Open Access Original Research Article

Activity of β-Amylase in Some Fungi Strains Isolated from Forest Soil in South-Western Nigeria

O. A. Oseni, M. M. Ekperigin

Biotechnology Journal International, Page 1-9
DOI: 10.9734/BBJ/2014/4686

Aims: The aim of this study was to isolate fungi species from Omo natural forest soil in Ogun State of Nigeria and study the amylases from the fungi species which are digestive enzymes that hydrolyze glycosidic bonds in starch to glucose, maltose, maltotriose and dextrin; and in particular determine the activities of β-amylase from the forest soil.

Study Design: Nine different species of fungi were isolated from the Omo natural forest reserve soil (Gonatobotrrys simplex, Aspergillus niger, Spiromyces minutus, Aspergillus flavus, Articulospora inflata, Botrytis cenera, Penicillium italicum, Aspergillus fumigatus and Aspergillus flavus). Four species of the fungi (Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus and Penicillum italicum) exhibited amylolytic activities maximally were obtained and screened for the production of beta-amylase (1,4-α-D-glucan maltohydrolase) for five days in liquid medium using 2% starch as carbon source. All the strains of fungi produced β-amylase optimally within the first 24 hours with progressive decreased production as the days gone by.

Place and Duration of Study: Department of Biochemistry, Federal University of Technology, Akure, Ondo State, Nigeria, between February 2010 and March 2011.

Methodology: We isolated many fungi species from forest reserve soil, four species (Aspergillus flavus, Aspergillus niger, Aspergillus fumigantus and Penicillum itallicum) were identified and assayed for β-amylase activity. 

Results: All the organisms produced β-amylase activity favorably at 40ºC; all were observed to be thermally stable at between 30ºC and 40ºC with optimal pH in alkaline medium between pH 7.00 and 9.00.

Conclusion: The results obtained in this study however showed that all the fungi strains are promising sources of β-amylase for potential applications in food and pharmaceutical industries and for biotechnological and industrial applications.

Open Access Original Research Article

Morphological Anomalies in Somatic Embryo Structure in Catharanthus roseus: Improving Embryo Germination by Amending Plant Growth Regulators, Activated Charcoal and Sucrose Level

Dipti ., Samar Fatima, Abdul Mujib

Biotechnology Journal International, Page 10-20
DOI: 10.9734/BBJ/2014/4809

The present study describes the incidence of somatic embryo (SE) irregularities in Catharanthus roseus (L.) G. Don. In this ornamental and anti-cancerous medicinal plant, SEs were developed at a very high rate from hypocotyl callus on MS medium, amended with  1.0 mg l-1 naphthalene acetic acid (NAA) and 1.5 mg l-1benzylaminopurine (BA). But at high (1.5 mg l-1) plant growth regulators (PGRs) levels severe embryo developmental abnormalities were caused. The major aberrant SE types have been described in the present communication. In several cases, the in vitro raised SEs had underdeveloped root ends or with aborted root axis. These irregularities in embryo structure appreciably reduced embryo germination rate and plantlet recovery. Here, we evaluated the effect of various PGRs, activated charcoal (AC), and various levels of sucrose in order to improve SE quality and germination. Medium added with 4.0 % sucrose improved rooting ability and growth. The addition of AC improved plantlet conversion significantly by promoting rooting.

Open Access Original Research Article

Effects of Utilization of Crushed, Boiled and Fermented Roselle Seeds (Hibiscus sabdariffa) on the Performance of Broiler Chickens

Maikano Mohammed Ari, Danlami Moses Ogah, Idris Danladi Hassan, Ibrahim Suleiman Musa-Azara, Nuhu Dalami Yusuf, Samuel Emmanuel Alu

Biotechnology Journal International, Page 21-29
DOI: 10.9734/BBJ/2014/6061

Aim of Study: The study examines the effects of utilization of crushed (CRRS), boiled (HTRS) and fermented (FRS) Roselle seeds (Hibiscus sabdariffa) on the performance of broiler chickens

Study Design: A total of 135 Anak day-old broiler chicks were randomly assigned to three (3) experimental groups of three (3) replicates using completely randomized design, data collected were subjected to ANOVA using SPSS and Likert scaling technique.

