Open Access Original Research Article

Partial Purification of Cellulase Enzyme from Fusarioum oxysporium by Using Egg White Matrix

Jagdish Singh, Manpreet Kaur

Biotechnology Journal International, Page 108-115
DOI: 10.9734/BBJ/2014/6772

Aim: Protein matrix possesses a capacity to bind organic and inorganic matter. Egg white matrix as an adsorbent was utilized for partial purification of cellulase enzyme in detailed batch study.

Methodology: Effect of pH, matrix size, enzyme concentration, contact time and temperature on the binding of cellulase on egg white matrix was studied. Desorption studies was carried with variable pH, sodium chloride and ammonium sulphate system to desorbs the cellulase for the egg white matrix.

Results: Equilibrium isotherms for the adsorption of the cellulase enzyme on egg white matrix were well fitted to Freundlich and Langmuir isotherm models. The adsorption process has been found endothermic in nature and thermodynamic parameters, Gibb’s free energy (∆Gº), change in enthalpy (∆Hº) and change in entropy (∆Sº) have been calculated as -13276.21, 2025.47 and 698.64 (KJ mol-1) respectively. Thermodynamic studies revealed that the adsorption process is spontaneous and endothermic in nature. In desorption studies it was concluded that maximum elution (66.8% was obtained for cellulase enzyme with 0.3M (NH4)2SO4 with respect to different pH and sodium chloride solution. Cellulase enzyme obtained after desorption process has maximum purification fold of 4.25 with 60% recovery.

Conclusion: The results from the current research suggest that egg white can be used for partial purification of cellulase enzyme with less cost.

Open Access Original Research Article

Agrobacterium-Mediated Transfоrmation of Maize with Antisense Suppression of the Proline Dehydrogenase Gene by an In Planta Method

Y. M. Moiseeva, V. A. Velikov, I. V. Volokhina, Yu. S. Gusev, O. S. Yakovleva, M. I. Chumakov

Biotechnology Journal International, Page 116-125
DOI: 10.9734/BBJ/2014/6504

Aims: The aim of this study was to estimate the efficiency of T-DNA transfer during Agrobacterium-mediated transformation of maize (Zea mays L.) at different temperatures. In addition, the way of T-DNA transfer was studied after application of an Agrobacterium suspension at maize pistil filaments.

Study Design: Transgenic maize plants were obtained with an antisense suppressor of the proline dehydrogenase gene (ASPG) by using the binary vector pBi2E. Temperatures of 28-35ºC were used to establish suitable conditions for transformation in planta.

Place and Duration of Study: Laboratory of Bioengineering, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences; between May 2008 and May 2013.

Methodology: A. tumefaciens strain LBA4404 (pBi2E), containing the marker gene and the ASPG from Arabidopsis thaliana was used for maize transformation. The presence of T-DNA in the maize genome was detected by PCR. The proline concentration in transgenic hybrids of maize lines was determined colorimetrically.

Results: T-DNA carrying the marker genes (nptII, gus) and the ASPG construct was detected in the maize genomes after Agrobacterium-mediated transformation. PCR analysis of total DNA isolated from 409 kanamycin-resistant diploid F1 seedlings revealed T-DNA insertions in the genomes of 30 plants. Expression of the ASPG in the maize genome led to a 4.5-fold increase (P=0.05) in free proline content in the transformed plants. Temperatures above 3ºC blocked the T-DNA transfer.

Conclusion: The transfer of the ASPG by Agrobacterium T-DNA into the maize genome was achieved with a frequency of 0.3-2.3% at temperatures not higher than 31ºC. The PCR-positive maize plants had a statistically significant increase in the proline concentration in leaf tissues as compared with the non-transformed control. T-DNA may be transported into the maize egg cell by the growing pollen tube after the pistil filaments are inoculated with an Agrobacterium suspension.

