532597140ENSusan Mirnajd GeramiInfectious and Tropical Disease Research Center, Tabriz University of Medical Sciences, P.O. Box 51656-65813, Tabriz, I.R. Iran.Safar FarajniaBiotechnology Research Center, Tabriz University of Medical Sciences, P.O. Box 51656-6581,Tabriz, I.R. Iran. Drug Applied Research Center, Tabriz University of Medical Sciences, P.O. Box 51656-65811, Tabriz, I.R.Iran.Feridoun MahboudiBiotechnology Research Center, Pasteur Institute of Iran, P.O. Box 13164, Tehran, I.R.Iran.Hossein BabaeiDrug Applied Research Center, Tabriz University of Medical Sciences, P.O. Box 51656-65811, Tabriz, I.R. Iran.Journal Article20111001Low molecular size additives such as L-arginine and the redox compounds have been used both in the culture<br />medium and in vitro refolding to increase recombinant proteins production. Additives increase protein<br />refolding and yield of active proteins by suppressing aggregate formation or enhancing refolding process.<br />In this work, a comparative study was performed on refolding of recombinant plasminogen activator (rPA)<br />in the presence of different concentrations of denaturants and additives. Escherichia coli-expressed rPA<br />inclusion bodies were solubilized in chaotropic denaturants and subjected to protein refolding by dilution<br />method. The effects of various additives, the impact of pH, residual Guanidin Hydrochloride (Gn-HCl) and<br />Dithiothreitol (DTT) on refolding process were investigated. The refolding process was assessed by determination of protein solubility and biological assay. The results of the study demonstrated that the best condition for solubilizing the rPA inclusion body was 6M guanidine hydrochloride at pH=10. In refolding step, Larginine showed increasing effect on suppression of aggregation at concentrations of 200-1000 mM.<br />Glutathione pairs (GSH-GssG) showed refolding enhancer effect in a range of 2-20 mM. The highest refolding yield was obtained in 500 mM L-arginine and reduced/oxidized glutathione 10:1 ratio in pH 10. In conclusion, the results show that L-arginine plays an important role in the refolding of human PA, preventing the aggregation of folding intermediate, and glutathione pair is essential for the correct refolding. The results also revealed that higher solubility in the presenceof higher concentration of L-arginine (> 500 mM) or pH (>10) is not associated with higher activity.https://www.ijbiotech.com/article_7140_a7651af40f069fcfee468002418e0520.pdfNational Institute of Genetic Engineering and Biotechnology of IranIranian Journal of Biotechnology1728-30439420111001Flux distribution in Bacillus subtilis: inspection on plurality of optimal solutions2602667148ENEhsan MotamedianBiotechnology Research Laboratory, School of Chemical Engineering, Iran University of Science and Technology, P.O. Box 16846-13114, Tehran, I.R. Iran.0000-0001-8750-2879Fereshteh NaeimpoorBiotechnology Research Laboratory, School of Chemical Engineering, Iran University of Science and Technology, P.O. Box 16846-13114, Tehran, I.R. Iran.Journal Article20111001Linear programming problems with alternate solutions are challenging due to the choice of multiple strategies<br />resulting in the same optimal value of the objective function. However, searching for these solutions is a<br />tedious task, especially when using mixed integer linear programming (MILP), as previously applied to<br />metabolic models. Therefore, judgment on plurality of optimal metabolic flux distributions (solutions) a priori<br />to applying MILP approach could prevent unnecessary computations. In this work for the first time, the<br />reduced cost coefficients for the non-basic variables in a current solution of a metabolic model were utilized to<br />inspect the possibility of multiple optimal flux distributions. If there exists at least one non-basic variable<br />with zero reduced cost coefficient, multiplicity of optimal solution may occur where MILP can be used to<br />find these solutions. This approach was implemented on a metabolic network of Bacillus subtilis aiming to<br />reduce the cell energy requirement. Solving the model at fixed specific growth rate of 0.4 1/h resulted in minimum energy requirement of 12.67 mmol/g-h. Inspection of reduced cost coefficients showed that six<br />non-basic variables had zero reduced cost coefficients at current solution, which shows that there can exist<br />multiple optimal solutions. Subsequently, by applying MILP, five optimal flux distributions at minimized energy requirement were identified, among which one showing no acid production and minimum glucose<br />consumption rate was selected as the superior solution.