Place and Duration of Study: Livestock Complex, College of Agriculture, Lafia, Nasarawa state, Nigeria: February 2012 to April 2012.

Methodology: The effects of inclusion of differently processed Roselle seeds on performance traits of experimental birds were evaluated through feeding trials (1- 28 d) and (29- 50 d) at starter and finisher phases. Dietary treatments were as follows: D1, D2 and D3 representing Crushing of Raw Rosselle Seeds (CRRS); Hydrothermally Processed Rosselle Seeds (HTRS) and Fermented Rosselle Seeds (FRS) base diets.

Results: No significant (P=0.05) difference in the following parameters: initial weight, feed intake, FCR and survival percentage in the starter phase while the finisher phase significantly (P=0.05) differ only the performance index. Overall scoring of performance parameters showed those birds in D3 group were better than D2 and D1 in that order

Conclusion: Roselle seeds inclusion in broiler diets provides effective mechanism for the improvement in performance traits of broilers and fermentation of rosselle (Hibiscus sabdarif) is best processing method.

Open Access Original Research Article

Screening of Filamentous Fungi for Xylanases and Cellulases Not Inhibited by Xylose and Glucose

L. F. C. Ribeiro, L. F. Ribeiro, J. A. Jorge, M. L. T. M. Polizeli

Biotechnology Journal International, Page 30-39
DOI: 10.9734/BBJ/2014/6066

Aims: Screening different filamentous fungi for thermostable xylanases and cellulases that would not be inhibited by xylose and glucose, respectively.

Methodology: Samples of fungi collected in the Atlantic forest region, Minas Gerais, Brazil, and some fungi from our Culture Collection were used in this screening. All fungi were grown in liquid media containing 1% sugar cane bagasse (SCB). After that, an aliquot of the crude broth was incubated at different temperatures (from 4 to 60 °C) in carboxymethyl cellulose (CMC) or xylan-media plates, for 12 hours. After this period, the plates were stained with Congo Red. Fungi that presented the best results (larger halos) were tested for the effect of adding xylose and glucose in the xylanase and cellulases activities, respectively. Crude extracts obtained from fungi grown in SCB were used for laccase and lichenase assay.

Results: The screening on agar plates with CMC/xylan presented halos of different sizes. From all tested fungi, the best cellulase producer was Malbranchea pulchella, which also presented the most thermostable xylanase. Penicillium griseofulvum presented bigger halos at all temperatures tested, but the xylanase lost almost 14% of its stability in higher temperatures. The effect of xylose and glucose on the enzymatic activities recorded dose-dependent. It was observed that 20% activation of the enzymes produced by M. pulchella with 30 mM glucose or 20 mM xylose to cellulase and xylanase, respectively. It was observed a loss of less than 20% for P. griseofulvum xylolytic activity using 50 mM xylose. Lichenase was detected in some fungi prospected but laccase was not detected.

Conclusion: Malbranchea pulchella was a good producer of xylanase and cellulase tolerant to xylose and glucose, respectively. Other studies must be performed with this fungus so that it can be used in the future for biotechnological purposes.

Open Access Original Research Article

Identification of Genes Putatively Involved in the Biosynthesis of Antitubercular Peptide in Streptomyces ribosidificus NRRL B-11466

Khaled M. Aboshanab, Nisreen M. Okba, Tarek S. El-banna, Ahmed A. Abd El-Aziz

Biotechnology Journal International, Page 40-50
DOI: 10.9734/BBJ/2014/5210

Aims: To determine the potential antitubercular activity of Streptomyces ribosidificus NRRL B-11466 both on genotypic and phenotypic levels. 

