Research Journal of Biotechnology

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Vol. 11(4) April 2016

Optimization of culture medium for rifamycin SV production by Amycolatopsis kentuckyensis 22-187

Mou HuiYan, Wang Yingdong, Sun Guizhi, Dai Jianlu, Wang Guanglin, Wang Yiguang and Zhang Huitu

A new strain harboring 3-amino-5-hydroxy benzoic acid (AHBA) synthase gene was isolated from China soil and taxonomically was identified as Amycolatopsis kentuckyensis 22-187. It was confirmed that the 22-187 strain mainly produced rifamycin SV and B by chemical identification. The culture medium for Amycolatopsis kentuckyensis 22-187 was optimized on a shake-flask scale by using Plackett-Burman design and response surface methodology for enhanced rifamycin SV production. The Plackett-Burman design indicated that the main factors that positively affect rifamycin SV production by Amycolatopsis kentuckyensis 22-187 were the concentration of glucose and peptone in the fermentation medium. A final concentration of 11.02% (w/v) glucose and 0.98% (w/v) of peptone was found to be best for rifamycin production. Using an optimized fermentation medium (citrate NH4 0.5%,K2HPO4 0.1%,MgSO4 0.1%,CaCO3 0.5%, glucose 11.02%, peptone 0.89%), the titer of fermentation of rifamycin SV was increased two folds comparing with that in the initial medium by HPLC analysis but the rifamycin B production keeps the same as in initial medium.

Optimization of keratinase activity produced by mutant Streptomyces sp. IF5 using Response surface methodology

Gandhirajan Priya, Radha Venkatesh Nagarajan, Karunanithi Jeyannathann and Ramakrishnan Jayapradha

In this study, we employed a two-step protocol to fine tune the enzymatic activity of the keratinase isolated from a mutant strain of Streptomyces sp. IF5. First, we identified physical factors (like temperature, pH) required for optimal enzymatic activity (through one-factor-at-a-time method, OFAT) at treatment stage and coupled these results with response surface methodology (RSM) using central composite design (CCD) to determine their mid-values. Through OFAT, we arrived at parameter ranges such as temperature (30-70°C), pH (4-13) and calcium salt concentration (25 to 75 mM). In RSM, we included substrate concentration, inoculum size, pH (for production of enzyme) alongside Ca2+ ion concentration, temperature (and pH of borate buffer for activity) and simulated response patterns through a 20 full-factorial rotatable CCD. The optimized variables were 1% substrate concentration, 5% inoculum size, pH 12.0 (for production), 75mM Ca2+, pH 8.0 and 40°C (for enzyme activity). Employing these variables in production media followed by purification of crude extract with DEAE-Sephadex-A50 column, specific enzyme activity was estimated at 17.168 U/mg of keratin and the purified enzyme had a molecular weight of ~66 kDa.

Statistical optimization of cellulase production from a new strain of Bacillus subtilis VS15 by central composite design and artificial neural network

Soujanya Lakshmi Ega, Ramya Krishna Kanamarlapudi, Manda R.NarasingaRao and Sudhamani Muddada

In the present study, a new species of Bacillus, Bacillus subtilis VS15 (GenBank accession number-KT210118) that is capable of producing cellulase was isolated from the decomposing logs of textile industry and optimized for enzyme production. One-factor-at-a-time method was employed to screen the key factors responsible for enzyme activity, further optimization and prediction were carried out by Central Composite Design (CCD) and Artificial Neural Network (ANN). The effects of different carbon sources like Carboxymethylcellulose (CMC), Cellobiose, Filter paper were examined for cellulase production at different conditions of incubation period (8–24 h), temperature (35°C–55°C), substrate concentration (0.5-2.5%) and pH (4.5–8.5). CMC was found to be the best carbon source for cellulase production followed by Cellobiose in this bacterial strain. Optimal conditions for cellulase production were found to be at pH 7.5, temperature at 50°C, substrate concentration at 2% (w/v) and incubation time of 20h.Under these optimum conditions, maximum CMCase activity was observed to be 0.5 IUml-l against the predicted values of RSM 0.49 IUml-l and ANN 0.5 IUml-l. Statistical studies revealed that ANN model was more accurate in prediction over RSM with regression coefficient (R2), absolute average deviation (AAD) and root mean square error (RMSE) values 0.9732, 0.027% and 0.0146 for ANN and 0.9593, 0.049% and 0.179 for RSM.

