Indian Journal of Experimental Biology

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VOLUME 52

NUMBER 11

NOVEMBER 2014

CODEN: IJEB (A6) 52 (11) 1017-1152 (2014)

ISSN: 0019-5189 (Print); 0975-1009 (Online)

CONTENTS

 

Special Issue on Advances in Biotechnology and Bioinformatics

Preface

1019

Neelu Nawani, Ashok Pandey, Samir Khanal & Parmeswaran Binod

 

 

 

Enzyme Technology

 

 

 

Production, purification and properties of fungal chitinases—A review

1025

 

 

Narayanan Karthik, Karthik Akanksha, Parameswaran Binod & Ashok Pandey

 

 

 

Purified dextransucrase from Pediococcus pentosaceus CRAG3 as food additive

1036

 

 

Rishikesh Shukla & Arun Goyal

 

 

 

Kinetic constraints and features imposed by the immobilization of enzymes onto solid matrices:
A key to advanced biotransformation

1045

 

 

A Foukis, P-Y Stergiou, M Filippou, M Koukouritaki, M Parapouli, L G Theodorou,
E Hatziloukas, A. Afendra, A. Pandey, E M Papamichael

 

 

 

Environmental Biotechnology

 

 

 

Differential toxicological endpoints of di(2-ethylhexyl) phthalate (DEHP) exposure in MCF-7 and MDA-MB-231 cell lines: Possible estrogen receptor α (ERα) independent modulations

1052

 

 

Mihir Tanay Das, Manoj Kumar Singh & Indu Shekhar Thakur

 

 

 

Industrial Biotechnology

 

 

 

Synthesis, DNA Cleavage and antimicrobial activity of 4-thiazolidinones-benzothiazole conjugates

1062

 

 

Meenakshi Singh, Mayank Gangwar, Gopal Nath & Sushil K Singh

 

 

 

Development of bioconjugate from Streptomyces tyrosinase and gold nanoparticles for rapid
detection of phenol constituents

1071

 

 

Mazhari Bi Bi Zainab, D N Madhusudhan , H Raghavendra, Syed G Dastager & Agsar Dayanand

 

 

 

 

Dilute acid pretreatment and enzymatic hydrolysis of sorghum biomass for sugar recovery—
A statistical approach

1082

 

 

Karthik Akanksha, Arjun Prasad, Rajeev K Sukumaran, Madhavan Nampoothiri K, Ashok Pandey, Rao S S & Parameswaran Binod

 

.

Medical Biotechnology

 

 

 

Assessment of phagosomes infected with Mycobacterium tuberculosis as a vaccine candidate
against tuberculosis

1090

 

 

Anjana Sharma  Pankaj Parihar & Juhi Sharma

 

 

Modulation of small intestinal homeostasis along with its microflora during acclimatization at simulated hypobaric hypoxia

1098

 

 

Atanu Adak, Kuntal Ghosh & Keshab Chandra Mondal

 

 

 

Nanobiotechnology

 

 

 

Development of bioprocess for the production of laccase by Pleurotus ostreatus MTCC 1802 using evolutionary optimization technique

1106

 

 

Jayanti Kumari & Sangeeta Negi

 

 

 

Plant Biotechnology

 

 

 

In vitro regeneration and ploidy level analysis of Eulophia ochreata Lindl

1112

 

 

Varsha Shriram, Vikas Nanekar, Vinay Kumar & P B Kavi Kishor

 

 

 

In vitro callus induction and estimation of plumbagin content from Plumbago auriculata Lam.

1122

 

 

Jyoti Deshpande, Dinesh Labade, Kalaiselvi Shankar, Navin Kata, Manoj Chaudhari, Minal Wani & Madhukar Khetmalas

 

 

 

Mannitol-induced drought stress on calli of Trigonella foenum-graecum L. Var. RMt-303

1128

 

 

Naveen C Pant, Ruchi Agarrwal & Sanjeev Agrawal

 

 

 

Anti-fungal potentials of extracellular metabolites of Western Ghats isolated Streptomyces sp.
NII 1006 against moulds and yeasts

1138

 

 

Himani Jayamurthya, Kuttavan Valappil Sajna, Syed G Dastagar & Ashok Pandey

 

 

 

Potential of Microbispora sp. V2 as biocontrol agent against Sclerotium rolfsii, the causative agent of southern blight of Zea mays L (Baby corn)–in vitro studies

1147

 

 

N N Patil, M S Waghmode, P S Gaikwad, M H Gajbhiye, A B Gunjal, N N Nawani &
B P Kapadnis

 

 

Author Index

Adak Atanu

1098

Afendra A

1045

Agarrwal Ruchi

1128

Agrawal Sanjeev

1128

Akanksha Karthik

1025,1082

 

 

Binod Parameswaran

1025,1082

 