Methodology: Standard methods and software programs were used for nucleotide/protein sequence analysis and phenotypic detection of antitubercular activity.

Results: Analysis of the submitted DNA segment (accession code = AJ744850) harbouring the ribostamycin biosynthetic gene cluster showed that the respective gene cluster was flanked in the upstream region by three open reading frames (ORFs), encoding putative type II thioesterase (SribL03.14c) and two nonribosomal peptide synthases (SribL03.14c and SribL03.14c). These ORFs were of high amino acid similarities (about 80%) to those located in the viomycin and related antibiotic biosynthetic gene clusters. A DNA segment harbouring three ORFs, putatively involved in capreomycidine biosynthesis was submitted into the GenBank database under the accession code HQ327309. Comparative analysis of the respective DNA segment with viomycin and related antibiotic biosynthetic gene clusters showed: firstly, location of the respective DNA segment in the neighbourhood and upstream to the ribostamycine biosynthetic gene cluster; secondly, conservation of six ORFs: SriC (putative L-arginine hydroxylase); SriD (putative L-capreomycidine synthase), SriE (putative permease) located on our submitted DNA fragment; and SribL03.14c, SribL03.15c, SribL03.16c located on the DNA fragment harboring ribostamycin biosynthetic gene cluster, among the tested biosynthetic gene clusters. Phenotypically, S. ribosodificus inhibited growth of Mycobacterium smegmatis ATCC 19420 and Mycobacterium phlei ATCC 11758.

Conclusion: Streptomyces ribosidificus NRRL B-11466 produces antimycobacterial agents and this was confirmed genotypically via detection of 6 ORFs with high amino acid similarities (about 80%) to those located in the viomycin and related antibiotic biosynthetic gene clusters as well as phenotypically by determining its inhibitory activity against Mycobacterium smegmatis ATCC 19420. This is the first report about identification of genes putatively involved capreomycidine biosynthesis in Streptomyces ribosidificus NRRL B-11466.

Open Access Original Research Article

Establishment of an Efficient and Practical Virus-free Seedling Supply System by Means of Culture of Shoot Apexes, RT-PCR and Clonal Propagation in Sweet Potato (Ipomoea batatas)

Lanzhuang Chen, Chenti Xu, Zhaosheng Du, Takuro Hamaguchi, Toru Sugita, Ryutarou Nagata, Liming Guan

Biotechnology Journal International, Page 51-63
DOI: 10.9734/BBJ/2014/6235

Aims: Sweet potato (Ipomoea batatas) cv. “Miyazakibeni” was used as material for shoot apex culture, reverse transcription-polymerase chain reaction (RT-PCR) and clonal propagation to establish an efficient and practical virus-free seedling supply system in production of vegetatively reproductive plants.

Study Design: At first, efficient plant regeneration was achieved from shoot apex culture of sweet potato. Secondly, RT-PCR method was used to detect the sweet potato feathery mottle virus (SPFMV) viral infection of tuber surface of edible sweet potato using the RNAs from the plants obtained from shoot apex culture. Finally, the virus-free plants verified by RT-PCR were propagated clonally by culture of suckers cut from stems of the virus-free plants.

Place and Duration of Study: Faculty of Environmental and Horticultural Science, Minami Kyushu University, between June 2008 and December 2012.

Methodology: The best efficiency for material sterilization was tested using different concentrations (0.1% - 1.5%) of sodium hypochlorite solution (SHS) and the treated times (5 min – 20 min). Theshoot apexes less than 0.3mm in size were cultured on Komamine and Nomura (1998) (KN) medium and Murashige and Skoog (1962) (MS) medium. The regenerated plants were used for RNA extraction and then, used for RT-PCR for detection of SPFMV. Based on the result of RT-PCR, the suckers cut from stems of virus-free plants were cultured and propagated clonally and routinely within a short period.