A New Strategy in the Synthesis of Hollow γ-Al2O3 Nanosphere using Alginate Gel Casting Process

Sadjadi M.S. and Rostamizadeh N.

In this work, we report fabrication of hollow γ-Al2O3 nanospheres with ca. 250-350 nm in diameter and 20 nm in shell thickness by water in oil inverse microemulsion method at room temperature using cyclo-hexane containing span85 nonionic emulsifier and sodium alginate as coagulating agent. Al(NO3)3•9H2O was used as precursor and cross-section builder between the polymer chains, (NH4)2SO4 or ammonia as pH tuning and calcination of the sample was finally performed at 650 oC for 3 hours. Characterization of the samples was then performed using X-ray diffraction (XRD) pattern and Fourier transform infrared (FTIR) spectroscopy. The surface morphology, hollow sphere diameter and wall thickness of the synthesized samples were examined by scanning electron microscopy (SEM). The specific surface area, average pore size and total pore volume of the samples were determined using BET technic and indicated higher specific surface area and total pore volume of the sample to be 226.25 m²/g and 0.3865 ml/g respectively. From the higher specific surface area and other high characteristics of hollow γ-Al2O3, we can clearly infer about suitability of the hollow γ-Al2O3 in a wide range of applications such as filters, membranes, sensors, catalyst carriers, piezoelectric ceramics, biomedical and construction materials.

Molecular cloning, sequence characterization of a novel pepper gene CaNDPK and its effect on cytoplasmic male sterility

Lv Jun-Heng, Zhao Kai, Huo Jin-Long and Deng Ming-Hua

Nucleoside diphosphate kinase (NDPK) is a key enzyme which catalyzes the transfer of the gamma phosphates from NTP to NDP. NDPK is considered as housekeeping enzyme involved in energy metabolism and homeostasis of intracellular NTP pools. The complete coding sequence (CDS) of the CaNDPK gene was amplified using a reverse transcriptase PCR based on the conserved sequence information of the tomato and other Solanaceae plants and known highly homologous pepper ESTs. 148 amino acids were encoded by the 447 long cDNA of the pepper CaNDPK gene. Nucleotide sequence analysis showed that the encoded amino acids were highly homologous with the seven species, Nicotiana tomentosiformis (97%), Nicotiana sylvestris (97%), Solanum lycopersicum (95%), Coffea canephora (94%), Flaveria bidentis (93%), Jatropha curcas (92%) and Camellia sinensis (90%). The result of the tissues expression showed that the pepper CaNDPK gene is over expressed in placenta, moderately in seed, weakly in stem, leaf, flower and pericarp. During the abortion stages, expression levels of CaNDPK in anthers of the sterile line were different with that in the maintainer and the CaNDPK gene expression level of transcripts in the maintainer line is much more stable than that in the sterile line. The unusual and the instable expression levels of CaNDPK may disturb the balance of energy metabolism in the sterile line indicating that stable transcripts of CaNDPK are necessary to maintain energy metabolism at a normal level. The CaNDPK gene plays an important role in keeping the balance of the energy metabolism within normal levels during microspore development.