 

Chaudhari Manoj

1122

 

 

Das Mihir Tanay

1052

Dastagar Syed G

1071,1138,

Dayanand Agsar

1071

Deshpande Jyoti

1122

 

 

Filippou M

1045

Foukis A

1045

 

 

Gaikwad P S

1147

Gajbhiye M H

1147

Gangwar Mayank

1062

Ghosh Kuntal

1098

Goyal Arun

1036

Gunjal A B

1147

 

 

Hatziloukas E

1045

 

 

Jayamurthya Himani

1138

 

 

K Madhavan Nampoothiri

1082

Kapadnis B P

1147

Karthik Narayanan

1025

Kata Navin

1122

Kavi Kishor P B

1112

Khetmalas Madhukar

1122

Koukouritaki M

1045

Kumar Vinay

1112

Kumari Jayanti

1106

 

 

Labade Dinesh

1122

Madhusudhan D N

1071

Mondal Keshab Chandra

1098

 

 

Nanekar Vikas

1112

Nath Gopal

1062

Nawani N N

1147

Negi Sangeeta

1106

 

 

Pandey A.

1045

Pandey Ashok

1025,1082,1138

Pant Naveen C

1128

Papamichael E M

1045

Parapouli M

1045

Parihar Pankaj

1090

Patil N N

1147

Prasad Arjun

1082

 

 

Raghavendra H

1071

 

 

S S Rao

1082

Sajna Kuttavan Valappil

1138

Shankar Kalaiselvi

1122

Sharma Anjana

1090

Sharma Juhi

1090

Shriram Varsha

1112

Shukla Rishikesh

1036

Singh Manoj Kumar

1052

Singh Meenakshi

1062

Singh Sushil K

1062

Stergiou P-Y

1045

Sukumaran Rajeev K

1082

 

 

Thakur Indu Shekhar

1052

Theodorou L G

1045

 

 

Waghmode M S

1147

Wani Minal

1122

 

 

Zainab Mazhari Bi Bi

1071

 

Keyword Index

Anti-fungal activity

1138

Anti-oxidants

1128

Apoptosis

1090

 

 

BCG

1090

Benzothiazoles

1062

Bioconjugate

1071

Biocontrol

1138

Biocontrol agent

1147

Biofuel

1082

 

 

Callus culture

1122,1128

 

 

Cell cycle

1052

Cell line

1090

Cellulase

1082

Chitin

1025

Chitinase

1025

 

 

DEHP

1052

Dextransucrase

1036

DNA cleavage

1062

DNA ploidy

1112

Drought stress

1128

 

 

Enzymatic hydrolysis
    Pretreatment


1082

Enzyme kinetics

1045

Epithelial barrier

1098

Esterification

1045

Estrogen receptors-α

1052

Eulophia

1112

EVOP

1106

Extracellular anti-fungal
    metabolites


1138

 

 

Fenugreek

1128

Flow cytometry

1112

Fungal sources

1025

 

 

Gel filtration

1036

Gene cloning

1025

Gold nanoparticles

1071

Growth direction index

1098

 

 

Hypoxia

1098

 

 

Immobilized enzymes

1045

 

 

Label-free quantification

1052

Laccase

1106

 

 

Mannitol

1128

MIC

1062

Microbispora sp.V2

1147

Microflora

1098

Milk

1036

Multiple shoots

1112

Mycobacterium tuberculosis

1090

 

 

Non-solvent systems

1045

Orchids

1112

 

 

Pediococcus pentosaceus

1036

Peroxidases

1128

Phenol detection

1071

Pleurotus ostreatus

1106

Plumbagin

1122

Plumbago auriculata

1122

Polyethylene glycol

1036

Protocorm-like-bodies

1112

Purification and Characterization

1025

 

 

Sclerotium rolfsii

1147

Secretome

1052

Small intestine

1098

Solid sate fermentation

1106

Sorghum biomass

1082

Spectrophotometric analysis

1122

Streptomyces

1071,1138

Systemic inflammation

1098

 

 

4-thiazolidinones

1062

Tyrosinase

1071

 

 

Vaccine

1090

 

 

Zea mays

1147

 

 

 

Preface

 

Biotechnology with its diverse application in almost each and every aspect of human life has been considered as the most important area for scientific and industrial research all over the world. There has been considerable effort for the production of health care products using the principles of chemistry and biology and in the exploitation of the micro-organisms for the over-production of various value-added products and processes such as food, feed, enzymes, antibiotics, organic acids, mycotoxins, vaccines and other drugs, ethanol and other solvent, waste disposal, and so on. Biotechnology has been proved as eco-friendly tool for development of society. With increased awareness on environmental pollution and its impact on life on earth, today’s society has great concern about their measure and control. Concept of eco-friendly processes and green-technologies has attained much attention globally. Scientific results obtained have shown that even the hazardous chemicals disposed in the environment could be effectively treated by means of biotechnology.