<br /><br />https://www.ijbiotech.com/article_7148_45574c4a50e0842bd9e47df5bcadac12.pdfNational Institute of Genetic Engineering and Biotechnology of IranIranian Journal of Biotechnology1728-30439420111001Amylase production from Aspergillus oryzae LS1 by solid-state fermentation and its use for the hydrolysis of wheat flour2672747138ENMohamed Abdel Fattah FaridDepartment of Natural and Microbial Products, National Research Centre, Dokki, Cairo, Egypt.Hoda Mohamed Abdel Halim ShataDepartment of Microbial Chemistry, National Research Centre, Dokki, Cairo, Egypt.Journal Article20111001Nine Aspergillus and three of Trichoderma strains were grown on wheat bran (WB) medium under solid state fermentation (SSF) for amylase production. Aspergillus oryzae LS1 produced the highest level of the enzyme. The thermal stability profile of its crude enzyme revealed the half-life time of more than 2 h at 50 and 60ºC. The enzyme production was affected by strain type, incubation periods, level of moisture content and carbon source supplementation. Maximum enzyme production of about 14249 IU/g WB was obtained under optimum conditions with an incubation period of 120 h, an initial moisture content of 54.5% and in the presence of sucrose (1 g/100g WB) at 30ºC. Of substrates tested, soluble starch was the best one hydrolyzed by the crude enzyme. Corn starch, dextrin and potato starch were also hydrolyzed to a lesser extent. The enzyme exhibited maximum activity at 55ºC. Moreover, the enzyme was also able to hydrolyze wheat flour under optimized conditions with efficiency of 89%.<br /> https://www.ijbiotech.com/article_7138_d23212f100ecabd1b3476447a419c019.pdfNational Institute of Genetic Engineering and Biotechnology of IranIranian Journal of Biotechnology1728-30439420111001Cloning and enhanced expression of an extracellular alkaline protease from a soil isolate of Bacillus clausii in Bacillus subtilis2752807129ENSeyed Mohsen Abbasi-HosseiniDepartment of Microbiology, Faculty of Biological sciences, Shahid Beheshti University, P.O. Box 19395-4716,
Tehran, I.R. Iran.Fereshteh EftekharDepartment of Microbiology, Faculty of Biological sciences, Shahid Beheshti University, P.O. Box 19395-4716,
Tehran, I.R. Iran.Bagher YakhchaliDepartment of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, I.R. Iran.0000-0003-4031-877xDariush Minai-TehraniDepartment of Microbiology, Faculty of Biological sciences, Shahid Beheshti University, P.O. Box 19395-4716,
Tehran, I.R. Iran.Journal Article20111001in the detergent industry. In this study, the extracellular alkaline serine protease gene, aprE, from Bacillus<br />clausii was amplified by PCR and further cloned and expressed in B. subtilis WB600 using the pWB980 expression vector. Protease activity of the recombinant B. subtilis WB600 harboring the plasmid pWB980/aprE<br />reached up to 1020 U/ml, approximately 3-folds higher than the native B. clausii strain. Characterization of the recombinant alkaline protease by SDS-PAGE and zymogram analyses indicated a molecular weight of<br />31 kDa. DNA sequence analysis and the deduced amino acid sequence revealed 98% homology with the<br />extracellular alkaline serine protease from B. clausii KSM-K16.<br /><br />https://www.ijbiotech.com/article_7129_45ea7dbb7127ac72c4ecbf2eae9cf6f0.pdfNational Institute of Genetic Engineering and Biotechnology of IranIranian Journal of Biotechnology1728-30439420111001Haplotype block partitioning and tagSNP selection under the perfect phylogeny model2812897132ENChangiz EslahchiFaculty of Mathematical Sciences, Shahid Beheshti University, G.C., P.O. Box 198396-3113, Tehran, I.R. Iran.0000000000000000Ali KatanforoushFaculty of Mathematical Sciences, Shahid Beheshti University, G.C., P.O. Box 198396-3113, Tehran, I.R. Iran.Hamid PezeshkSchool of Mathematics, Statistics and Computer Sciences, Center of Excellence in Biomathematics, College of Science, University of Tehran, P.O. Box 6455-14155, Tehran, I.R. Iran. School of Computer Science, Institute for Research in Fundamental Sciences, P.O. Box 19395-5746, Tehran, I.R. Iran.Narjes AfzalySchool of Mathematics, Statistics and Computer Sciences, Center of Excellence in Biomathematics, College of Science, University of Tehran, P.O. Box 6455-14155, Tehran, I.R. Iran.Journal Article20111001Single Nucleotide Polymorphisms (SNPs) are the most usual form of polymorphism in human genome.