ORIGINAL_ARTICLE
Overexpression of full-length core protein of hepatitis C virus by Escherichia coli cultivated in stirred tank fermentor
The 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 overallproductivity of 51±1 mg HCVC173/l/h were obtained in the stirred-tank fermentor. The recombinantPelB::core protein was overexpressed as the inclusion body (IB) form, higher than the expected level whencompared to the HCVC173, which was also showed by the analysis of secondary structure of mRNAs andcalculation 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 matureHCVC173 in E. coli. Therefore, the fusion form of the mature HCV core protein and the conditions defined inthis study provide an alternative strategy for HCVC173 production in high cell density culture of E. coli.
https://www.ijbiotech.com/article_7128_45c6632f59efadc1fa8df487b69e343e.pdf
2011-10-01
245
252
Hepatitis C Virus
core protein
Overexpression
Recombinant protein
Jafar
Hemmat
j.hemmat@gmail.com
1
National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, I.R. Iran.
AUTHOR
Bagher
Yakhchali
bahar@nigeb.ac.ir
2
National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, I.R. Iran.
LEAD_AUTHOR
Khosro
Khajeh
khajeh_k@yahoo.com
3
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 14115-154, Tehran, I.R. Iran.
AUTHOR
Ali Akbar
Moosavi-Movahedi
4
Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box 13145-1384, Tehran, I.R. Iran
AUTHOR
Ali Asghar
Karkhane
karkhane@nigeb.ac.ir
5
National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, I.R. Iran.
AUTHOR
ORIGINAL_ARTICLE
Optimizing refolding condition for recombinant tissue plasminogen activator
Low molecular size additives such as L-arginine and the redox compounds have been used both in the culturemedium and in vitro refolding to increase recombinant proteins production. Additives increase proteinrefolding and yield of active proteins by suppressing aggregate formation or enhancing refolding process.In this work, a comparative study was performed on refolding of recombinant plasminogen activator (rPA)in the presence of different concentrations of denaturants and additives. Escherichia coli-expressed rPAinclusion bodies were solubilized in chaotropic denaturants and subjected to protein refolding by dilutionmethod. The effects of various additives, the impact of pH, residual Guanidin Hydrochloride (Gn-HCl) andDithiothreitol (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.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.pdf
2011-10-01
253
259
Refolding
Recombinant Plasminogen Activator
arginine
Glutathione
Susan
Mirnajd Gerami
1
Infectious and Tropical Disease Research Center, Tabriz University of Medical Sciences, P.O. Box 51656-65813, Tabriz, I.R. Iran.
AUTHOR
Safar
Farajnia
farajnia@yahoo.com
2
Biotechnology Research Center, Tabriz University of Medical Sciences, P.O. Box 51656-6581,Tabriz, I.R. Iran.
LEAD_AUTHOR
Feridoun
Mahboudi
3
Biotechnology Research Center, Pasteur Institute of Iran, P.O. Box 13164, Tehran, I.R.Iran.
AUTHOR
Hossein
Babaei
4
Drug Applied Research Center, Tabriz University of Medical Sciences, P.O. Box 51656-65811, Tabriz, I.R. Iran.
AUTHOR
ORIGINAL_ARTICLE
Flux distribution in Bacillus subtilis: inspection on plurality of optimal solutions
Linear programming problems with alternate solutions are challenging due to the choice of multiple strategiesresulting in the same optimal value of the objective function. However, searching for these solutions is atedious task, especially when using mixed integer linear programming (MILP), as previously applied tometabolic models. Therefore, judgment on plurality of optimal metabolic flux distributions (solutions) a priorito applying MILP approach could prevent unnecessary computations. In this work for the first time, thereduced cost coefficients for the non-basic variables in a current solution of a metabolic model were utilized toinspect the possibility of multiple optimal flux distributions. If there exists at least one non-basic variablewith zero reduced cost coefficient, multiplicity of optimal solution may occur where MILP can be used tofind these solutions. This approach was implemented on a metabolic network of Bacillus subtilis aiming toreduce 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 sixnon-basic variables had zero reduced cost coefficients at current solution, which shows that there can existmultiple 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 glucoseconsumption rate was selected as the superior solution.
https://www.ijbiotech.com/article_7148_45574c4a50e0842bd9e47df5bcadac12.pdf
2011-10-01
260
266
Bacillus subtilis
flux balance analysis
metabolic reaction network
multiple optimal flux distribution
reduced cost coefficient
mixed integer linear programming
Ehsan
Motamedian
motamedian@modares.ac.ir
1
Biotechnology Research Laboratory, School of Chemical Engineering, Iran University of Science and Technology, P.O. Box 16846-13114, Tehran, I.R. Iran.