Assessment of antioxidant and in vitro inhibitory potential against key enzymes catalyse for hyperglycemia and prostate inflammation

Azhagu Saravana Babu Packirisamy, Vajiha Aafrin Basheer , Sudharsan Kasirajan and Muthusamy Sukumar

The present study focussed on evaluation of nutraceutical properties of the bioactive components from the extracts of Cucumis sativus seed (CSS). The antioxidant property of CSS extracts were carried out by estimating total phenolic and flavonoid contents and radical scavenging evaluations. The results revealed that the aqueous extract of CSS rich in phenolic (88.5 ± 0.1 mg GAE /g) and ﬂavonoid (23.5 ± 0.2 mg QE/g) compounds than alcoholic extracts. The inhibitory activity against α- amylase and α- glucosidases enzyme linked to type- 2 diabetes was evaluated and the aqueous extract was exhibited maximum amylase and glucosidase inhibition of 95.1 ± 0.2 and 92.6 ± 0.3 % respectively. The bioactive compounds had significant therapeutic effect against benign prostate hyperplasia (BPH) by inhibiting the key enzyme lipoxygenase. Lipoxygenases inhibitors had antiproliferative effects against prostate cancer cells, therefore their presence in the extracts of CSS could possible exert protective action against prostate hyperplasia. The natural antioxidant and polyphenols present in the extracts showed abundant nutraceutical value and it may be incorporated in to food to enhance the functional characteristics and the healthy diet.

Assessment of genetic diversity among Chickpea (Cicer arietinum L.) genotypes using EST-SSR markers and SDS-PAGE

Kumar Ashwani, Chaudhary Sorabh, Sagar Sushma, Kumar Vinay and Kumar Mukesh

Chickpea is a major and economically important cool season grain legume crop in the world and is widely distributed in the arid and semi-arid regions. Knowledge of genetic diversity and relationship within and between the cultivated chickpea genotypes and its wild relatives are of paramount importance and may ensure the long-term success of chickpea improvement programs. The development of expressed sequence tags (ESTs) has provided a reliable source for the mining of simple sequence repeat (SSRs) markers for investigation of genetic diversity. The effectiveness of EST-SSR markers and total seed protein profiling (SDS-PAGE) were investigated to assess the genetic diversity among and within twenty Cicer arietinum L. genotypes. Out of 20 EST-SSR primer pairs, 17 pairs showed amplification in which thirteen primer pairs (76.5%) were monomorphic and 4 primer pairs (23.5%) were polymorphic in the chickpea genotypes. Polymorphism information content (PIC) values ranged from 0.99 (locus ICCeV0008) to 0.05 (locus ICCeV0006) with an average of 0.47. The resolving power of the EST-SSR primers varied from 0.5 to 3.2. Based on UPGMA clustering method, all genotypes were clustered in three groups which indicated the probable origin and region, similarity of landraces and local Indian landraces over the other cultivars and wild species. SDS-PAGE results revealed that seeds of chickpea genotypes are rich in storage proteins with a number of stable bands in the gel. The major components of all the species were in the molecular weight ranged from 95 to 11 kDa, with the variation in relative mobility values. Cluster analysis sorted the genotypes into two major clusters. Genetic diversity detected in this study can be useful for selective breeding programme and in enhancing the genetic base of breeding programs.

Exploitation of microorganisms for the production of industrial enzyme- A tool for Biotechnological applications

Sivakumar G.S. and Perumal Raj R.

The study focussed on the production and purification of laccase enzyme from Trametes versicolor and the optimum parameters were analyzed. The effects of carbon and nitrogen sources, initial pH and incubation temperature on laccase activity produced by Trametes versicolor were studied. The optimization study of various parameters including temperature, pH, substrate and inhibitors was done and the molecular weight was obtained at 64 kDa using SDS- PAGE and the SDS-PAGE showed a band with a molecular weight of 64kDa and the ammonium sulfate precipitation showed a specific activity of 6.28U/mg protein and the yield was 78.5%. The laccase reached its maximum activity at the pH of 3- 4 and the activity was more at the temperature between 25- 35°C. The maximum activity of laccase was observed for sucrose and glucose and the laccase was highly inhibited by sodium hydrogen sulphate and sodium meta sulphate. The study revealed that the Trametes versicolor produced maximum amount of laccase with cost effective manner and the enzyme has more industrial applications.

Effect of methanolic root extract of Blepharispermum subsesssile DC in controlling arthritic activity

Das Soni and Sureshkumar P.

