Research Journal of Biotechnology

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Vol. 15(5) May 2020

Improved Cellulase Production through RSM by using Aspergillus tubingenesis MN239975 in Solid State Fermentation

Patel Srushti Prahaladbhai and Duggirala Srinivas Murty

Page No: 1-10

Abstract: Cellulose is the most important constituent of the plant cell wall and the cellulase enzymes catalyze it to glucose. Fungal cellulases have been produced on different agro residues through solid state fermentation which is one of the most cost-effective methods. In the present study, cellulase producing Aspergillus flavus, Aspergillus tubingenesis, Rhizopus oryzae and Aspergillus fumigatus were isolated and identified by 18 S rRNA sequencing. Aspergillus tubingenesis MN239975 was found to be the most potent isolate for cellulase production in SSF. Various agro residues such as sorghum straw, sorghum hay, maize straw, wheat straw and paddy straw were used as a substrate for cellulase production in SSF. The fermentation conditions such as temperature, pH, moisture and inoculum size were optimized by response surface method (RSM) CCD for improved cellulase production by Aspergillus tubingenesis MN239975 with minimal mineral salt medium and sorghum straw as a substrate.

In the optimization experiment, the highest CMCase and FPase production were 271.6 U/g and 104 U/g respectively at 70.5 % moisture content, 5.5 pH, 27.5°C temperature and 7.5 % inoculum size. It was observed that moisture and temperature had a more significant effect on cellulase production as compared to pH and inoculum.

Isolation, Screening and Identification of a potent tannase producing bacteria from the rhizospheric soil of Casia Species

Dhiman Sunny, Shukla Yamini, Dhiman Ruby and Mukherjee Gunjan

Page No: 11-18

Abstract:A potent tannase producing bacterial strain was isolated from the rhizospheric soil of Casia species and was identified as Bacillus haynesii strain SSRY4 (Genbank Accession Number MN031245). Various environment samples were inoculated in enrichment liquid media containing 1% (w/v) tannic acid for 48 hrs. Upon plating of appropriately diluted culture aliquots on tannic acid agar plates, only few of them developed bacterial colonies showing clearly visible zone of tannic acid hydrolysis around them. Zones of hydrolysis were measured in mm. Eight bacterial colonies showing maximum tannic acid hydrolysis zone around them were selected. Pure cultures were developed by repeated streaking on nutrient agar plates. The isolates were then screened for their ability to produce tannase in submerged conditions.

Upon quantitative estimation of tannase production, only three isolates SSRY1, SSRY2 and SSRY4 showed considerably good enzyme activities of 1.37 U/ml, 1.29 U/ml and 1.56 U/ml respectively. Various morphological and microscopic features of the bacterial isolate SSRY4 were studied and a gram positive, rod shaped bacterium was confirmed. 16s rRNA sequencing results confirmed its identity as Bacillus haynesii strain SSRY4 (Genbank Accession Number MN031245).

Morphological and Genetic Diversity of Fusarium spp. associated with Panama wilt disease of Banana in Bihar

Niwas Ram, Chand Gireesh and Azad Chandrashekhar

Page No: 19-23

Abstract:The Bihar State played a most important role in the area and production of banana in India and the most of farmers are totally dependent on the cultivation of those crops. In the current scenerio these crops suffer from a number of diseases caused by fungi, bacteria, viruses, nematodes, and also it is affected by abiotic factors.

The present study was conducted in the Laboratory of Plant Pathology, BAU, Sabour for evaluation of morphological and genetic diversity of Fusarium wilt of banana. After extensive survey in different banana growing area of Bihar, diseased samples were collected from Grande Naine cultivar of banana which is susceptible. Successfully 4 isolates of Fusarium were isolated. The great variations were found in the size, shape of conidia, colony color, and sporulation pattern. The second experiment was conducted for genetic diversity Fusarium by using to primer ITS 4 and ITS 5. Un weighted Pair Group Method with Arithmetic Average (UPGMA) cluster analysis based on similarity cofficient showed Fusarium isolates into one cluster with similarity value of 100% in PCR-RAPD of ITS4 and ITS5. The results indicate that Fusarium isolates were closely related to the banana and location.

Identification of the most suitable reference genes for gene expression normalization in safflower (Carthamus tinctorius L.) treated with gibberellin

Elkholy Shereen F.