Open Access Original Research Article

Heavy Metals Concentration in Fish Mugil cephalus from Machilipatnam Coast and Possible Health Risks to Fish Consumers

P. V. Krishna, K. Madhusudhana Rao, V. Swaruparani, D. Srinivas Rao

Biotechnology Journal International, Page 126-135
DOI: 10.9734/BBJ/2014/4317

Heavy metals are dangerous to aquatic organisms and it can be bioaccumulated in the food chain leading to diseases in humans. Cumulative effects of metals or chronic poisoning may occur as a result of long term expore even to low concentrations. The accumulation of heavy metals conditions depending upon the species, environmental conditions and inhibitory processes. Considering the human health risk due to the consumption of fish, the concentration of heavy metals (Zn, Pb, Mn, Cu, Cr and Hg) are investigated in fish samples collected from the Machilipatnam coast. The fish was examined for metal constituents are the basis on the human nutrition in the study area. These metal concentrations were exceeding the limits set by the world health organization (WHO). The study provides an insight into the potential impact of increased levels of metals in the environmental as well as estimated of the contaminated of fish tissues with metals.

Open Access Original Research Article

Effect of Desiccation and Chilling Treatment on Somatic Embryo Development and Germination in Rough Lemon (Citrus jambhiri Lush)

Balwinder Singh

Biotechnology Journal International, Page 136-148
DOI: 10.9734/BBJ/2014/6241

The goal of this study was to evaluate the effect of desiccation and chilling treatments on somatic embryogenesis of rough lemon (Citrus jambhiri Lush.). Styles were cultured on seven culture media (MS I-MS VII) containing Benzylaminopurine (BAP), Kinetin (KN) and Malt Extract for cell proliferation and somatic embryo development.  Cell proliferation was maximum on MS IV media but maximum cultures showing somatic embryogenesis (52.08 %) was observed on MS VII media. Embryogenic callus proliferated on MS VII media was subjected to desiccation and chilling treatment for 24, 48, 72 and 96 hours. Embryogenic callus desiccated for 24 and 48 hours in sterile petriplates showed 58.33 and 56.94 % somatic embryogenesis respectively as compared to undesiccated callus (51.98%). Average number of cotyledonary embryos (6.80/culture) in embryogenic cultures from desiccated callus (48 hrs) was more as compared to untreated callus (2.26/culture). There was significantly less number of abnormal embryos (0.60-0.53/culture) in desiccated callus for 48, 72 and 96 hours as compared to untreated callus (7.20/culture). Chilling treatment also improves the average number of cotyledonary embryos and reduces the abnormal development of embryos. Among all the treatments desiccation of embryogenic callus for 48 hrs proved beneficial for improvement of somatic embryo development and germination.

Open Access Original Research Article

Histopathological Effects of Glyphosate and Its Toxicity to the Earthworm Nsukkadrilus mbae

Oluah Ndubuisi Stanley, Ochulor Amarachi Joy

Biotechnology Journal International, Page 149-163
DOI: 10.9734/BBJ/2014/6727

This study was carried out to investigate the toxicity of Roundup, aglyphosate-containing herbicide to the earthworm Nsukkadrilus mbae and the associated histological changes. One hundred and sixty five earthworms were randomly divided into five groups and exposed to different concentrations of glyphosate (0.0, 13.0, 52.0, 117.0 and 207 mg/Kg soil) for 96 h. The mortality was recorded every 24h and after 96 h, sections of the earthworm from each treatment group were prepared for microscopic examination. There was no mortality in the control group and the survivors in the treatment groups were weak. The percentage mortality was least after 24 h and highest after 96 h and the mortality was significantly different (P = 0.05) in the groups except after 24h. The survival rate was generally concentration and duration dependent. The 96 h LC50 value was 10.4 mg/Kg soil. The observed histopathological changes included rupture of the cuticullar wall, tissue necrosis and prominent vacuolations. The circular and longitudinal muscle layers had altered structural integrity. These histopathological end points could be useful biomonitors of glyphosate toxicities in ecotoxicological studies involving earthworms.