Results: The combination of 0.3% of SHS and 10 and/or 20 min gave the best result (100%) of surviving rate for material sterilization. The culture of shoot apexes less than 0.3 mm in size gave plant regenerating rates of 82% and 65% on KN and MS medium, respectively. The results of RT-PCR of RNAs from plants obtained from shoot apex culture and plants of SPFMV infection showed that SPFMV virus was clearly removed by shoot apex culture conducted in this study. For clonal propagation, 80-100% of suckers cut from the stems of the virus-free plants detected grew into complete plants after 6 weeks of culture, indicating that the virus-free plants could be routinely propagated 5 times in number each time and repeatable by the short circle. The sweet potato produced in field showed no symptom called as russet crack-like symptom (RC-LS) even after cultivation two seasons.

Conclusion: Overall, an efficient and practical virus-free seedling supply system was established in sweet potato by the three steps of 1) virus-free plant regeneration from shoot apex culture, 2) quick detection of SPFMV using RNA of the regenerated plants by RT-PCR, and 3) the verified virus-free plants were propagated clonally and routinely within a short period using culture of suckers cut from the stems of virus-free plants.

Open Access Original Research Article

Different Methods for DNA Extraction from Yeast-Candida famata Isolated from Toddy

T. Santra, S. K. Ghosh, A. Chakravarty

Biotechnology Journal International, Page 64-73
DOI: 10.9734/BBJ/2014/4884

Aims: Isolation and biochemical characterization of yeasts from toddy and standardization of best method for DNA extraction from yeast.

Study Design: Biochemical characterization of yeast and genomic DNA extraction by manual and kits methods.

Place and Duration of Study: Department of Microbiology, Institute of Genetic Engineering, Badu, kol-128, India and  Molecular Mycopathology Lab, P. G. Department of Botany, Ramakrishna Mission Vivekananda Centenary College, Rahara, kol-118, India, from November 2012 –April,2013.

Methodology: Toddy was collected in sterilized polythene bags from palm tree (Borassus flabellifer L; Family: Arecacea) in the morning, from Badu, 24-parganas (N) India. Isolation of yeasts was done by the method of Beech and Davenport [15] using MA (Malt extract) medium. Biochemical Identification was performed by using basal medium and procedure [1,2,15]. Genomic DNA extraction was done by manual and kits methods (UniflexTM DNA isolation Kit). Quality of extracted DNA was checked by the absorbance ratio (A260 / A280) ranged from 1.8 to 2.0.

Results: By performing morphological, microscopical and biochemical characterization the isolated yeast from toddy was identified as Candida famata consulting with the key of yeast published [1,2].

The UniflexTM DNA isolation Kit method is much more convenient way to get pure and high quality DNA than the manual methods.                      

Conclusion:  Isolated yeast from toddy was identified as Candida famata. The genomic DNA of Candida famata was extracted purely by UniflexTM DNA isolation Kit. This method was better and more convenient than manual method.

Open Access Original Research Article

In Vitro Propagation of Alternanthera sessilis L. from Internode Explant

Dimpy Das, P. K. Borua

Biotechnology Journal International, Page 74-80
DOI: 10.9734/BBJ/2014/6261

An efficient protocol was developed for micropropagation of Alternanthera sessilis L. from internode explant. Callus was developed from the cut ends and surface of the internode explants when the Murashige and Skoog (MS) medium was supplemented with 0.5mg/L or 1.0mg/L 2,4-D. The greatest mean number of shoots (124) with the highest mean shoot length (9.3cm) was obtained when calli were cultured on the MS medium with 1.0mg/L each of 6-Benzylaminopurine and adenine sulfate. Early and maximum root induction was noted when shoots were cultured in half strength MS basal medium. In vitro regenerated plants were successfully hardened in a mixture of soil and cow dung in ratio of 3:1 and transferred to field. Cent percent plants survived in field condition, morphologically identical to parent plant and showed normal flowering.