Page No: 24-32

Abstract: Safflower (Carthamus tinctorius L., Asteraceae) is an important oil crop and medicinal plant which is of considerable interest for the expression of agronomically important genes. Semi-quantitative reverse transcription PCR (semi-qRT-PCR) is a powerful tool for studying gene expression, but it requires appropriate reference genes that can reduce variability. To identify safflower reference genes, four candidate housekeeping genes were investigated: CtPP2A (serine/threonine-protein phosphatase), CtE1F4A (eukaryotic initiation factor 4A), CtUBI (ubiquitin) and Ct60S (60S acidic ribosomal protein). 32 samples of safflower seedlings were treated with different gibberellin (GA) concentrations (0 ppm, 50ppm, 100ppm and 150 ppm; eight replicates each). Expression stability across samples was then investigated using semi-qRT-PCR.

GA at 150ppm is observed as an optimal concentration for promoting safflower growth with superior plant heights, stem diameters, numbers of branches and flowers per plant. Additionally CtUBI and Ct60S were identified as the most suitable reference genes for evaluating the expression of gibberellin-biosynthesis genes including GA20-oxidase (GA20ox), GA 3-oxidase (GA3ox) and GA 3-beta-hydroxylase (GA4). The results indicate that both genes are appropriate as internal controls to normalize safflower gene expression.

Direct and Indirect Organogenesis in Hippophae salicifolia D. Don., A Nutraceutically Important Plant

Thakur Ajay, Pant Manu and Lal Ankita

Page No: 33-41

Abstract:An in vitro propagation protocol of a medicinally and nutraceutically important plant Hippophae salicifolia has been developed. Induction of shoots was achieved from cotyledon and hypocotyl explants derived from in vitro established seedlings of H. salicifolia. MS medium containing BAP (5µM), NAA (3µM) and activated charcoal gave the best results of direct shoot induction from cotyledonary explants while a combination of BAP (7.5µM) and NAA (1.5µM) proved to be the best for callus induction from hypocotyl segments.

In vitro shoots regenerated via direct and indirect organogenesis were multiplied in separate sets of experiments. MS media containing BAP, NAA and adjuvant AdS were optimal for micro-shoot multiplication. In vitro developed shoots were rooted on half strength MS medium devoid of agar and supplemented with 1.5µM IAA. The plantlets so developed were hardened in vitro and shifted to polyhouse for further growth and development.

Isolation and Characterization of Microalgae Isolated from Freshwater Sources in Karnataka for Biofuel Production

Dhanya Ranjit V. and Kumudini Belur Satyan

Page No: 42-49

Abstract:Rapid depletion of fossil fuels coupled with emission from their combustion has resulted in the search for alternate sources of energy. Microalgae are emerging as a promising source of energy as they contain large amount of lipids which further can be trans-esterified into biodiesel. The present study was aimed at screening of potential algal isolates from Karnataka, India for increased lipid production. Of the ten isolates shortlisted, JUCHL_9 was considered for further studies based on biomass yield and lipid content which was identified to be C. sorokiniana by sequencing using 18S r RNA.

Cellulolytic fungi from central Morocco: comparative analysis of enzyme activities, in silico prediction of physico-chemical properties and molecular docking

Nait M’Barek Hasna and Hajjaj Hassan

Page No: 50-60

Abstract:Fungal cellulases are important biocatalysts implicated in the conversion of lignocellulose for the production of second-generation bioethanol. Predicting their catalytic properties and bonding ability to substrate using computational tools is a prominent way to better assign their industrial use. Cellulolytic potential of two wood decaying fungi from central Morocco is studied. Comparison is made between cellulolytic activities in the presence of microcrystalline cellulose as the sole carbon source, results of in silico computation of physico-chemical properties and molecular docking of enzymes to substrate.

The strain Penicillium brasilianum (Pb) shows a pertinent cellulolytic potential producing 1.1 IU/ml total cellulase activity, 3.2 IU/ml endoglucanase activity and up to 41.7 IU/ml Xylanase activity. It yields 63.6% hydrolysis. Trichoderma atroviride (Ta) isolate is less cellulolytic and hydrolyses the polysaccharide only to 1.26%. Computational analysis shows that Pb cellulases are particularly stable and dock well to microcrystalline cellulose with 2.65Å to 2.92Å hydrogen bond distances. The studied cellulase of Ta is also stable and has strong docking properties to substrate. Reporting these results to quantitative activities shows evidence of the proteome diversity among isolates of the same Ta species mainly controlled by the gene expression under the surrounding environmental conditions.