<br />Analyses of genetic variations have revealed that individual genomes share common SNP-haplotypes. The<br />particular pattern of these common variations forms a block-like structure on human genome. In this work,<br />we develop a new method based on the Perfect Phylogeny Model to identify haplotype blocks using<br />samples of individual genomes. We introduce a rigorous definition of the quality of the partitioning of haplotypes into blocks and devise a greedy algorithm for finding the proper partitioning in case of perfect and<br />semi-perfect phylogeny. It is shown that the minimum number of tagSNPs in a haplotype block of Perfect<br />Phylogeny can be obtained by a polynomial time algorithm. We compare the performance of our algorithm<br />on haplotype data of human chromosome 21 with other previously developed methods through simulations.<br />The results demonstrate that our algorithm outperforms the conventional implementation of the Four<br />Gamete Test approach which is the only available method for haplotype block partitioning based on<br />Perfect Phylogeny.https://www.ijbiotech.com/article_7132_68d5eb3d68a319ff1fa0e43777a8b118.pdfNational Institute of Genetic Engineering and Biotechnology of IranIranian Journal of Biotechnology1728-30439420111001Bioaffinity based immobilization of almond (Amygdalus communis) β-galactosidase on Con A-layered calcium alginate-cellulose beads: Its application in lactose hydrolysis in batch and continuous mode2903017149ENShakeel Ahmed AnsariDepartment of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh-202002, India.Qayyum HusainDepartment of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh-202002, India.Journal Article20111001In this study, immobilization of partially purified almond (Amygdalus communis) β-galactosidase on Con A layered calcium alginate-cellulose beads was investigated. Immobilized β-galactosidase retained 72% of the<br />initial activity after crosslinking by glutaraldehyde. Both soluble and immobilized enzyme exhibited the same<br />pH and temperature optima at pH 5.5 and 50ºC, respectively. However, the immobilized enzyme<br />showed a remarkable broadening in pH and temperature-activity profiles as compared to the native<br />enzyme. Immobilized enzyme was significantly more stable against thermal denaturation at 60ºC.<br />Immobilized β-galactosidase exhibited 67% residual activity in the presence of 5% D-galactose while its soluble counterpart retained only 35% activity under identical conditions. Soluble enzyme showed 69% residual<br />activity after exposure to pepsin (0.15 mg/ml) for 1 h whereas the immobilized β-galactosidase was more<br />stable and retained nearly 84% activity under identical experimental conditions. The activity of immobilized<br />enzyme was enhanced to 156% whereas soluble β-galactosidase showed an enhancement upto 134%<br />when exposed to trypsin (0.1 mg/ml) for 1 h. Moreover, immobilized β-galactosidase exhibited greater<br />enhancement in enzyme activity against exposure to various ions present in milk such as Na+, K+, Ca+2,<br />Mg+2 and citrate ions. The higher concentration of lactose was hydrolyzed from whey as compared to the<br />hydrolysis from milk by immobilized enzyme at 50ºC and 60ºC in batch processes. Lactose was hydrolyzed<br />to 86% and 78% after 20 days continuous operation of reactors at the flow rates of 20 ml/h and 30 ml/h,<br />respectively. In view of its stability and utility in batch and continuous processes, such preparation could be<br />exploited for the hydrolysis of lactose from milk and whey in a more convenient and cheaper way in dairy<br />industries.