All over the world, as in India as well, there has been enormous effort put on R & D on bio-based economy. Large number of academic and research institutes and universities are engaged in high quality research and the trend has been turning for industrial products/processes oriented research. In recent years, there has been good growth on the industrialization of biotechnological processes in the areas covering Agricultural Biotechnology, Industrial Biotechnology, Food & Beverages, Medical Biotechnology and Energy & Environment. However, there is much gap on need and availability of technologies and awareness about these. Also, there appears to be much gap in collaboration and networking among the academic researchers, policy planners and industrial entrepreneurs. Yet another important aspect is about the concept of ‘global village’, which aims to bring together all countries at one forum for all purposes.

This special issue of Indian Journal of Experimental Biology comprises the selected peer-reviewed papers presented in the International Conference on Advances in Biotechnology and Bioinformatics 2013 and X Convention of the Biotech Research Society, India, organized by the Biotech Research Society, India (www.brsi.in) and Dr. D. Y. Patil Vidyapeeth, Pune, India during November 25–27, 2013 at Pune. The conference was attended by nearly 650 participants from 22 countries. Scientific deliberations were made by 78 eminent scientists and academicians from US, UK, Europe, Australia, Africa and Asia. Poster sessions were organized in six theme areas, viz., Microbial & Industrial Biotechnology, Animal & Microbial Biotechnology, Systems Biology & Bioinformatics, Biotechnology Business & IPR, Nano-Biotechnology, and Plant Biotechnology. The conference emphasized the significance of modern biotechnological tools and innovations with precision and safety for the development of eco-friendly and sustainable bioprocesses and products and their availability at affordable costs for the common population of the country. It also presented the industry’s perception on Biotechnology and hoped that biotechnological innovations will truly reach the industrial level. In this context, it is hoped that the BRSI would continue to disseminate scientific knowledge to the society through its national and international network.

The articles published in this issue give insight to new technologies, alternative technologies or processes and improved products for meeting increasing demand of energy. There are total fifteen papers in this issue, which were presented in the conference and have been selected based on the peer-review process of the journal. These have been classified in different areas such as Environmental Biotechnology (one), Enzyme Technology (three), Industrial Biotechnology (three), Medical Biotechnology (two), Nano-Biotechnology (one) and Plant Biotechnology (five).

The Guest Editors thank authors for their contribution and also thank CSIR-NISCAIR, New Delhi for promoting biotechnology through the publication of Indian Journal of Experimental Biology special issues as this, which genuinely reflect the advancement in biological sciences and engineering, providing a common digital platform for scientific exchange. Special thanks are due to Mr. Rajiv Mathur, Editor of the journal, and Director, CSIR-NISCAIR, New Delhi for providing special volume of the journal to bring out this special issue. Thanks are also due to the reviewers for their efforts in completing the review process in a timely manner.

 

 

Guest Editors

Dr Neelu Nawani

Prof Ashok Pandey

Prof Samir Khanal

Dr Parmeswaran Binod

Department of
Biotechnology

Dr DY Patil Institute of Biotechnology and
Bioinformatics

Pune 411 033, India

 

Centre for Biofuels &
Biotechnology Division

CSIR-National Institute of Science and Technology Trivandrum 695 019, India

 

Department of Molecular
Biosciences and Bioengineering

University of Hawaiii at Mānoa

Honolulu, HI 96822,
USA

 

Centre for Biofuels &
Biotechnology Division

CSIR-National Institute of Science and Technology

Trivandrum 695 019, India

 

            Correspondent author is marked by *

 

 

Indian Journal of Experimental Biology

Vol. 52, November 2014, pp. 1025-1035

 

 

 

Review Article

 

 

Production, purification and properties of fungal chitinases—A review

Narayanan Karthik, Karthik Akanksha, Parameswaran Binod* & Ashok Pandey

CSIR-National Institute of Interdisciplinary Science and Technology (NIIST), Thiruvanathapuram 695 019, India

 

After cellulose, chitin is the second most abundant organic and renewable polysaccharide in nature. This polymer is degraded by enzymes called chitinases which are a part of the glycoside hydrolase family. Chitinases have many important biophysiological functions and immense potential applications especially in control of phytopathogens, production of chito-oligosaccharides with numerous uses and in treatment and degradation of chitinous biowaste. At present many microbial sources are being explored and tapped for chitinase production which includes potential fungal cultures. With advancement in molecular biology and gene cloning techniques, research on fungal chitinases have made fast progress. The present review focuses on recent advances in fungal chitinases, containing a short introduction to types of chitinases, their fermentative production, purification and characterization and molecular cloning and expression.