Challenges associated with Herpes Simplex Virus isolation using Vero cell culture

Dhull Divya, Sharma Vikrant, Sharma Yashika and Kaushik Samander

Page No: 61-66

Abstract: Virus isolation considered as a gold standard and reference is to compare the sensitivity and specificity of other diagnostic methods. There are large numbers of factors and conditions which affect the growth of the virus while culturing. The present study focuses on the various aspects which cause hurdle in the virus isolation, appearance of Cytopathic Effect (CPE) in cell-line due to the virus, contamination, long time. Contamination is one of the major barriers in promising outcome. Sterility assays were performed to ensure the cause of contamination and the measures were suggested to forbid it.

The virus was incapable of culture from Cerebrospinal fluid (CSF) samples of the Herpes Simplex Virus (HSV) positive cases where no ophthalmic fluid samples were found HSV positive by any of the methods and tissue biopsy HSV positive sample was successfully illustrate CPE in Vero cell line culture.

Role of 2,6 Dichloroisonicotinic acid inducing resistance in cotton against cotton leaf curl disease

Kumari Archana, Sangha Manjeet Kaur, Vasmatkar Pashupat, Akhatar Javed and Pathak Dharmender

Page No: 67-74

Abstract: Cotton (Gossypium hirsutum L.), an important fibre crop is challenged with large number of pest and pathogens leading to huge yield losses. To survive these threats, we aimed at investigating the effect of plant elicitor salicylic acid analogue 2,6 Dichloro isonicotinic acid on pathogenesis related (PR) proteins. The chemical was applied once through seed imbibition and thrice [5, 19 and 33 days after germination (DAG)] for soil drenching and foliar spray on three cotton genotypes viz. FDK124 (resistant), LH2076 (moderately resistant) and F1378 (suceptible). Our results depicted an overall increase in chitinase, β-1,3 glucanase, peroxidase activities and total protein content in response to INA treatment. All methods of application caused higher induction in PR proteins. Additionally, seed priming induced resistance proteins up to 35 DAG.

The study was upscaled by infecting both control and treated genotypes with Gemini virus, causing agent of Cotton leaf curl disease (CLCuD). Treated plants were reported with less disease incidence and disease index compared to their respective control genotypes. We concluded that INA not only induced the expression of PR proteins but also served as protective regime against CLCuD.

Obtaining new recombinant strains of Brevibacterium flavum containing heterologous Geobacillus stearothermophilus gene argJ

Koloyan Haykanush, Avetisyan Sona, Paronyan Marina, Aganyants Hovsep and Hovsepyan Anichka

Page No: 75-81

Abstract:Molecular cloning of the heterologous gene argJ of thermophilic bacteria Geobacillus stearothermophilus on Escherichia coli – Corynebacterim glutamicum shuttle expression vector pEC-XK99E in the cells of coryneform bacteria was carried out. To create a new arginine-producing strain, the designed recombinant plasmid pARGJ was used. Brevibacterium flavum HK-19A strain obtained by us earlier synthesizing up to 25.0 g/l L-arginine was used as a recipient. The obtained new recombinant strain containing the heterologous gene argJ of thermophilic bacteria provides higher yield of L-arginine compared to the recipient strain.

Isolation, Identification and Evaluation of anti-oxidant, anti-inflammatory and antimitotic properties of bio active pigment from Planococcus maritimus SCG23

Gadal Shivasharanappa Chandrappa and Naik Chandrashekhar

Page No: 82-94

Abstract:Microorganisms are prolific producers of pigments for different reasons like its survival under stress conditions, to shield themselves from phagocytosis, UV radiation, antibiotics agents and heavy metal resistance. In our examination, yellow-orange producing bacterium was isolated from pharmaceutical effluent to assess its antioxidant, anti-inflammatory and antimitotic properties. Morphological and molecular (16s rRNA) characterization delighted the confine as a gram positive bacilli Planococcus maritimus and assigned as SCG23. The 16s rRNA sequence was submitted to NCBI Gen Bank with accession number: KU995335 and gene ID: 1015917315. The phylogenetic investigation with most extreme probability technique indicated 100% closeness with Planoococcus maritimus APBSDSB141. The thin layer chromatography was done for extracted pigment with various solvents systems. Acetonitrile and water (8:2, v/v) was observed to be the best solvent system and indicated distinct separations.