<br /><br />https://www.ijbiotech.com/article_7149_0c9a0323a79a215adc9d1eefb212674d.pdfNational Institute of Genetic Engineering and Biotechnology of IranIranian Journal of Biotechnology1728-30439420111001Isolation of endophytic actinomycetes from Catharanthes roseus (L.) G. Don leaves and their antimicrobial activity3023067142ENAbdul KafurDepartment of Ecology and Environmental Sciences, Pondicherry University, Puducherry-605 014, India.Anisa Basheer KhanDepartment of Ecology and Environmental Sciences, Pondicherry University, Puducherry-605 014, India.Journal Article20111001Endophytic actinomycetes were isolated from surface sterilized leaves of Catharanthes roseus (L.) G. Don of<br />family Apocynaceae. A total of 38 endophytic actinomycetes were recovered on Starch Casein Agar.<br />Among the 38 isolates 20 morphologically different isolates were screened for antibacterial activity against<br />Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris and for antifungal activity against fungi Candida albicans, Botrytis cinerea, Curvularia lunata, Fusarium oxysporum, Fusarium solani and Rhizoctonia solani. Sixty five percent of the isolates exhibited antimicrobial activity. Among the 20 isolates tested two isolates Cr-12, Cr-20 exhibited highest activity against the test organisms. The selective isolation of endophytic actinomycetes in the present study indicates the richness of microbial diversity in Catharanthus roseus and screening for antimicrobial activity should be investigated for a comprehensive identification and potential use as source of bioactive agents.https://www.ijbiotech.com/article_7142_eeee2c3f2f3a84f07660d0a62cd9ee08.pdfNational Institute of Genetic Engineering and Biotechnology of IranIranian Journal of Biotechnology1728-30439420111001Improved protocol for isolation of genomic DNA from leaf tissues of Phyllanthus emblica Gaertn3073137146ENSangeetha Nagarajan NagarajanDepartment of Biotechnology, Plant Genetic Improvement Laboratory, Sri Paramakalyani Centre for
Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi 627 412, Tirunelveli District, Tamilnadu, India.Mercy SteephenDepartment of Biotechnology, Plant Genetic Improvement Laboratory, Sri Paramakalyani Centre for
Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi 627 412, Tirunelveli District, Tamilnadu, India.Kavitha MuruganDepartment of Biotechnology, Plant Genetic Improvement Laboratory, Sri Paramakalyani Centre for
Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi 627 412, Tirunelveli District, Tamilnadu, India.Rahul Raveendran NairDepartment of Biotechnology, Plant Genetic Improvement Laboratory, Sri Paramakalyani Centre for
Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi 627 412, Tirunelveli District, Tamilnadu, India.Thilaga SethuramanDepartment of Biotechnology, Plant Genetic Improvement Laboratory, Sri Paramakalyani Centre for
Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi 627 412, Tirunelveli District, Tamilnadu, India.Parameswari Alagar Alagar1Department of Biotechnology, Plant Genetic Improvement Laboratory, Sri Paramakalyani Centre for
Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi 627 412, Tirunelveli District, Tamilnadu, India.Doss GaneshDepartment of Plant Biotechnology, School of Biotechnology, Madurai Kamarai University, Palkalainagar, Madurai 625 021, India.Journal Article20111001Modified Cetyltrimethylammonium bromide (CTAB) protocol for DNA isolation was developed from leaf tissues<br />of Phyllanthus emblica for obtaining high quality genomic DNA. Fresh leaves of three different maturity<br />were analyzed for yield and quality of DNA. Acidity was determined in three different maturity of leaves viz.<br />tender, intermediate and mature and their influence on DNA quality was determined. Drastic reduction of pH<br />was the primary cause for poor quality of DNA. However, high quality DNA isolation was achieved by<br />stabilizing the pH by addition of NaOH during different stages of DNA isolation process. The present protocol<br />yielding high quality intact DNA for genetic fingerprinting as well as for amplification of chloroplast genes for<br />molecular analysis.https://www.ijbiotech.com/article_7146_62d3bfeb1f1a30b5060d0eb2d9584b40.pdf