Open Access Original Research Article

Production of Bovine Herpesvirus-1 Vaccine Strains in MDBK Cells Using BelloCell Bioreactor

Lekshmi S. Rajan, Aman Kamboj, Bikash R. Prusty, Tapan Palai, L. R. Ananthakrishna, Mohini Saini, Praveen K. Gupta

Biotechnology Journal International, Page 1-7
DOI: 10.9734/BBJ/2016/20401

Aim: To produce bovine herpesvirus-1 (BoHV-1) vaccine strains in MDBK cells using an oscillating bioreactor, BelloCell.

Place and Duration of Study: Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar-243122, India, between November 2013 and June 2015.

Methodology: MDBK cells were cultured in BelloCell bioreactor seeded with MDBK cells. After 6 days incubation at 37°C under 5% CO2 tension, the MDBK cells were infected with 0.001 multiplicity of infection (MOI) of either wild-type (wt)-BoHV-1 or glycoprotein E deleted (∆gE)-BoHV-1vaccine strains and incubated further for 96 h. To analyse the vaccine potential of the harvests, inactivated wt-BoHV-1 and ∆gE-BoHV-1 were inoculated in guinea pigs. Sera from immunized guinea pigs were collected at 90 days after primary immunization and analysed for virus neutralizing (VN) antibody response specific to wt-BoHV-1.

Results: Using BelloCell bioreactor, the MDBK cell density reached to a maximum number of         1.5 x 109 to 2.0 x 109 cells after 6 days cultivation. After infection with wt-BoHV-1 and ∆gE-BoHV-1 strains, the total virus yield at 96 h post-infection was 0.5 x 1011.25 and 0.5 x 1011.83, respectively. There was no significant difference in VN antibody response in sera collected from immunized guinea pigs.

Conclusion: This study indicated that BelloCell bioreactor can be used as a simple system for high population density cell culture and large scale production of vaccines.

Open Access Original Research Article

Purification and Characterization of Pectin Methylesterase Produced in Solid State Fermentation by Aspergillus tubingensis

Mukesh Kumar Patidar, Anand Nighojkar, Sadhana Nighojkar, Anil Kumar

Biotechnology Journal International, Page 1-10
DOI: 10.9734/BBJ/2016/23632

Aim: Purification and characterization of pectin methylesterase produced by Aspergillus tubingensis in solid state fermentation.

Study Design: Pectin methylesterase enzyme produced by A. tubingensis was extracted from the fermented solid medium and purified using chromatographic techniques. The purified enzyme was characterized for physico-chemical and kinetic properties.

Place and Duration of Study: Experiments were performed at the School of Biotechnology, Devi Ahilya University, Indore, INDIA and Maharaja Ranjit Singh College of Professional Sciences, Indore, INDIA, between October, 2014 and August, 2015.

Methodology: The enzyme was extracted and purified using ammonium sulphate fractionation, ion exchange chromatography (IEC) using CM- cellulose and gel filtration chromatography (GFC) using Sephadex G-100. The molecular weight of the purified enzyme was determined using native polyacrylamide gel electrophoresis (Native PAGE) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). The purified enzyme was characterized to determine the pH and temperature optima. Thermostability, pH stability and substrate kinetics were studied for purified pectin methylesterase.

Results: The acidic pectin methylesterase of Aspergillus tubingensis was purified to 20.3 fold with a 47.7% recovery through IEC on CM- cellulose and GFC using Sephadex G-100. The purified enzyme had a specific activity, 112.6 U/mg. The SDS-PAGE revealed that the enzyme was monomeric with a molecular weight of 45.7 kDa. The optimum pH and temperature were 4.6 and 50°C, respectively. This enzyme was stable over a wide pH range (3.0–8.0) and at relatively high temperature at 50°C for 1 h. The Km and Vmax values of pectin methylesterase towards citrus pectin were 33.3 mg/l and 251.2 µmol/ml/min, respectively. In addition, the enzyme activity increased by about 16% in the presence of 5 mM Mg2+.