AUTHOR
Fereshteh
Naeimpoor
fnaeim@iust.ac.ir
2
Biotechnology Research Laboratory, School of Chemical Engineering, Iran University of Science and Technology, P.O. Box 16846-13114, Tehran, I.R. Iran.
LEAD_AUTHOR
ORIGINAL_ARTICLE
Amylase production from Aspergillus oryzae LS1 by solid-state fermentation and its use for the hydrolysis of wheat flour
Nine 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%.
https://www.ijbiotech.com/article_7138_d23212f100ecabd1b3476447a419c019.pdf
2011-10-01
267
274
Amylase
Aspergillus oryzae
Solid state fermentation
production
Application
Mohamed Abdel
Fattah Farid
1
Department of Natural and Microbial Products, National Research Centre, Dokki, Cairo, Egypt.
LEAD_AUTHOR
Hoda Mohamed Abdel
Halim Shata
2
Department of Microbial Chemistry, National Research Centre, Dokki, Cairo, Egypt.
AUTHOR
ORIGINAL_ARTICLE
Cloning and enhanced expression of an extracellular alkaline protease from a soil isolate of Bacillus clausii in Bacillus subtilis
in the detergent industry. In this study, the extracellular alkaline serine protease gene, aprE, from Bacillusclausii 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/aprEreached 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 of31 kDa. DNA sequence analysis and the deduced amino acid sequence revealed 98% homology with theextracellular alkaline serine protease from B. clausii KSM-K16.
https://www.ijbiotech.com/article_7129_45ea7dbb7127ac72c4ecbf2eae9cf6f0.pdf
2011-10-01
275
280
Alkaline protease
Cloning
Expression
Bacillus clausii
Bacillus subtilis
Seyed Mohsen
Abbasi-Hosseini
1
Department of Microbiology, Faculty of Biological sciences, Shahid Beheshti University, P.O. Box 19395-4716, Tehran, I.R. Iran.
AUTHOR
Fereshteh
Eftekhar
f-eftekhar@sbu.ac.ir
2
Department of Microbiology, Faculty of Biological sciences, Shahid Beheshti University, P.O. Box 19395-4716, Tehran, I.R. Iran.
LEAD_AUTHOR
Bagher
Yakhchali
bahar@nigeb.ac.ir
3
Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, I.R. Iran.
AUTHOR
Dariush
Minai-Tehrani
4
Department of Microbiology, Faculty of Biological sciences, Shahid Beheshti University, P.O. Box 19395-4716, Tehran, I.R. Iran.
AUTHOR
ORIGINAL_ARTICLE
Haplotype block partitioning and tagSNP selection under the perfect phylogeny model
Single Nucleotide Polymorphisms (SNPs) are the most usual form of polymorphism in human genome.Analyses of genetic variations have revealed that individual genomes share common SNP-haplotypes. Theparticular pattern of these common variations forms a block-like structure on human genome. In this work,we develop a new method based on the Perfect Phylogeny Model to identify haplotype blocks usingsamples 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 andsemi-perfect phylogeny. It is shown that the minimum number of tagSNPs in a haplotype block of PerfectPhylogeny can be obtained by a polynomial time algorithm. We compare the performance of our algorithmon haplotype data of human chromosome 21 with other previously developed methods through simulations.The results demonstrate that our algorithm outperforms the conventional implementation of the FourGamete Test approach which is the only available method for haplotype block partitioning based onPerfect Phylogeny.
https://www.ijbiotech.com/article_7132_68d5eb3d68a319ff1fa0e43777a8b118.pdf
2011-10-01
281
289
Single Nucleotide Polymorphisms
Haplotype
tagSNP
perfect Phylogeny
Changiz
Eslahchi
ch-eslahchi@sbu.ac.ir
1
Faculty of Mathematical Sciences, Shahid Beheshti University, G.C., P.O. Box 198396-3113, Tehran, I.R. Iran.
LEAD_AUTHOR
Ali
Katanforoush
2
Faculty of Mathematical Sciences, Shahid Beheshti University, G.C., P.O. Box 198396-3113, Tehran, I.R. Iran.
AUTHOR
Hamid
Pezeshk
pezeshk@khayam.ut.ac.ir
3
School 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.
AUTHOR
Narjes
Afzaly
4
School 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.