 

Papers

 

Indian Journal of Experimental Biology

Vol. 52, November 2014, pp. 1036-1044

 

 

Purified dextransucrase from Pediococcus pentosaceus CRAG3 as food additive

Rishikesh Shukla & Arun Goyal*

Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, 781 039, India

Received 24 October 2013; revised 13 May 2014

The extracellular crude dextransucrase (0.67 U/mg) from P. pentosaceus CRAG3 (GenBank accession number JX679020) after PEG-1500 fractionation gave specific activity, 20.0 U/mg which by gel filtration resulted in 46.0 U/mg. The purified dextransucrase displayed molecular size of approximately, 224 kDa. The optimum assay conditions for dextransucrase activity were 5% sucrose in 20 mM sodium acetate buffer (pH 5.4) and 30 oC. The dextransucrase was stable up to 40 oC and at pH range of 5.4-7.0. The metal ions such as Co2+, Ca2+, Mg2+ and Zn2+ stimulated the dextransucrase activity by 56, 44, 14 and 12%, respectively. It was most stable at -20 oC with half-life of 307 days. Amongst various additives used, glycerol and Tween 80 provided significant stability to the enzyme with half-life 15.5 and 85.5 h, respectively as compared to control (6.9 h). The solidification of sucrose supplemented milk by purified dextransucrase due to dextran synthesis displayed its application as additive for improving the texture of dairy products.

 

 

Indian Journal of Experimental Biology

Vol. 52, November 2014, pp. 1045-1051

 

 

Kinetic constraints and features imposed by the immobilization of enzymes onto solid matrices: A key to advanced biotransformation

A Foukis1, P-Y Stergiou1, M Filippou2, M Koukouritaki2, M Parapouli2, L G Theodorou1, E Hatziloukas2,
A Afendra2, A Pandey3, E M Papamichael1,*

1University of Ioannina, Department of Chemistry, Group of Enzyme Biotechnology and Genetic Engineering, Ioannina, 45110, Greece.

2University of Ioannina, Department of Biological Applications & Technologies, Group of Enzyme Biotechnology and Genetic Engineering, Ioannina, 45110, Greece.

3CSIR-National Institute for Interdisciplinary Science & Technology (NIIST), Trivadrum 695 019, India

Received 15 December 2013

The kinetics of immobilized enzymes can not be analyzed by means of the simple Michaelis-Menten concept, which generally fails to describe the immobilized state due to both its probable barriers, and because the active concentration of the enzyme approaches, or even exceeds this of its substrate(s). In such cases, the various experimental data are usually treated by complex rate equations comprising too many parameters acquiring different natures and meanings, depending on both the properties of the immobilization state and the experimental conditions; thus, more likely, only apparent values of the Michaelis-Menten kinetic parameters can be estimated experimentally. Likewise, immobilization is often a key method in optimizing the operational performance of enzymes, in both laboratory and industrial scale, and affects considerably the kinetics in non-aqueous and non-conventional media due to several issues as the structural changes of the enzyme molecule, the heterogeneity of the system, and the partial or total absence of water. In this work a theoretical approach is described on the formulation of simplified rate equations, reflecting also the actual mass balances of the reactants, in the case where esterification synthetic reactions are catalyzed by immobilized lipases, in either a non-aqueous organic solvent or in a non-solvent system.

 

 

Indian Journal of Experimental Biology

Vol. 52, November 2014, pp. 1052-1061

 

 

Differential toxicological endpoints of di(2-ethylhexyl) phthalate (DEHP) exposure in MCF-7 and MDA-MB-231 cell lines: Possible estrogen receptor
α (ERα) independent modulations

Mihir Tanay Das, Manoj Kumar Singh & Indu Shekhar Thakur*

School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110 067, India

Received 25 December 2013; revised 19 May 2014

Wide spread use of Di-(2-ethylhexyl) phthalate (DEHP) has made it a ubiquitous contaminant in today’s environment, responsible for possible carcinogenic and endocrine disrupting effects. In the present investigation an integrative toxico-proteomic approach was made to study the estrogenic potential of DEHP. In vitro experiments carried out with DEHP (0.1-100 μM) induced proliferations (E-screen assay) in human estrogen receptors-α (ERα) positive MCF-7 and ERα negative MDA-MB-231 breast cancer cells irrespective of their ERα status. Further, DEHP suppressed tamoxifen (a potent anti-breast cancer drug) induced apoptosis in both cell types as shown by flowcytometric cell cycle analysis. Label-free quantitative proteomics analysis of the cell secretome of both the cell lines indicated a wide array of stress related, structural and receptor binding proteins that were affected due to DEHP exposure. The secretome of DEHP treated MCF-7 cells revealed the down regulation of lactotransferrin, an ERα responsive iron transport protein. The results indicated that toxicological effects of DEHP did not follow an ERα signaling pathway. However, the differential effects in MCF-7 and MDA-MB-231 cell lines indicate that ERα might have an indirect modulating effect on DEHP induced toxicity.