Conclusion: The pectin methylesterase enzyme of A. tubingensis has been purified up to homogeneity and found to be monomeric on SDS-PAGE. Enzyme characterization revealed that purified enzyme worked optimally in acidic conditions and was stable at wider pH range.

Open Access Original Research Article

Detection and Analysis of the Random Mutagenesis Site(s) of Xylanase Gene from Mutants of Bacillus subtilis subsp. spizizenii ATCC 6633

Hooi Ling Ho, Ajounmah Maryann Chinonso

Biotechnology Journal International, Page 1-20
DOI: 10.9734/BBJ/2016/23057

Aims: A total of five mutant strains of Bacillus subtilis subsp. spizizenii ATCC 6633 designated as the MXB 1, MXB 2, MXB 3, MXB 4 and MXB 5 were developed using random mutagenesis of ethyl methane sulfonate (EMS) and acridine orange (AO) in our previous study. Based on our present investigation, we identified, verified and sequenced xylanase gene of mutant strains of B. subtilis ATCC 6633 as the potent bacterial xylanase producers under submerged fermentation. Furthermore, amino acid analysis and comparison between the xylanases of the mutants and other xylanolytic bacteria were also elucidated. Overall, this study would provide gene and protein molecular information correlating nucleotide and amino acid structure related to the increased xylanase production by random mutagenesis. In respect to the objectives of this study, we compared the endoxylanase sequence of wild type B. subtilis ATCC 6633 with its mutants in order to determine their possible site(s) of mutagenesis and to analyse amino acid xylanase sequence of the mutants of B. subtilis ATCC 6633.

Methodology: After the verification of xylanase production by all mutants of B. subtilis ATCC 6633 on the xylan agar using Congo-red staining in the previous study, xylanase gene of the mutants was amplified from the genomic DNA to detect the mutagenesis site(s) by synthesizing primers directed against the sequence of xylanase gene obtained from the wild type of Bacillus subtilis subsp. spizizenii ATCC 6633.

Results: The comparison of xylanase genes from different mutants of B. subtilis and the wild type revealed the site(s) of mutagenesis. Interestingly, the mutations of the mutants of B. subtilis ATCC 6633 in this study were significantly reflected at the 5’ end of the mutants xylanase genes. The open reading frames (ORF) of the mutant xylanase genes ranged from 644 bp to 684 bp with translated encoding protein between 214 and 228 amino acid residues were obtained. On the other hand, predicted molecular mass from 23.94 kDa to 25.40 kDa and theoretical pI which ranged from 8.63 to 9.16 were attained from all of the mutant strains in this study. Based on the characteristics obtained, the mutant xylanases were suggested to belong to Glycosyl Hydrolase (GH) Family 11 with 98% homology to endo-1,4-beta-xylanase of B. subtilis subsp. spizizenii of W23. In fact, conserved regions, signal peptide, a cleavage site between the Ala28 and Ala29 residues and four Tyr residues specific to GH Family 11 xylanase were also observed and detected in all mutants. Indeed, two conserved glutamate residues of E94 and E183 that directly involved in the enzyme catalytic mechanism were also detected in the amino acid sequences of the mutants. The analysis of the deduced amino acid sequences revealed that the mutations in the signal peptide regions fostered increased hydrophobic core of xylanase residue. We suggested that these changes would probably be responsible for the increased extracellular xylanase yield in the mutants of B. subtilis. On the other hand, all the mutants of B. subtilis ATCC 6633 exhibited the tendency to be thermostable based on the Val, Ser and Thr frequency ratios which were almost identical to those of thermophiles. Furthermore, the increase of Thr to Ser ratio and presence of Arg residue found in the mutant strain of MXB 5 would enhance the polar interactions and hence improve the secondary structure stabilization that was usually one of the determining factors in the thermophilic proteins.