The aim of the study was to evaluate the in vitro and in vivo anti-arthritic potential in the roots of Blepharispermum subsessile DC (MEBS). In vitro models such as HRBC membrane stabilization (Human red blood cell), inhibition of protein denaturation and proteinase inhibitory activity was performed for the assessment of anti-arthritic activity. Three different in vivo models of arthritis namely Freund’s Complete Adjuvant (FCA)-, formaldehyde- and carragennan-induced models were also used in this study. The MEBS significantly inhibited the HRBC (95.00±0.03), protein denaturation (94.22±0.04) and proteinase inhibitory activity (85.44±0.45). The various biochemical parameters (Hb, Total WBC count, RBC count, ESR and C-reactive protein) histopathological analysis and radiographic examinations studies substantiated the antiarthrtic potential of MEBS.

Cytosine deamination rates analysis to determine species origin in ancient mixed and processed Animal tissues

Matar Rachel and Merheb Maxime

In living organisms, Cytosine deamination is one of the most common forms of DNA lesion where cytosine is converted to uracil. In such DNA molecules, DNA polymerases will incorporate deoxyadenosine as complementary to the uracil where at those positions a deoxyguanosine should normally have been incorporated, thus, C to T and G to A transitions will appear in the amplified DNA sequences. While such lesion is very abundant in ancient DNA, in the present study our goal in Cytosine deamination analysis is not only to check sequences authenticity but also to compare signatures of sheep and cattle mtDNA substrates in order to determine the species origin of an ancient leather and glue samples in a complicated mixture.

Excavation of Rice Ortholog of Metal Transporter Gene OsNAS2 in Micronutrient Dense Minor Millet Crops

Dubey Mahima, Patil Arun H. and Chandel Girish

Minor millets serve as alternative and healthy food choices for population with entire reliance on staple crops which are usually scanty in micronutrients. In the present investigation, the aid of advancing computational methods has been employed to identify more potent genes or orthologs their off for refining the improvement methods of nutritional quality traits. An ortholog of Nicotianamine synthase family gene NAS2 was identified through amplification, sequencing and characterization in ten minor millet genotypes belonging to four different categories. The genotypes were initially characterized for grain micronutrient levels and the estimation showed Barnyard millet genotypes to be a typically rich in Fe and Zn. In contrast, Finger millet was found to contain relatively lower levels of these elements. Structural analysis of millet NAS2 sequences revealed the presence of uninterrupted coding region (intronless gene with single exon) confirming the structural features of NAS genes of rice. Multiple sequence alignment revealed that a finger millet genotype BR- 36 bearing lesser amount of Fe and Zn content formed an out group with patches of insertion and deletion of size ranging from 2 to 12 nucleotides. This analysis also revealed high level sequence homology of millet NAS2 sequences (with an exception of BR-36) to the rice NAS2 gene. Further, computational based physico- chemical characterization of the predicted protein of millet NAS2 sequences showed their proteins to have better interaction with water. Out of ten millet genotypes studied, six millets showed the presence of NAS domain, a domain unique to this family of metal transporter genes. Protein modelling of these showed the presence of reliable amount of amino acid residues in the core region of the protein with good quality model confirming well to the stereochemistry. The predicted protein models showed remarkable similarity to the NAS2 protein of model plant genomes. This high degree resemblance establishes their orthology and provides significant insights into the molecular bases of NAS2 gene in metal ion homeostasis

Identification of suitable parents for the development of populations for mapping genomic regions controlling commercially favourable pomological traits in guava (Psidium. guajava)

Naga Chaithanya M. V. , Dinesh M. R., Ramesh S., Sailaja D., Vasugi C. and Aswath C.