 

 

Indian Journal of Experimental Biology

Vol. 52, November 2014, pp. 1062-1070

 

 

Synthesis, DNA cleavage and antimicrobial activity of
4-thiazolidinones-benzothiazole conjugates

Meenakshi Singha, Mayank Gangwarb, Gopal Nathb & Sushil K Singha*

aDepartment of Pharmaceutics, Indian Institute of Technology (BHU)

bDepartment of Microbiology, Institute of Medical Sciences

Banaras Hindu University, Varanasi 221 005, India

Received 4 November 2013; revised 16 May 2014

Antimicrobial screening of several novel 4-thiazolidinones with benzothiazole moiety has been performed. These compounds were evaluated for antimicrobial activity against a panel of bacterial and fungal strains. The strains were treated with these benzothiazole derivatives at varying concentrations, and MIC’s were calculated. Structures of these compounds have been determined by spectroscopic studies viz., FT-IR, 1H NMR, 13C NMR and elemental analysis. Significant antimicrobial activity was observed for some members of the series, and compounds viz. 3-(4-(benzo[d]thiazol-2-yl) phenyl)-2-(4-methoxyphenyl)thiazolidin-4-one and 3-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(4-hydroxy phenyl)thiazolidin-4-one were found to be the most active against E.coli and C.albicans with MIC values in the range of 15.6–125 μg/ml. Preliminary study of the structure–activity relationship revealed that electron donating groups associated with thiazolidine bearing benzothiazole rings had a great effect on the antimicrobial activity of these compounds and contributes positively for the action. DNA cleavage experiments gave valuable hints with supporting evidence for describing the mechanism of action and hence showed a good correlation between their calculated MIC’s and its lethality.

 

 

Indian Journal of Experimental Biology

Vol. 52, November 2014, pp. 1071-1081

 

 

Development of bioconjugate from Streptomyces tyrosinase and gold nanoparticles for rapid detection of phenol constituents

Mazhari Bi Bi Zainab, D N Madhusudhan, H Raghavendra, Syed G Dastagera & Agsar Dayanand*

Actinomycetes-Diversity and Bioprocess Technology Research Laboratory,

Department of Microbiology, Gulbarga University, Gulbarga 585 106, India

aNCIM Resource centre, Biochemical Sciences Division,
CSIR-National Chemical Laboratory (NCL), Pune 411 008, India

Received 4 November 2013; revised 23 May 2014

Most of the phenol compounds are toxic and have been considered as hazardous pollutants. Several physicochemical and biological methods are available to detect and monitor the phenol pollutants in water and soil. In the present study, phenol constituents of winery, paper and plastic industrial effluents were successfully detected employing tyrosinase-gold nanoparticles bioconjugate. The synthesis of extracellular tyrosinase and gold nanoparticles was achieved by a single isolate of Streptomyces sp. DBZ-39. Enhanced production (369.41 IU) of tyrosinase was produced in submerged bioprocess employing response surface method with central composite design. Extracellular gold nanoparticles synthesized (12-18 nm) by Streptomyces sp. DBZ-39 were characterized with TEM, EDAX and FTIR analysis. A rapid detection (within 10 min) of phenol constituents from winery effluents was achieved by bioconjugate, when compared to tyrosinases and gold nanoparticles independently. Streptomyces tyrosinase could exhibit relatively a better performance than commercially available mushroom tyrosinase in the detection of phenol constituents. Winery effluent has shown much higher content (0.98 O.D) of phenol constituents than paper and plastic effluents based on the intensity of color and U.V absorption spectra.

 

 

Indian Journal of Experimental Biology

Vol. 52, November 2014, pp. 1082-1089

 

 

Dilute acid pretreatment and enzymatic hydrolysis of sorghum biomass for
sugar recovery—A statistical approach

Karthik Akanksha1, Arjun Prasad1, Rajeev K Sukumaran1, Madhavan Nampoothiri K1, Ashok Pandey1,
Rao S S2 & Parameswaran Binod1*

1Centre for Biofuels, CSIR-National Institute for Interdisciplinary Science and Technology (NIIST), Trivandrum 695 019, India

2Directorate of Sorghum Research (DSR), Indian Council of Agricultural Research (ICAR)

Rajendranagar PO, Hyderabad 500 030, India

Received 12 December 2013; revised 20 May 2014

Sorghum is one of the commercially feasible lignocellulosic biomass and has a great potential of being sustainable feedstock for renewable energy. As with any lignocellulosic biomass, sorghum also requires pretreatment which increases its susceptibility to hydrolysis by enzymes for generating sugars which can be further fermented to alcohol. In the present study, sorghum biomass was evaluated for deriving maximum fermentable sugars by optimizing various pretreatment parameters using statistical optimization methods. Pretreatment studies were done with H2SO4, followed by enzymatic saccharification. The efficiency of the process was evaluated on the basis of production of the total reducing sugars released during the process. Compositional analysis was done for native as well as pretreated biomass and compared. The biomass pretreated with the optimized conditions could yield 0.408 g of reducing sugars /g of pretreated biomass upon enzymatic hydrolysis. The cellulose content in the solid portion obtained after pretreatment using optimised conditions was found to be increased by 43.37% with lesser production of inhibitors in acid pretreated liquor.