The pigment was additionally purified through silica gel column chromatography and purified pigment was exposed to GC-MS examination. GC-MS results revealed the presence of ~15 compounds out of which methyl 13-methyltetradecanoate (peak area % = 34.91) having cytotoxicity and anti-cancer capability, Pentadecanoic acid methyl ester (peak area% = 5.47) and n-hexadecanoic acid (peak area% =9.68), cis-10-Heptadecenoic acid, methyl ester (peak area% = 17.47) and cis-10-Heptadecenoic acid (peak area% = 10.94) having antiinflamatory and anti-mitotic properties.

Docking of PPAR α with VEGF, Nephrin and Hif 1 α

Awasthi Srikant and Srivastava Shivani

Page No: 95-98

Abstract:Through docking we have found the direct interaction of PPAR α with Hif 1α, VEGF and Nephrin. Docking studies showed that VEGF has more favourable interaction than Nephrin and Hif 1α with better docking score. Residues ASP301, ASN303 and VAL306 of PPAR α were found important residues for docking.

Isolation and in-silico studies on AP2/ERF domain containing gene DREB2A from pearl millet

Meena Rajendra Prasad, Vishwakarma Harinder and Padaria Jasdeep Chatrath

Page No: 99-108

Abstract:Drought stress tolerance is a complex phenomenon which is genetically determined and governed by a large number of genes which are sequentially and differentially expressed in response to drought stress. DREB (Dehydration Responsive Element Binding Protein) is one of the important genes for drought tolerance which regulates expression of many water stress inducible genes. Based on its upregulation in a previously constructed drought responsive SSH library, drought stress responsive DREB TF gene PgDREB2A from Pennisetum glaucum (L.) R. Br. was selected for further study. Validation by qRT-PCR revealed that expression of PgDREB2A increased 10.39-fold in response to 20 days of drought stress. Full length cDNA sequence of DREB2A from P. glaucum cv. 841B was deduced by using 5' and 3' RACE technique, amplified and then cloned. CDS of DREB2A (1002bp) (Accession number: MF370935) having a predicted protein of 333 amino acids shows maximum 99% identity with Cenchrus americanus dehydration responsive element binding protein 2A with 100% query coverage.

In-silico based studies, 3D protein structure, motifs and domain prediction analysis were carried out to decipher the role of DREB2A in drought stress tolerance in pearl millet. PgDreb2A can serve as a genomic resource for development of abiotic stress tolerant climate smart crops either through breeding or transgenic approach.

Production, Partial Purification and Characterisation of α-Amylase from Aspergillus niger using Aqueous Two Phase System (ATPS)

Naganagouda V. Kote, Manjula A.C., Vishwanatha T. and Gouda Patil Aravind

Page No: 109-114

Abstract:The enzymes purified by conventional methods such as precipitation, chromatographic techniques are economically not viable with laborious procedure and may lead to low yields and recovery of enzyme activity. The ATPS is able to give high recovery yield and is easy to scale up. It is also very economic and environment friendly method. Thus, the aim of this work was to find characterization of α-amylase produced from Aspergillus niger and partitioning behavior of partially purified enzyme was studied in aqueous two phase system (ATPS), The production of amylase was highest on the 5th day under submerged fermentation and the activity is found to be 2.511 U ml-1.

The optimum pH and temperature were found to be 7.5, 35 0C respectively. The Km and Vmax values were 0.6 mg/ml and 16.05mg/ml/min. The highest partition coefficient 0.326 was obtained in phase system with PEG 6000-Na2SO4 followed by the highest yield in the bottom phase 29.27%. The purity of enzyme was characterized by native and zymogram analysis. On the basis of the results obtained, purified α-amylase could be employed in the food processing industry.