Developing mapping populations to identify DNA markers linked to economically important traits in crop plants with no exception, requires identification of genotypes contrasting for target traits and for a larger number of DNA markers. Putative parents contrasting for pomological traits as well as at several SSR markers were identified from among the 52 guava accessions being maintained at Indian Institute of Horticultural Research (IIHR), Bengaluru, India. The pairs of accessions namely, Benaras and Seedless, Dhareedar and Seedless, Dhareedar and EC-147039, Abu Ishaqwala and Pati for fruit weight, Benaras and CIW1, Benaras and Surkha Chitti Neptuani, Sringeri Seedless and Surkha Chitti Neputanifor outer pulp thickness, Allahabad Safeda and CIW1, Hisar Safeda and Local 4, Hisar Safeda and CIW1, for total soluble solids and 9-35EC147036 and Florida Seedling, 9-35 EC-147036 and Arka Mridula, GR1 and Hisar Safeda, GR1and Florida Seedling for seed hardness were polymorphic at more number of SSR loci. These contrasting pairs of accessions are suggested for use as parents in developing mapping populations which can be utilized to identify SSR markers linked to genomic regions controlling economically important pomological characters in guava. Additionally, ten different SSR markers that can be used to distinguish five best performing guava accessions with respect to fruit weight have been identified.

QTL Mapping for Downy Mildew Resistance in Pearl Millet [Pennisetum glaucum (L.) R. Br.]

Sanghani A.O., Vaja M.B., Ramani H.R., Vadher K.J., Sanghani J.M., Bambharolia R.P., Garaniya N.H. and Golakiya B.A.

Pearl millet [Pennisetum glaucum (L.) R. Br.] is a multipurpose cereal grown for grain, stover and green fodder. Relative to other cereal species, it ranks fifth in annual world production. Use of molecular marker technology for pearl millet genetic improvement has been limited because of an insufficiently developed linkage map. The majority of the pearl millet molecular markers (RFLP and SSR) mapped to date are clustered around the seven pearl millet chromosome centromeres and only a few marker loci are mapped to more distal regions. There is a need for more evenly distributed markers throughout the genome linkage and greater marker coverage of the gaps in earlier established maps. The objective of this research was to develop a PCR-based molecular marker linkage map of downy mildew concened molecular markers of pearl millet on the basis of a segregating F2 population resulting from a cross between J-2480 (Resistant) and J-2372 (Susceptible). This mapping effort involved 125 F2 populations and 24 polymorphic PCR-based SSR. The average genetic distance between markers was 10 cM, varying from 4.9 to 21.2 cM. The number of markers in each linkage group (LG) ranged from 2 to 7. The resulting map consisted of seven linkage groups that spanned about 240.8 cM. The majority of the markers used in this study to construct the genetic linkage map were randomly distributed in each LG which makes it useful in pearl millet mapping and breeding.

The adaptor protein MyD88 essential for EHEC induced inflammatory responses in HT-29 cells

Jinbo Wang and Lili Qi

Intestinal epithelial cells (IECs) not only represent a physical barrier to the pathogens but also participate in immune and inflammatory responses. The immune inflammatory response of IECs plays important roles in the intestine disease induced by Enterohaemorrhagic Escherichia coli (EHEC). The synthesis and release of the pro-inflammatory cytokines IL-6, IL-8 and TNF-α were elevated by EHEC infection in HT-29 cells. EHEC infection significantly induced the translocation of NF-κB(p65) into the nuleus and activated the NF-κB signaling pathway. MyD88 silently and significantly inhibited NF-κB activation and pro-inflammatory cytokine production stimulated by EHEC. EHEC could activate the inflammatory response by a MyD88-dependent signaling pathway in HT-29 cells.

Impact of anthropogenic disturbances on genetic diversity in Tectona grandis L. f. - analysis using nuclear gene markers

Nair Pramod N. and Palanivel Hemalatha

Genetic resources of teak (Tectona grandis L.f.) have been very much altered during the past several years through anthropogenic disturbances and there has been much concern over the protection of the remaining natural teak populations. The repercussions of human interference are also to be evaluated to formulate strategies for effective conservation and sustainable management of teak genetic resources. Hence, the impact of human disturbances on the genetic diversity of natural teak populations in India was analysed using three highly polymorphic nuclear gene markers. Seven paired natural teak populations, disturbed and protected, were selected in the states of Kerala, Madhya Pradesh, Gujarat and Orissa. A total of 26 alleles were identified for the three nuclear gene markers G3PDHcy1, G3PDHcy2 and CAT. The study showed a reduction in gene diversity and other genetic parameters such as allelic richness, number of rare alleles and heterozygosity in the plots affected by human disturbances. Out of the ten rare alleles observed in the protected populations, six were absent in the disturbed stands. The significant reduction in allelic richness and loss of rare alleles will lead to changes in gene flow and mating patterns and may affect the fitness and evolutionary adaptability of teak.