 

 

Indian Journal of Experimental Biology

Vol. 52, November 2014, pp. 1090-1097

 

 

Assessment of phagosomes infected with Mycobacterium tuberculosis as a vaccine candidate against tuberculosis

Anjana Sharma*, Pankaj Parihar & Juhi Sharma

Bacteriology Laboratory, Department of P.G Studies and Research in Biological Sciences, R.D University, Jabalpur 482001, India

Received 4 November 2013; revised 15 May 2014

The present study describes a novel and simple vaccination strategy that involve culturing of M. tuberculosis in the macrophage cells. Isolation of phagosome from macrophage (cell line J774) infected with M. tuberculosis (H37) and
M. bovis (BCG) at early and late phase of infection was done ensuing the identification and characterization of these phagosome. In vitro study of apoptosis induced by phagosome infected with (H37) and (BCG) was performed. The vaccine candidate with H37 MOI- 1:10 at 3 h, MOI- 1:20 at 1, 1.5, 2.5 and 3 h and BCG MOI- 1:20 at 3.5 h showed percentage apoptosis as 38.64, 39.93, 34.66, 22.56,34.59 and 37.81% respectively. The results designates that macrophages provide cellular niche during infection and illustrate considerable immunogenic property. Novel antigens expressed or secreted by H37 in infected macrophages can provide evidence to be a successful vaccine candidate as it endures enhanced immune response than BCG.

 

 

Indian Journal of Experimental Biology

Vol. 52, November 2014, pp. 1098-1105

 

 

Modulation of small intestinal homeostasis along with its microflora during acclimatization at simulated hypobaric hypoxia

 

Atanu Adak, Kuntal Ghosh & Keshab Chandra Mondal *

Department of Microbiology, Vidyasagar University, Midnapore 721 102, India

Received 4 November 2013; revised 20 May 2014

At high altitude (HA) hypobaric hypoxic environment manifested several pathophysiological consequences of which gastrointestinal (GI) disorder are very common phenomena. To explore the most possible clue behind this disorder intestinal flora, the major player of the GI functions, were subjected following simulated hypobaric hypoxic treatment in model animal. For this, male albino rats were exposed to 55 kPa (~ 4872.9 m) air pressure consecutively for 30 days for 8 h/day and its small intestinal microflora, their secreted digestive enzymes and stress induced marker protein were investigated of the luminal epithelia. It was observed that population density of total aerobes significantly decreased, but the quantity of total anaerobes and Escherichia coli increased significantly after 30 days of hypoxic stress. The population density of strict anaerobes like Bifidobacterium sp., Bacteroides sp. and Lactobacillus sp. and obligate anaerobes like Clostridium perfringens and Peptostreptococcus sp. were expanded along with their positive growth direction index (GDI). In relation to the huge multiplication of anaerobes the amount of gas formation as well as content of IgA and IgG increased in duration dependent manner. The activity of some luminal enzymes from microbial origin like α-amylase, gluco-amylase, proteinase, alkaline phosphatase and β-glucuronidase were also elevated in hypoxic condition. Besides, hypoxia induced in formation of malondialdehyde along with significant attenuation of catalase, glutathione peroxidase, superoxide dismutase activity and lowered GSH/GSSG pool in the intestinal epithelia. Histological study revealed disruption of intestinal epithelial barrier with higher infiltration of lymphocytes in lamina propia and atrophic structure. It can be concluded that hypoxia at HA modified GI microbial imprint and subsequently causes epithelial barrier dysfunction which may relate to the small intestinal dysfunction at HA.

 

 

Indian Journal of Experimental Biology

Vol. 52, November 2014, pp. 1106-1111

 

 

Development of bioprocess for the production of laccase by Pleurotus ostreatus MTCC 1802 using evolutionary optimization technique

Jayanti Kumari & Sangeeta Negi*

Motilal Nehru National Institute of Technology, Allahabad 211 004, India

Received 4 November 2013: revised 15 May 2014

For cost effective production of laccase enzyme (benzenediol: oxygen oxidoreductase) from P. ostreatus MTCC 1802 through solid sate fermentation, physico-chemical parameters such as temperature (20-35 ºC), incubation period (9-17 days) and substrate (Neem bark and wheat bran, in various ratios, w/w) were optimized first by one parameter at time approach and then obtained optimum conditions were considered as zero level in evolutionary optimization factorial design technique. At statistically optimized conditions yield of laccase was found 303.59+16.8) U/gds after 13 days of incubation at 25 ºC taking wheat bran and neem bark as substrate at a ratio of 3:2 (w/w). The results obtained could be a base line for industrial scale production of laccase.

