Open Access Short Research Article

Domain Analysis and Isolation of Coniferyl Alcohol Dehydrogenase Gene from Pseudomonas nitroreducens Jin1

Praveen P. Balgir, Dinesh Kalra

Biotechnology Journal International, Page 684-695
DOI: 10.9734/BBJ/2014/7877

Aim: The Aim of present study is to analyse conserved functional Short Dehydrogenase        Reductase (SDR) domain from bacteria. Based on the domain analysis selection of coniferyl alcohol dehydrogenase gene for isolation from Pseudomonas nitroreducens Jin1.

Place and Duration of Study:  Department of Biotechnology, Punjabi University, Patiala. From July, 2012 to November, 2012.

Methodology: Bioinformatics tools were used to analyse various calA genes from bacteria based on the presence of conserved domain in members of SDR family. Based on insilico analysis, Pseudomonas nitroreducens Jin1 calA was selected. PCR was used for amplification of the gene from the genome of Pseudomonas nitroreducens Jin1.

Result: Multiple sequence alignment results for conserved domains amongst members of SDR family identified presence of all domains of Short Dehydrogenase Reductase members in Pseudomonas nitroreducens Jin1 calA gene. Amongst the various sequences compared the P. nitroreducens Jin1, calA was found to be the smallest in size. The locus of calA in the genome resides at 103513-104280 bases. It was amplified from the genome of Pseudomonas nitroreducens Jin1. The calA gene that was amplified is of size 768bp.

Conclusion: calA gene isolated from Pseudomonas nitroreducens Jin1 is a small gene with all the functional domains and can be used for biotransformation of coniferyl alcohol to coniferyl aldehyde.

Open Access Short Research Article

Production and Evaluation of Biosurfactant by Serratia marcescens UCP 1549 Using Industrial Wastes

T. S. Alves, J. P. Salgado, R. F. S. Andrade, D. Montero-Rodríguez, W. B. Ferreira, M. M. Almeida, G. M. Campos Takaki, H. W. C. Araújo

Biotechnology Journal International, Page 708-719
DOI: 10.9734/BBJ/2014/9774

Production and Evaluation of Biosurfactant by Serratia marcescens UCP 1549 Using Industrial Wastes

Aims: Biosurfactants are surface-active agents of microbial origin that have a property of lowering the surface tension between two liquids. This study aimed to the production of biosurfactant by Serratia marcescens UCP 1549 in medium containing agro-industrial wastes for making possible its industrial application in the near future and to propose its environmental applications.

Place and Duration of Study: Center of Science and Technology  from State University of Paraíba - UEPB, Campina Grande – PB, Brazil and Nucleus of Research in Environmental Sciences and Biotechnology (NPCIAMB), Catholic University of Pernambuco - UNICAP, Recife-PE, Brazil between June 2011 and July 2012.

Methodology: According to the proposed factorial design, the culture medium was developed and used for the production of biosurfactant and incubated at 28ºC, 155 rpm during 48 h. The produced biosurfactant was evaluated by emulsifying index, emulsifying activity and surface tension using hydrophobic substrates as vegetable oils after frying, n-hexadecane and diesel.

Results: The best results for emulsification index were found between 79.92 and 100% of emulsification and for activity were values between 2.304 and 5.000 EA (emulsification activity) using vegetable oil after frying as substrate. In relation of the surface tension, the best value obtained was 33.10mN/m in the condition of the central point of the experimental design.

Conclusion: The results show that Serratia marcescens UCP 1549 was capable of producing a biosurfactant with emulsifying property from industrial wastes in the studied conditions in this work.

Open Access Original Research Article

Effects of External Carbon Source Concentration on Sulphate Removal by Selected Bacterial and Fungal species

O. B. Akpor, S. O. Dahunsi, R. Aransiola

Biotechnology Journal International, Page 640-648
DOI: 10.9734/BBJ/2014/10664

The aim of this study was to investigate the role of initial external carbon source concentration on sulphate removal by four bacterial and four fungal isolates under shake flask conditions. The test wastewater was filtered and supplemented with sodium acetate as the external carbon source at respective concentrations of 5g/L, 10g/L, 15g/L and 20g/L, before dispensing in 200mL quantity in 250mL capacity conical flasks, sterilised and inoculated with the test microbial isolates. Prior inoculation and at 24h interval, for 96 h for the estimation of sulphate concentration in the wastewater using standard methods. The results revealed remarkable sulphate removal in the absence of the sodium acetate and on its 5g/L addition. An increase in the concentration of the sodium acetate caused a corresponding decrease in the level of sulphate removal. Percentage sulphate removals in presence of the test isolates were observed to range from 47.01 to 57.81%, 18.66 to 51.66%, -1.64 to 11.03%, 5,38 to 22.37% and -3.59 to 5.18%, at sodium acetate concentrations of 0g/L, 5g/L, 10g/L, 15g/L and 20g/L, respectively. This trend was irrespective of the isolates used for investigation. The study was able to provide an insight to the role of carbon concentration on sulphate removal by the test microbial isolates.