Postbiotics: A potential approach towards eradication of biofilm of various pathogens

Sharma Komal and Gupte Shilpa

Page No: 115-120

Abstract:Probiotics are live microorganisms exerting health beneficial effects on the host when taken in adequate amount. Despite of proven health benefits of probiotics in certain cases like immuno-compromised patients, probiotics are not as beneficial as in normal inhabitants. Therefore, as an alternative, active compounds released by the probiotics termed as postbiotics can be analyzed for biofilm removal. The postbiotics refer as products or metabolic byproducts secreted by live bacteria or released after lysis. The study includes postbiotics derived from probiotics, Lactobacillus clausaii, Lactobacillus rhamnous, Lactobacillus sporogenes and Saccharomyces boulardii investigated for antibiofilm activity on Pseudomonas aeruginosa PAO1. Among them, Saccharomyces boulardii postbiotics were most effective in removing biofilms.

Cell free supernatants, exopolysaccharide and combination of them which were used as postbiotics have been used for treating biofilms of Pseudomonas aeruginosa, Staphylococcus aureus, Klebsilla pneumoniae and Enterococcus faecalis. Among all pathogens, maximum biofilm removal efficacy (86.06%) was observed in case of S.aureus during pretreatment while biofilm removal was observed in range of 65-76% for other pathogens. In case of post-treatment study of postbiotics, only 50% of S.aureus biofilm removal was observed within 2h and no further rise was observed in biofilm removal efficacy with time.

Cytotoxic and genotoxic effects of 4-methylimidazole on Allium cepa root tips

Liman Recep

Page No: 121-127

Abstract:4-Methylimidazole (4-MeI or 4-MEI) is widely used chemical as cleaning and agricultural chemical, drugs, rubber, dyes and pigments. It is also formed in manufacturing process of class III or IV caramel. In this study, Allium cepa root tips were used for cyto-genotoxic effects of 4-MeI by using Allium ana-telophase and comet assays. Changes in mitotic index (MI), mitotic phase frequencies, chromosome aberrations (CAs) and DNA damage were evaluated after Allium bulbs exposed to three different concentrations (25, 50 and 100 ppm) of 4-MeI at four different time intervals (24, 48, 72 and 96 h) for comparison with a negative (distilled water) and positive control (Methyl methane sulfonate, MMS-10 ppm). 4-MeI was found cytotoxic by reducing root growth and MI and was also found genotoxic by increasing CAs and DNA damage. Additional molecular toxicological studies are necessary to verify the cyto-genotoxicity of 4-MeI on plants.

RAPD Profile Reproducibility of Pogostemon benghalensis and Dysophylla stellata DNA

Lodha Swati and Deshmukh Sanjay

Page No: 128-132

Abstract:Regenerating conditions of random amplified polymorphic DNA (RAPD) markers using the polymerase chain reaction (PCR) for Pogostemonbenghalensis and Dysophylla stellate DNA were optimised. The importance of this technique is to obtain reproducible RAPD patterns. The standard reaction developed included: 100-200ng of DNA extracted,10pmol primer, 10X PCR buffer, Taq DNA polymerase (5.0 units/μl), MgCl2 (50mM) and dNTPs mix(0.5mM) in 25µl of PCR reaction.

In silico analysis and molecular docking studies of potential anti-Helicobacter pylori compounds

Kumar Raman, Bansal Poonam, Dalal Sunita and Dhanda Suman

Page No: 133-136

Abstract:The objective of this study was to analyze the novel compounds for targeting Helicobacter pylori by computational docking studies. Colonization of Helicobacter pylori in stomach is the major causative factor of gastrointestinal illness and gastric cancer. Urease, vacuolating cytotoxin A (VacA) and cytotoxin-associated gene antigen (CagA) are main virulence factors responsible for Helicobacter pylori pathogenesis.

Effective therapeutic molecules are immediately required to combat such infections. Though, Food and Drug Administration (FDA) approved some natural products mainly from plant extract as pharmaceutical candidates for Helciobacter pylori eradication and related gastric disease prevention. Present in-silico study is focused on molecular docking analysis to identify potential compounds of plant source from literature. Study revealed that only two molecules i.e. Epigallocatechin and Idebenone (out of 28 screened) bind very effectively with target proteins (virulence factors) and thus can be used as novel therapeutics to target Helicobacter pylori borne illness.