 

 

Indian Journal of Experimental Biology

Vol. 52, November 2014, pp. 1112-1121

 

 

In vitro regeneration and ploidy level analysis of Eulophia ochreata Lindl

Varsha Shriram1*, Vikas Nanekar1, Vinay Kumar2 & P B Kavi Kishor3

1Department of Botany, Prof. Ramkrishna More College (University of Pune), Akurdi, Pune 411 044, India

2Department of Biotechnology, Modern College (University of Pune), Ganeshkhind, Pune 411 016, India

3Department of Genetics, Osmania University, Hyderabad 500 007, India

Received 16 October 2013; revised 12 May 2014

Various parameters including explant-type, medium compositions, use of phytohormones and additives were optimized for direct and indirect regeneration of E. ochreata, a medicinal orchid under threat. Protocorm-like-bodies (PLBs) proved to be the best explants for shoot initiation, proliferation and callus induction. Murashige and Skoog’s (MS) medium containing 2.5 mg L-1 6-benzylaminopurine (BAP), 1.0 mg L-1 kinetin (Kin) and additives (adenine sulfate, arginine, citric acid, 30 mg L-1 each and 50 mg L-1 ascorbic acid) was optimal for shoot multiplication (12.1 shoots and 7.1 PLBs per explant with synchronized growth), which also produced callus. Shoot number was further increased with three successive subcultures on same media and ~40 shoots per explant were achieved after 3 cycles of 30 days each. Additives and casein hydrolysate (CH) showed advantageous effects on indirect shoot regeneration via protocorm-derived callus. Optimum indirect regeneration was achieved on MS containing additives, 500 mg L-1 CH, 2.5 mg L-1 BAP and 1.0 mg L-1 Kin with 30 PLBs and 6 shoots per callus mass (~5 mm size). The shoots were rooted (70% frequency) on one by fourth-MS medium containing 2.0 mg L-1 indole-3-butyric acid, 200 mg L-1 activated charcoal and additives. The rooted plantlets were hardened and transferred to greenhouse with 63% survival rate. Flow-cytometry based DNA content analysis revealed that the ploidy levels were maintained in in vitro regenerated plants. This is the first report for in vitro plant regeneration in E. ochreata.

 

 

Indian Journal of Experimental Biology

Vol. 52, November 2014, pp. 1122-1127

 

 

In vitro callus induction and estimation of plumbagin content from
Plumbago auriculata Lam.

Jyoti Deshpande1, Dinesh Labade2, Kalaiselvi Shankar2, Navin Kata1, Manoj Chaudhari1,
Minal Wani1 & Madhukar Khetmalas1*

1Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Tathawade, Pune 411 033, India

2Rise n’ Shine Biotech Pvt. Ltd., Theur, Pune 412 110, India

Received 4 November 2013; revised 27 May 2014

The medicinal plant Plumbago contains a very potent secondary metabolite, plumbagin having many therapeutic properties. Callus culture was induced using explants, leaf, stem and shoot apex, from P. auriculata. Murashige and Skoog media fortified with various growth hormones like NAA, IAA, IBA and 2, 4-D individually and in various combinations were checked for callus induction. Among the growth hormones used, 1 mg/L 2, 4-D showed best callusing. The hormonal combinations of 1 mg/L IAA and 1.5 mg/L NAA in the media exhibited best callus induction using stem internode as an explant. Plumbagin content from root, stem, leaf and callus was analyzed by using thin layer chromatographic technique. The callus derived from stem showed comparable plumbagin content to the in vivo plant parts. Quantitative spectrophotometric analysis of plumbagin from plant samples and callus indicated that plumbagin content was maximum in roots which was followed by callus, stem and leaf samples respectively. Generation of in vitro sources for plumbagin, for therapeutic applications will serve as a continuous supply and will contribute to preserve the natural plant recourses.

 

 

 

Indian Journal of Experimental Biology

Vol. 52, November 2014, pp. 1128-1137

 

 

Mannitol-induced drought stress on calli of Trigonella foenum-graecum L.
Var. RMt-303.