Open Access Original Research Article

Histopathological, Hemochromatotic, Hypercholesterolemic, and Androgenic Effects of Escravos Crude oil on the Testis in Male Chinchilla Rabbits

J. O. Okoye, T. Ude, S. N. Ibekailo, C. J. Awalu

Biotechnology Journal International, Page 649-658
DOI: 10.9734/BBJ/2014/10808

Histopathological, Hemochromatotic, Hypercholesterolemic, and Androgenic Effects of Escravos Crude oil on the Testis in Male Chinchilla Rabbits

Aim: This study aimed to investigate the effects of Escravos crude oil on serum concentrations of testosterone, total cholesterol, body weight, relative weight and micro-anatomical architecture of the testis using male Chinchilla Rabbits.

Place and Duration of Study: This study was carried out at the Department of Medical Laboratory Science, Nnamdi Azikiwe University, Nnewi Campus between May and June 2013 (28 days). 

Methodology: A total of thirty male Chinchilla Rabbits aged 12 to 14 weeks and weighing 1.2kg to 1.45kg was used. Crude oil was administered orally at the doses of 15, 20, 25 and 30mg/kg body weight to groups designated B, C, D and E respectively for 28 days, while group A was given normal saline. Serum concentration of testosterone and total cholesterol were estimated using the microplate enzyme immunoassay and enzymatic end point methods, respectively. The SPSS software (version 20) was used for the statistical analysis and the result expressed in mean ± SD.

Result: The results showed dose dependent effects on the hormone and biochemical assays, especially at the concentration of 25mg/kg body weight of the administered crude oil. Significant increases in serum concentrations of testosterone (0.32+/-0.05 to 0.46+/- 0.14) and total cholesterol (1.35+/-0.17 to 1.76+/-0.15) concentrations (p≤.05), and insignificant increase (p≥.05) in the relative weight of the testis (4.80+/-0.40 to 6.50+/- 0.90) were observed. The histology of the testes revealed hypertrophy of the seminiferous tubules, atrophy of the basal lamina and interstitial cells, and hemochromatosis. The histological findings agree with the hormonal and biochemical findings.

Conclusion: These findings suggest that Escravos crude oil might be a potential endocrine disruptor and toxic substance which can affect the micro-anatomical architecture of the testis.

Open Access Original Research Article

Integration of Reticuloendotheliosis Virus in Most of Tanzanian Fowl Pox Virus Isolates is not attributed to Imported Commercial Fowl Pox Vaccines

S. N. Masola, A. Mzula, C. J. Kasanga, P. N. Wambura

Biotechnology Journal International, Page 659-669
DOI: 10.9734/BBJ/2014/10703

Integration of Reticuloendotheliosis Virus in Most of Tanzanian Fowl Pox Virus Isolates is not attributed to Imported Commercial

Fowl Pox Vaccines

Aim: To investigate integration of reticuloendotheliosis virus (REV) in the Tanzanian fowlpox virus (FWPV) field isolates, and the imported commercial fowl pox vaccines currently used in the country.

Study Design:  Experimental.

Place and Duration of Study: Faculty of Veterinary Medicine, Sokoine University of Agriculture, Morogoro, Tanzania; between June 2012 and October 2013.

Methodology: Fifty five samples of FWPV isolates from naturally infected chickens, and two isolates of FWPV from samples of the imported commercial fowl pox vaccines were analyzed for integration of REV envelope (env) gene and REV 5'long terminal repeat (5'LTR). The study involved polymerase chain reaction (PCR) amplification of FWPV P4b gene, REV env gene, and REV 5'LTR; agarose gel electrophoresis of PCR products, purification of PCR products, sequencing of the purified PCR products, and sequence analysis using standard procedures. 

Results: Out of 55 analyzed field isolates 53 (96.36%) were found to have REV inserts. Most of them [38 (69.09%)] contained both REV env gene and REV 5'LTR inserts, 10 (18.18%) contained inserts of REV env gene only, and 5 (9.09%) contained inserts of REV 5'LTR only. Two isolates (3.64%) were found to be integrated with neither REV env gene nor REV 5'LTR. None of the screened FWPV isolates from the imported commercial vaccines was found to have REV inserts. Sequence analysis revealed that genomic fragments of REV integrated in the Tanzanian FWPV isolate were closely related (99–100% identity) to REV sequences integrated in FWPV isolates from other countries.     