AUTHOR
ORIGINAL_ARTICLE
Bioaffinity 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 mode
In 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 theinitial activity after crosslinking by glutaraldehyde. Both soluble and immobilized enzyme exhibited the samepH and temperature optima at pH 5.5 and 50ºC, respectively. However, the immobilized enzymeshowed a remarkable broadening in pH and temperature-activity profiles as compared to the nativeenzyme. Immobilized enzyme was significantly more stable against thermal denaturation at 60ºC.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% residualactivity after exposure to pepsin (0.15 mg/ml) for 1 h whereas the immobilized β-galactosidase was morestable and retained nearly 84% activity under identical experimental conditions. The activity of immobilizedenzyme was enhanced to 156% whereas soluble β-galactosidase showed an enhancement upto 134%when exposed to trypsin (0.1 mg/ml) for 1 h. Moreover, immobilized β-galactosidase exhibited greaterenhancement in enzyme activity against exposure to various ions present in milk such as Na+, K+, Ca+2,Mg+2 and citrate ions. The higher concentration of lactose was hydrolyzed from whey as compared to thehydrolysis from milk by immobilized enzyme at 50ºC and 60ºC in batch processes. Lactose was hydrolyzedto 86% and 78% after 20 days continuous operation of reactors at the flow rates of 20 ml/h and 30 ml/h,respectively. In view of its stability and utility in batch and continuous processes, such preparation could beexploited for the hydrolysis of lactose from milk and whey in a more convenient and cheaper way in dairyindustries.
https://www.ijbiotech.com/article_7149_0c9a0323a79a215adc9d1eefb212674d.pdf
2011-10-01
290
301
β-galactosidase
almond
lactose hydrolysis
cellulose-alginate beads
Batch process
Shakeel Ahmed
Ansari
saansari@kau.edu.sa
1
Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh-202002, India.
AUTHOR
Qayyum
Husain
2
Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh-202002, India.
LEAD_AUTHOR
ORIGINAL_ARTICLE
Isolation of endophytic actinomycetes from Catharanthes roseus (L.) G. Don leaves and their antimicrobial activity
Endophytic actinomycetes were isolated from surface sterilized leaves of Catharanthes roseus (L.) G. Don offamily Apocynaceae. A total of 38 endophytic actinomycetes were recovered on Starch Casein Agar.Among the 38 isolates 20 morphologically different isolates were screened for antibacterial activity againstBacillus 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.pdf
2011-10-01
302
306
Antimicrobial activity
Catharanthes roseus
endophytic actinomycetes
leaves
Abdul
Kafur
1
Department of Ecology and Environmental Sciences, Pondicherry University, Puducherry-605 014, India.
AUTHOR
Anisa
Basheer Khan
2
Department of Ecology and Environmental Sciences, Pondicherry University, Puducherry-605 014, India.
LEAD_AUTHOR
ORIGINAL_ARTICLE
Improved protocol for isolation of genomic DNA from leaf tissues of Phyllanthus emblica Gaertn
Modified Cetyltrimethylammonium bromide (CTAB) protocol for DNA isolation was developed from leaf tissuesof Phyllanthus emblica for obtaining high quality genomic DNA. Fresh leaves of three different maturitywere analyzed for yield and quality of DNA. Acidity was determined in three different maturity of leaves viz.tender, intermediate and mature and their influence on DNA quality was determined. Drastic reduction of pHwas the primary cause for poor quality of DNA. However, high quality DNA isolation was achieved bystabilizing the pH by addition of NaOH during different stages of DNA isolation process. The present protocolyielding high quality intact DNA for genetic fingerprinting as well as for amplification of chloroplast genes formolecular analysis.
https://www.ijbiotech.com/article_7146_62d3bfeb1f1a30b5060d0eb2d9584b40.pdf
2011-10-01
307
313
Phyllanthus emblica
chloroplast DNA
restriction enzymes
trnL (UAA) intron sequences
gene specific primers
Sangeetha Nagarajan
Nagarajan
1
Department of Biotechnology, Plant Genetic Improvement Laboratory, Sri Paramakalyani Centre for Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi 627 412, Tirunelveli District, Tamilnadu, India.
AUTHOR
Mercy
Steephen
2
Department of Biotechnology, Plant Genetic Improvement Laboratory, Sri Paramakalyani Centre for Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi 627 412, Tirunelveli District, Tamilnadu, India.
AUTHOR
Kavitha
Murugan
3
Department of Biotechnology, Plant Genetic Improvement Laboratory, Sri Paramakalyani Centre for Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi 627 412, Tirunelveli District, Tamilnadu, India.
AUTHOR
Rahul
Raveendran Nair
4
Department of Biotechnology, Plant Genetic Improvement Laboratory, Sri Paramakalyani Centre for Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi 627 412, Tirunelveli District, Tamilnadu, India.
AUTHOR
Thilaga
Sethuraman
5
Department of Biotechnology, Plant Genetic Improvement Laboratory, Sri Paramakalyani Centre for Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi 627 412, Tirunelveli District, Tamilnadu, India.
AUTHOR
Parameswari Alagar
Alagar
6
1Department of Biotechnology, Plant Genetic Improvement Laboratory, Sri Paramakalyani Centre for Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi 627 412, Tirunelveli District, Tamilnadu, India.
AUTHOR
Doss
Ganesh
7
Department of Plant Biotechnology, School of Biotechnology, Madurai Kamarai University, Palkalainagar, Madurai 625 021, India.
LEAD_AUTHOR