Naveen C Pant, Ruchi Agarrwal* & Sanjeev Agrawal

Department of Biochemistry, College of Basic Sciences and Humanities, GBPUA&T Pantnagar, 263 145, India

Received 4 November 2013; revised 26 May 2014

Different explants of fenugreek, T. foenum-graecum L. (Var. RMt-303), were compared for their callus induction and subsequent shoot regeneration capabilities on Murashige and Skoog media supplemented with different phytohormones in varying concentration. The highest percentage of callus induction frequency was observed in 1ppm benzylaminopurine (BAP). Maximum shoots were induced on media supplemented with 0.5ppm BAP using leaf and stem tissues as explants. However, root tissues showed only callusing with no subsequent shooting. Cotyledonary node responded better than hypocotyls in terms of shoot induction on media supplemented with thidiazuron (0.1ppm). The callus was subjected to drought stress as simulated by reduced water potential of growth media due to addition of mannitol. Calli could withstand -2 MPa water potential till 30 days indicating that the drought stress tolerance mechanisms are functional in this variety. Chlorophyll a and b and total chlorophyll, proline and total phenolic contents, total peroxidase and catalase activities increased under stress conditions suggesting the tolerance of callus to drought stress. However, ascorbate peroxidase, guaiacol peroxidase activities were found to decrease slightly. Malondialdehyde and H2O2 contents were found to decrease while only a slight disturbance was found in membrane stability index. These results underline the mechanisms that are crucial for drought stress tolerance in fenugreek.

 

 

Indian Journal of Experimental Biology

Vol. 52, November 2014, pp. 1138-1146

 

 

Anti-fungal potentials of extracellular metabolites of Western Ghats isolated Streptomyces sp. NII 1006 against moulds and yeasts

Himani Jayamurthya*, Kuttavan Valappil Sajnaa, Syed G Dastagarb & Ashok Pandeya

aBiotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology (NIIST), Trivandrum 695 019, India

bNCIM Resource center, CSIR-National Chemical Laboratory (NCL), Dr. Homi Bhabha Road, Pune 411 008

Received 4 November 2013

Realization of hazardious effects of chemical fungicides has led to an interest in the usage of biocontrol agents.
The present study, therefore, evaluates the biocontrol efficacy of Western Ghats (India) soil bacterial isolates.
A potential strain NII 1006 was evaluated for its antagonistic property against a diverse range of moulds and yeasts. The strain
was characterized morphologically, biochemically and molecularly, which revealed the isolate belonged to Streptomyces genus. Organic solvent extracts of NII 1006 culture filtrates inhibited the growth of the test pathogens indicating that
growth suppression was due to extracellular anti-fungal metabolites present in the culture filtrates. The strain
produced extracellular chitinase enzyme in addition to some stable partially purified anti-fungal compounds. Morphological changes such as hyphae degradation into debris and abnormal shapes were observed in test fungi and yeast grown on potato dextrose broth that contained the NII 1006 culture filtrate. The cell free supernatant has a tolerance to wide range of pH, temperature and enzymes such as lipase and protease. The biocontrol potential of NII 1006 strain may be correlated significantly with their ability to produce antibiotics as well as extracellular hydrolytic enzymes particularly chitinolytic enzyme.

 

 

Indian Journal of Experimental Biology

Vol. 52, November 2014, pp. 1147-1151

 

 

Potential of Microbispora sp. V2 as biocontrol agent against Sclerotium rolfsii,
the causative agent of southern blight of Zea mays L (Baby corn)–in vitro studies

N N Patil1*, M S Waghmode1, P S Gaikwad1, M H Gajbhiye2, A B Gunjal2, N N Nawani3 & B P Kapadnis2

1Department of Microbiology, PDEA’s Annasaheb Magar Mahavidyalaya, Hadapsar, Pune 411 028, India

2Department of Microbiology, University of Pune, Ganeshkhind, Pune 411 007, India

3Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D.Y. Patil Vidyapeeth, Pune 411 033, India

Received 4 November 2013; revised 16 May 2014

The study was undertaken with the aim of exploring novel and beneficial agro activities of rare actinomycetes like Microbispora sp. V2. The antagonistic activity of Microbispora sp. V2 was evaluated as a biocontrol agents against Sclerotium rolfsii, a soil-borne fungal plant pathogen. The methodology performed for evaluation of biocontrol agent was in vitro evaluation assay which comprised of three tests viz., cellophane overlay technique, seed germination test and Thiram (fungicide) tolerance of Microbispora sp. V2. The isolate was found to inhibit the fungal pathogen Sclerotium rolfsii to 91.43% in cellophane assay. In seed germination assay, Microbispora sp. V2 treated seeds resulted in 25.75% increased germination efficiency, as compared to seeds infected by Sclerotium rolfsii. The isolate Microbispora sp. V2 could tolerate 1000 µg mL-1 of Thiram (fungicide). The in vitro assay studies proved that Microbispora sp. V2 can be used as antifungal antagonist and thus posses’ great potential as biocontrol agent against southern blight caused by Sclerotium rolfsii in Zea mays L (Baby corn) which causes large economical losses.