Conclusion: In a nutshell, the properties of B. subtilis mutant xylanases particularly mutant MXB 5 revealed its relevant potential in the biotechnology applications in bio-bleaching, textile, paper and pulp industries that commonly require high temperature usage in xylanase applications.

Open Access Original Research Article

Frequency of Procedural Errors during Root Canal Treatment Performed by Interns

Syed Ajlal Akhtar, Fahad Ata Siddiqui, Abu Bakar Sheikh, Saqib Rashid, Zohaib Khurshid, Shariq Najeeb, Muhammad Sohail Zafar

Biotechnology Journal International, Page 1-8
DOI: 10.9734/BBJ/2016/23768

Aims: Chemo-mechanical preparation continues to be one of the most challenging steps in root canal treatment procedures. The aim of this study was to examine the frequency of procedural errors during root canal treatment performed by interns.

Study Design: Cross sectional descriptive study.

Methodology: A total of 200 patients scheduled for root canal treatment in the permanent first molar were selected and pre-operative radiographs were taken before the procedure. After achieving a straight line access, the interns performed conventional step back technique to prepare the canals and irrigation was done using 2.5% sodium hypochlorite solution. After completion of the instrumentation procedure, two experienced endodontists evaluated the cases both clinically and radiographically.

Results: Results showed that a total of 78 (39%) cases received procedural errors, and the remaining cases received appropriate instrumentation procedures. Apical Transportation (12%) presented the highest percentage for procedural errors followed by ledge formation (10%), strip perforation (5%), apical perforation (5%), instrument separation (4%) and perforation during access (3%).

Conclusion: The present study suggests a high frequency of procedural errors 39% in all cases performed by interns. This reflects the amount of clinical knowledge and skill possessed and applied by operator during the course of the treatment.

Open Access Original Research Article

Genetic Diversity of Native Bacillus thuringiensis Strains Isolated from Soil of Different Localities in Mali and their cry Gene Profile

R. Fané, D. Traoré, A. Kassogué, F. Samaké, A. H. Dicko, A. Hamadoun, F. H. Valicente, A. H. Babana

Biotechnology Journal International, Page 1-8
DOI: 10.9734/BBJ/2016/20432

Aims: To determine the genetic diversity of Bacillus thuringiensis strains isolated from soils of different locality in Mali, and select strains with cry1F, cry1B and cry1C genes to control caterpillars and strains with cry2 gene against African rice gall midge.

Study Design: Strains of Bacillus thuringiensis (Bt) used in this study belong to collection of Laboratory of Research in Microbiology and Microbial Biotechnology (Laborem-Biotech) and that isolated from different ecological environment of Mali. Bt strains used as positive controls were kindly provided by Dr. Fernando Hercos Valicente from Embrapa Milho e Sorgo (Brazil).

Methodology: The total DNA of the native B. thuringiensis and reference strains were extracted from overnight grown culture. Gene identification was performed by amplification (PCR) of DNA of Bt strains using specific primers. The gel revelation was performed using ethidium bromide and the gel photography was performed using an E-Box VX2 system, Version 15.06.

Results: Native B. thuringiensis strains studied, showed high genetic diversity. 48,3% of the studied B. thuringiensis strains of the collection carry cry1 gene, 49,06% of them harbor both cry1 and cry2 gene, 5.7% of the native Bt strains didn’t react with any cry1 and cry 2 specific primers and 94.3% of the strains produce different PCR products. The analysis of cry1 positive B. thuringiensis showed sub-group frequencies of 7.6% for cry1F and 3.8% for cry1B and cry1C. In native Bt strains of our collection, the cry2 gene was always present with one or two cry1 sub-group(s).

Conclusion: In this study, high genetic diversity in the native Bt strains from the bacterial collection in Mali was observed. Most of the native Bt strains studied harbor the cry1 gene only. In the cry1 gene profile, the cry1F sub-group was found to be the most frequently detected. None of studied Bt strains harbored the cry2 gene only.