Salt and pH stress tolerating plant growth promoting rhizobacteria from multivarietal mango orchards

Radha T.K., Chinthana K.L., Ganeshamurthy A.N. and Rupa T.R.

Page No: 137-142

Abstract:The adverse effects of biotic and abiotic stress on various horticultural crops can be overcome by inoculating the roots with stress tolerating plant growth promoting bacteria. In our study, 7 bacteria were isolated from multivarietal mango orchards of Chittoor district of Andhra Pradesh and characterized based on morphological and biochemical tests. Among all the isolates, K1 and K3 were able to tolerate up to 10% NaCl concentration at all the pH (7, 8 and 9). P solubilzation of the isolates ranged from 9.70 to 25.39 µg/ml; ZnCO3 from 0.61 to 2.64, ZnO from 1.76 to 2.55, Zn3(PO4)2 from 1.07 to 2.47 mg/L; IAA, GA3 and ABA production ranged from 607 to 906 ng/ ml, 308 to 1096 ng/ ml and 189 to 762 ng/ ml of broth respectively. Isolate K3 was found to be more effective against Fusarium oxysporum and Colletotrichum gloeosporioides with the maximum inhibition of 46.42% and 58.46% respectively.

Molecular confirmation of strains K1 showed 99 % similarity with Providencia vermicola (Accession no: MK942853) and K3 with 98% similarity of Providencia rettgeri (Accession no: MH588071). The identified strain K1 and K3 from our study had a high potential for use particularly under stress conditions.

NIPAH –Virus: A review

Joshi Mahavir, Kaur Sukhminderjit, Dabas Vipin and Middha Aastha

Page No: 143-153

Abstract:Zoonotic diseases have the potential to cause an epidemic in a short time period and due to changing ecological and environmental conditions, zoonoses have been on the rise posing a threat to human health. The emergence of Nipah virus in Malaysia in 1998-99 is a classical example of emerging zoonosis. Following the outbreak in Malaysia, there were major cases of NIPAH in India and Bangladesh throughout the past two decades with high fatality rate. The natural host of the virus was found to be a species of fruit eating bats i.e. Pteropus bat also known as flying fox. The major sources of infection amongst humans were raw date palm sap and pigs which were infected by fluids present inside Pteropus bats. The most affected were Asian countries but similar virus was also found in bats across African subcontinent. Nipah infection can be diagnosed in laboratory throughout the critical and rejuvenating stages of the infection by appointing various test combinations. NiV infections in humans and animals are diagnosed by various tests such as virus isolation, serological tests and nucleic acid amplification tests. Since Nipah is highly dangerous and potentially pathogenic contaminant, appropriate cautions have to be appointed while assorting, submitting and handling the sample.

All the work related to Nipah has to be carried out in physical contaminant level. Till date no vaccine has been produced against Nipah virus due to limited experimental conditions, so certain measures could only be taken to prevent against Nipah virus, however the studies are in progress. A vaccine has been developed in Australia for protection of horses against the similar virus i.e. Hendra virus. The vaccine uses Hendra G protein and produces cross protective antibodies against HeV and NiV. Due to no licensed antibodies, only precautions can be taken to prevent infection by Nipah virus.

Osmoadaptation in halophilic bacteria and archaea

Mukhtar Salma, Malik Kauser Abdulla and Mehnaz Samina

Page No: 154-161

Abstract:Microbial diversity characterized from saline environments shows a great adaptation ability to grow under a wide range of salt concentrations. Halophiles are salt loving organisms that grow in salt affected environments and can tolerate a wide range of salt concentrations. According to their salt requirements, they can be categorized into slight, moderate and extreme halophiles. These organisms have a unique ability to survive and grow at high salt concentration and thus could serve as tremendous model systems to understand the molecular basis of high salt adaptation.

The physiology of the halophiles is affected by change in salt concentration, pH, growth temperature and nature of available nutrients. Halophilic bacteria and archaea have evolved two strategies to deal with high osmotic stress. Most of the bacteria use ‘compatible solute strategy’ in which they accumulate different organic compounds like proline, ectoine, glycine betaine and glutamine. Halophilic archaea and anaerobic bacteria cope with high salt stress by ‘salt in strategy’ in which they maintain their internal environment by accumulation of high concentration KCl ions.