Open Access Original Research Article

3D Structure Modeling of Human Telomere Repeat Binding Factor 2 and DNA-Protein Docking Studies

Koel Mukherjee, Dev Mani Pandey, Ambarish Saran Vidyarthi

Biotechnology Journal International, Page 81-95
DOI: 10.9734/BBJ/2014/6381

Aims: Human telomere repeat binding factor (hTRF2) is a double stranded telomere binding protein that plays key role in protecting the chromosome ends and a necessary building block of telomere structure maintenance. The aim of the present study was to focus on the modeling of 3D structure of hTRF2 (500 residues long) and its interaction studies with DNA in silico.

Study Design: The overall work was designed in different steps starting with the modeling of the concerned protein, its physiochemical properties study, modeling of 3D-DNA with specific length and varying bend angle, docking studies of modeled DNA and hTRF2 protein.

Place and Duration of Study: Bioinformatics Lab, Department of Biotechnology, Birla Institute of Technology, Mesra, India. November 2012- July 2013.

Methodology: 3D structure of hTRF2 was modeled through I-TASSER method. The modeled structure was verified by 5ns of simulation run in solvent (water) condition and also evaluated with different bioinformatics tools. Physiochemical properties were calculated through CLC Protein Workbench.  DNA 3D structure was modeled with the conserved nucleotide sequence motif, TTAGGG with varying bend angles of 0° to 60°. The DNA-protein docking studies were carried out through HADDOCK easy interface for each bend angle.

Results: The best model was selected depending on minimum RMSD value and C-Score and the Stereochemical quality of that model was verified with different tools, as the Molprobity score (>1) of hTRF2 was predicted 4.2 and Ramachandra favored residue was 80.56%. The selected model protein and DNA structure was docked and among all the docking results the best orientation of DNA and hTRF2 was at 60° DNA bend angle with lowest RMSD and maximum Z-value. The amino acids which are directly involved in the interaction were also selected.

Conclusion: In future further study will be planned with further bend angle for getting better information on DNA-protein interactions. In silico studies will also be helpful for the researchers to study the complex structure in vitro.

Open Access Original Research Article

In vitro Propagation and GC-MS Studies of Ocimum basilicum Linn. var. pilosum (Willd.) Benth

Gaddaguti Venu Gopal, Srideepthi Repalle, Venkateswara Rao Talluri, Srinivasa Reddy Ronda, Prasada Rao Allu

Biotechnology Journal International, Page 96-107
DOI: 10.9734/BBJ/2014/6225

An in vitro propagation method is outlined for Ocimum basilicum Linn. var. pilosum (Willd.) Benth., a wild aromatic plant belongs to Lamiaceae family. Shoot buds were used as source of explants on MS media supplemented with different concentrations of growth regulators for callus growth, induction of multiple shoots and roots respectively. MS media with 1.5 mg/L of kinetin and 0.5 mg/L of NAA showed 95.5% shooting, maximum number of shoots (7.33) and relatively better shoot lengths (4.15 cm). Excised shoots were carefully transferred to half-strength MS medium supplemented with 1.0 mg/L indole-3-butyric acid (IBA) for root induction and it yields 86.6% rooting.  Whereas, average root length and number of roots observed were 1.73 cm and 3.31 respectively per explants. Rooted plantlets were hardened and successfully established in natural soil, where they grew and matured normally. GC-MS studies in methanolic leaf extract of naturally grown Ocimum species yielded 15 compounds.  Two compounds viz. cis-9-Hexadecenal (35.06%) and n-Hexa decanoic acid (21.6%) accounted for the major share (56.66%). On the other hand, 2-hydroxy-6-methylbenzaldehyde (10.99%) as well as 4H-1-Benzopyran-4-one and 5-Hydroxy-6, 7-Dimethoxy-2-(4-Methoxyphenyl) (7.75%) represented only 18.74%. Establishment of reliable in vitro propagation protocol, with phytochemical profile of hitherto unreported Ocimum species further widens the scope to evaluate its therapeutic properties.