Conclusion: Currently there is a heterogeneous population of FWPV in Tanzania comprising of REV-integrated FWPV strains and REV-free FWPV strains. Since strain(s) of REV-integrated FWPV are more virulent than strain(s) of REV-free FWPV, further studies on the REV-integrated Tanzania FWPV isolates aiming at obtaining the appropriate isolate for development of autogenous fowl pox vaccine are highly recommended.

Open Access Original Research Article

Isolation and 16S rRNA-Based Identification of Benomyl-Degrading Bacteria

Adil A. El-Hussein, Randa H. Elsalahi, Awad G. Osman, Ashraf M. Sherif, Marmar A. El Siddig

Biotechnology Journal International, Page 670-683
DOI: 10.9734/BBJ/2014/10633

Laboratory experiments were conducted to isolate and identify Benlate- (Benomyl) degrading microorganisms from two soil types collected from different locations in Khartoum State, Sudan. Benomyl degradation was studied at two temperatures (28 and 40ºC) in soil treated with three Benomyl concentrations (0.032, 3.2 and 8.0mg Benomyl/g soil) and incubated for 360 days. Potential degraders were also tested in mineral salt liquid medium using Benomyl as a sole carbon source. Degradation percentages were then determined and the most efficient Benomyl degraders were identified by amplification with 16S rRNA gene, sequencing and alignment with deposited sequences in the international gene bank. A total of 64 isolates were recovered from the two soil types, with 59 (92.2%) isolates recovered from the clay soil. Thirty four isolates were recovered from clay soil treated with 8.0mg Benomyl/g soil and incubated at 28ºC. The most efficient Benomyl degraders, with degradation percentages in the range of 44-59, were identified as Pseudomonas stutzeri (Two different isolates), Pseudomonas putida, Acinetobacter johnsonii, Brevibacillus invocatus, Bacillus clausii, Lysinibacillus sp. and Agrobacterium radiobacter.

Open Access Original Research Article

Synseed Production in Spilanthes mauritiana DC. for Short-Term Storage and Germplasm Exchange

Shiwali Sharma, Anwar Shahzad

Biotechnology Journal International, Page 696-707
DOI: 10.9734/BBJ/2014/9652

Aims: The present study provides an efficient protocol for short-term storage and germplasm exchange of a potent medicinal herb, Spilanthes mauritiana using encapsulated nodal segments.

Study Design: For in vitro conversion of synseeds, 5 beads were placed in each flask having Murashige and Skoog (MS) medium supplemented with different combinations of plant growth regulators (PGRs). While for ex vitro conversion, 5 synseeds per thermocol cups having different planting substrates were directly sown. The data for each experiment were collected after 6 weeks. All the experiments were conducted with a minimum of 20 replicates per treatment and each experiment was repeated thrice.

Place and Duration of Study: Plant Biotechnology Lab, Department of Botany, AMU, July 2012 to November 2013.

Methodology: Concentration of two different manufacture grade of Na-alginate (purchased from Central Drug House and Loba Chemie) were compared for the production of ideal synseeds. Conversion of synseeds was tested under in vitro and ex vitro conditions. A low temperature storage (4ºC) experiment was also carried out to understand the explants’ ability to revive physiological activity leading to plantlet development.

Results: A gelling matrix of 4% Na-alginate (CDH) or 3% (Loba Chemie) with 100 mM calcium chloride (CaCl2∙2H2O) was found most suitable for the production of ideal Ca-alginate bead. However, CDH grade Na-alginate (74.4% conversion) was found to be better than Loba Chemie (62% conversion) in terms of in vitro conversion of synseeds into complete plantlets when cultured on MS basal medium. Supplementation of Plant Growth Regulators (PGRs) to the MS basal medium further enhanced the conversion frequency of the synseeds. Maximum conversion (83%) was recorded on MS basal medium supplemented with 5.0 µM 6-benzyladenine (BA) and 0.5 µM indole-3-acetic acid (IAA). Synseeds, stored at 4ºC for 1-8 weeks showed successful sprouting with variable percent in successive weeks of transfer, followed by development into complete plantlets when returned to regeneration medium. Ex vitro conversion of synseeds also occurred when synseeds were sown directly into SoilriteTM moistened with quarter-strength MS salts. Plantlets regenerated from non-stored and stored synseeds were successfully hardened, acclimatized and established in soil with a success of 90%. While plantlets recovered from direct sowing of synseeds exhibited 80% survivability.

Conclusion: Being small in size, synseeds provide an effective tool for conservation, storage and exchange of this valuable medicinal plant species, potentially overcoming many of the difficulties associated with long-distance transport of plant germplasm.