Open Access Original Research Article

In vitro Propagation of Caralluma diffusa (Wight) N.E.Br

K. Kalimuthu, K. Kalaiyarasi, R. Prabakaran, T. Sasikala

Biotechnology Journal International, Page 164-172
DOI: 10.9734/BBJ/2014/7488

An efficient protocol is described for the rapid in vitro multiplication of an endangered medicinal plant, Caralluma diffusa, via enhanced axillary bud proliferation from nodal explants collected from young shoots of six-months-old plant. The physiological effects of growth regulators Benzylamino purine (BAP), Thidiazuron (TDZ), Kinetin (KIN), α-Naphthalene acetic acid (NAA) full strength of Murashige and Skoog’s (MS) medium on in vitro propagation were investigated. The highest number of shoots in initiation (4.16±0.30) and in subculture (17.33±0.33) and the maximum average shoot length (4.17±0.03cm) were recorded on MS medium supplemented with BAP (4.44 μM) and TDZ (0.90 μM) at pH 5.8. Rooting was best achieved on MS medium augmented with NAA (5.37 μM). The plantlets regenerated in vitro with well-developed shoot and roots were successfully established in pots containing decomposed coir waste, vermiculite and garden soil (1:1:1ratio) and grown in a shade house with 93.83% survival rate.

Open Access Original Research Article

Exploration of Actinobacteria from Mangrove Ecosystems of Nizampatnam and Coringa for Antimicrobial Compounds and Industrial Enzymes

Usha Kiranmayi Mangamuri, Muvva Vijayalakshmi, Sudhakar Poda

Biotechnology Journal International, Page 173-184
DOI: 10.9734/BBJ/2014/6424

Aim: A study was made to examine the kinship between the seasonal distribution of actinobacteria and the physico-chemical properties of the mangrove sediments of Nizampatnam and Coringa located along the South East coast of Andhra Pradesh, India.

Place and Duration of Study: Department of Botany and Microbiology, between April 2010 to February 2011.

Methodology: Seasonal enumeration of actinobacteria from two different stations 1 (Nizampatnam) and 2 (Coringa) accorded by four different pre-treatments of soil sediments followed by plating onto three different media showed high incidence of actinobacteria in the month of February and least in December. Pretreatment with calcium carbonate and plating on starch casein agar yielded maximum number of actinobacteria. The strains were identified based on the morphological characteristics such as aerial mycelium, substrate mycelium, diffusible pigments and micro morphological features.

Results: The present investigation revealed that majority of the mangrove actinobacteria (69%) belongs to Streptomyces spp. Among the 55 isolates screened for antimicrobial compounds, 28 were found to be potential producers. The isolates could also produce commercially important enzymes such as L-asparaginase, cellulase and amylase. In addition the statistical study also revealed that positive correlation between the distribution of the actinomycetes and influence of physico-chemical parameters and the organic matter of the soil.

Conclusion: Our study revealed that the unexplored regions like Nizampatnam and Coringa mangrove ecosystems are proved as potential sites for antimicrobial and industrial enzyme producing actinobacteria.

Open Access Original Research Article

Structural Analysis and Docking of Stilbene Synthase Protein from Chinese Grape Vine Vitis pseudoreticulata

K. Divya, G. Venkata Ramana, K. V. Chaitanya

Biotechnology Journal International, Page 185-203
DOI: 10.9734/BBJ/2014/3835

Aims: The present work aims to perform the molecular modeling of stilbene synthase protein from Chinese grape vine Vitis pseudoreticulata.

Place and Duration of Study: The study has been performed in the Department of Biotechnology, GITAM Institute of Technology, GITAM University, Visakhapatnam, India for a period of 8 months.

Methodology: The sequence of Vitis STS protein was obtained by BLAST search from DFCI web server using Arabidopsis Stilbene synthase sequence. To read the amino acid pattern among these sequences, Multiple Sequence alignment have been performed using clustal W. The secondary and 3D structures were predicted for the protein and the stability of the structures was determined through Ramachandran plot and PROSA analysis. 3D structure obtained using Swiss model workspace was utilized for docking studies.

Results: In the multiple sequence alignment except Gossypium and Ipomea remaining sequences were aligning well. The secondary structure of the protein is possessing helices, coils and sheets respectively and most of the protein structure is coiled. The predicted model was subjected to evaluation by PROSA with a Z score of -10.1. Ramachandran plot revealed that the predicted that 96.6% residues were in favoured region, 2.6% were in allowed region and 0.8% were in outlier region proving that the predicted model is acceptable. Docking STS protein with secondary metabolite ligands elucidated that anethole, ascorbic acid and arbutin have good binding affinity.

Conclusion: The structural model of Vitis pseudoreticulata stilbene synthase has been determined, and in silico docking studies have elucidated that this protein has docked with some of the essential secondary metabolites like anethole, ascorbic acid and arbutin which might enhance the performance when they enter into a biological system.

Open Access Original Research Article

Listeria spp. in Raw Cow and Goat Meat in Port Harcourt, Nigeria

O. C Eruteya, S. A Odunfa, J. Lahor

Biotechnology Journal International, Page 204-214
DOI: 10.9734/BBJ/2014/6750

Aims: To determine the occurrence of Listeria monocytogenes and other Listeria spp. in raw cow and goat meat.

Study Design: This work was based on a completely randomized design with two replication and the average values calculated for mean comparison.

Place and Duration of Study: Department of Microbiology, University of Port Harcourt, Nigeria and Nigeria Institute of Medical Research, Yaba, Lagos, Nigeria. Isolation of Listeria spp. was between March 2011 and February 2012.

Methodology: In this study, a total of 240 raw cow and goat meat samples were analyzed for the presence of Listeria monocytogenes and other Listeria species. The techniques recommended by the United State Department of Agriculture (USDA) revised and the Health Products and Food Methods of the Government of Canada were employed using Fraser broth and polymixin acriflavin lithium chloride ceftazidime aesculin mannitol (PALCAM) agar.

Results: The results of conventional and polymerase chain reaction (PCR) characterization of the isolates revealed that 81 samples (33.75%) were positive for Listeria spp. The highest prevalence of Listeria was found in cow flesh samples (19 of 36 samples; 52.78%) followed by cow intestine samples (8 of 20; 40%) and least was goat kidney samples (5 of 28; 17.86%). Out of the 310 characterized Listeria spp., L. monocytogenes were 4(1.29%). Other species isolated were L. innocua 20(6.45%), L. ivanovii 4(1.29%), L. seeligeri 72 (23.23%), L. welshimeri 139 (44.84%) and L. grayi 71(22.90%). No L. monocyogenes was isolated from cow samples. A higher incidence was noted during the raining season 216 (69.68%) than the dry season 94(30.32%).

Conclusion: This study demonstrated the occurrence and distribution of Listeria species in retail raw cow and goat meat in Port Harcourt, Nigeria.

Open Access Original Research Article

Ficus religiosa L.: Callus, Suspension Culture and Lectin Activity in Fruits and In vitro Regenerated Tissues

Varsha Parasharami, Priya Yadav, Shilpa Mandkulkar, Sushama Gaikwad

Biotechnology Journal International, Page 215-227
DOI: 10.9734/BBJ/2014/7338

Ficus religiosa L. is a tree of immense cultural heritage in Asian countries. It is respected by followers of many religions and faiths. Fruits of Ficus religiosa L. are the ‘figs’ and possess many medicinal properties reported in ethnomedicinal and pharmacological studies. These medicinal properties range from antidiabetic, anticancer, anticonvulsant, antimicrobial, antiviral and antioxidant activities. In the figs, pollination takes place with ‘wasp’. Till date no work on ‘fruit tissue culture’ has been reported in this species. For the first time the callus cultures have been developed using ‘fig fruits’. Fruit callus was multiplied on solid medium using 2 mm to 3 mm diameter fruits. During the present study, lectin/hemagglutinin activity was detected in fruits and fruit callus extracts for the first time. Both In vivo fruits (figs) and In vitro fig callus were used to assay the hemagglutinin activity using pronase treated and untreated rabbit blood erythrocytes. Fruit extract showed 4-8 times more hemagglutination activity in presence of pronase treated erythrocytes. It is the first report of callus/suspension culture and detection of thermostable (up to 70ºC) hemagglutinin/lectin from fruits in this species. Preliminary biochemical characterization of the lectin activity e.g. metal ion requirement, EDTA, pH and temperature stability was carried out during the present study.