Open Access Original Research Article

Arachis hypogaea L. Direct Organogenesis and SDS - PAGE Analysis in Groundnut

S. Palanivel, S. Parvathi, V. Muniappan, C. Deepa, A. Abuthahir

Biotechnology Journal International, Page 720-732
DOI: 10.9734/BBJ/2014/9237

The present research work was carried out to observe the effect of various concentrations of Benzyl amino purine (BAP) in terms of multiple shoot induction with special emphasis on qualitative and quantitative changes of proteins during multiple shoot formation. From whole embryonated cotyledons of groundnut. The shoot induction medium containing different concentrations of Benzyle amino purine ranged from 5.0 to 25.0mg/l along with lower concentration of Indole acetic acid (IAA).The multiple shoots were observed in all the BAP concentrations at a varying levels. Among the various levels of BAP, 25.0mg/l of BAP plus 0.50mg/l of IAA was found to be the most effective in terms of multiple shoot induction. The growth parameters like plantlet height, fresh and dry weight also highly influenced by the concentration of the BAP. The root induction was achieved in micropropagated shoots by using Indole butyric acid (IBA) at a concentrations of 1.0 to 5.0mg/l. The mean number of roots, root length was highly influenced by IBA. In all the concentrations of IBA the rooting was associated with moderate and heavily associated basal callusing. The quantitative estimation of protein content in regenerated shoots also increased with higher concentrations of BAP. The SDS-PAGE analysis showed that there was a nine visible bands with 82.2, 76.4, 57.5, 27.6, 23.7, 21.4, 18.2, 16.5 and 12.8 kDa were observed in multiple shoots derived from all the concentrations of BAP. Whereas in whole embryonated cotyledonary explants there was a entirely different protein banding pattern with 82.2, 76.4, 57.5, 27.6, 23.7, 21.4, 18.2, 16.5 and 12.8 kDa were recorded. 

Open Access Original Research Article

Rapid micropropagation of Cucumis sativus var. Dastgerdi (Iranian cultivar) by Node Culture Technique

Otroshy Mahmoud, Mokhtari Arash

Biotechnology Journal International, Page 733-739
DOI: 10.9734/BBJ/2014/9476

Rapid micropropagation of Cucumis sativus var. Dastgerdi (Iranian cultivar) by Node Culture Technique

Tissue culture can be used in developing an efficient method for production of valuable and enduring indigenous cultivars. A short-term protocol for plantlet proliferation by using nodal segments was developed for Cucumis sativus, local cultivar from Isfahan; known as Dastgerdi as a tolerant cultivar to the root-knot nematode. Nodal explants were cultured on MS medium containing various concentrations of KIN (0, 0.5, 1 and 1.5mg.l-1) in combination with IBA (0, 0.025 and 0.5mg.l-1). The best response for proliferation rate (100%), mean number of nodes per plantlet (6.01) and percentage of rooted In vitro propagated shoots were observed on medium supplemented by KIN (1.5mg.l-1) after about 3-4 weeks. The maximum length (5.43cm) of micro-shoots obtained on MS medium with KIN (1mg.l-l) + IBA (0.025mg.l-1).

Open Access Original Research Article

Mass Spectrometric Determination of the Primary Structure of β Amylase Isolated from Amylolytic Nigerian Maize Cultivar

O. A. Awoyinka, O. Daini, K. Satisha, Dipankar Chatterji

Biotechnology Journal International, Page 740-750
DOI: 10.9734/BBJ/2014/10063

Mass Spectrometric Determination of the Primary Structure of β Amylase Isolated from Amylolytic Nigerian Maize Cultivar

This study was carried out after a five day germination period on TZEE*TZEE-W*DEMARSCUS*TZEE-W one of the most recommended high amylolytic Nigerian maize cultivar. Purification steps comprising of fractional precipitation by ammonium sulphate, gel filtration and anion exchange chromatography, was used respectively to purify β amylase (EC 3.2.1.2) from the malt. The sensitivity of the beta amylase indicated that it has serine at its active site. An apparent 60KDa monomeric protein was detected by one dimensional sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE). Identity assigned to the purified protein by Matrix-assisted laser desorption ionization time –of- flight mass spectrometry ( MALDI-ToF) reference to electronic protein data base is a 58542Da high putative beta amylase – Q9AV88-ORSA from Oryza sativa. Complete primary structure thereafter deduced with the aid of MS/MS MALDI- ToF showed a signature of a highly conserved ubiquitous not yet reported beta amylase. This study paved an insight to the gene encoding the β amylase in TZEE*TZEE-W*DEMARSCUS*TZEE-W and better understanding of the activity of the enzyme at molecular level.