A draft genome sequencing using next generation sequencing technology in black sesame (Sesamum indicum L.)

Vaja M. B. and Golakiya B. A.

Sesame (Sesamum orientale L.) is one of the most ancient and important oil seed crop, also known as S. indicum L. The diploid chromosome number of sesame is 2n=26 and it is usually self-pollinated. Sesame belongs to the Pedaliaceae family. Sesame GT-10 (Black sesame) oil is good source of some phyto-nutrients. In present study, ABI SOLiDTM and Ion Torrent Whole Genome Sequencing (WGS) technologies were used to generate sesame draft genome. For the measurement of the genome size, flow cytometer (Accuri C6) was used, found approximately 375Mb. The total raw sequence generated by SOLiD genome analyzer was 61.07 Gb. PGM generated 1.8 Gb data. The de novo assembly (using SOLiD analyzer) yielded assembled reads of 2,962,553,296 and number of contings was 63,785,429. de novo assembly of PGM (combined data four runs) yielded assembled reads of 48,729,832 with number of contings count as 62178.5. In the assembly the N25, N50, N75 conting size was 737.75, 533.5 and 396.5 bp respectively. In Blast 2GO analysis, total 94752 sequence were functionally annotated out of which 94705 (99.95%) showed positive interpro. While 71160 (75.10%) got Blast hits, 73.32% (69474) and 29.59% (28044) sequence were mapped and annotated while in gene ontology, total 101098 GO IDs were found among which 27.61% (36201), 49.70% (65165) and 22.67% (29732) were grouped into biological process, cellular components and molecular function, respectively. Using BatchPrimer3 (V 2.0), total 406 SSRs were identified with Tm and GC % range of 62.23-50.15 and 66.11-33.00 respectively.

The static extraction of lipid from microalgae Desmodesmus sp. MCC34

Nagappan Senthil and Verma Sanjay Kumar

The commonly used technique for extraction of lipids from dry or wet biomass involves energy intensive steps such as cell lysis, high temperature and cell mixing causing substantial energy burden on the process. In present work, we report our finding on using a static method of mixing standard solvent with dry algal biomass without stirring. This extraction procedure was found to depend on the ratio of solvent volume to the biomass (SBR) and surface area factor (SAF). The kinetic study suggests that the static extraction followed Patricelli model of bi-phasic lipid extraction, consisting of a rapid washing step followed by the diffusion step. The results also suggest that the rate of lipid extraction in static process at optimum SBR and SAF matched the rate of extraction obtained when lysed biomass was used or in the case where biomass was mixed (stirred) with solvent.

Computational inhibitor screening on human APEX1 (Multifunctional DNA repair enzyme) for Glioma

Rohini Karunakaran and Srikumar Padmalayam Sadanandan

Glioma, a malignant tumor arises from glial cells of brain. The process of DNA base excision repair (BER) modulation plays a vital role in chemotherapy response of glioma. Among the BER enzymes, human APEX1 (apurinic/apyrimidinic endonuclease 1) is directly involved in repair of DNA damage through base excision. APEX1’s potentially cytotoxic abasic sites (AP sites), is a promising new target in cancer. Hence, our study was focused on screening novel inhibitors against human APEX1 for glioma using computational approach. The structure based virtual screening was applied for the human APEX1 (PDB code: 1HD7) for the glioma treatment. Combined methodology of virtual screening and molecular docking were followed for the computational study. Initial virtual screening was carried out using the large library of ZINC database in I-screen server. Two steps re-docking were performed in AutoDock Vina and AutoDock for the lead candidate identification. Finally the lead candidates were optimized by Lipinski’s rule of five. In our results, top 200 hits with best score were screened based on the rapid docking process from the ZINC database. The strong inhibition of human APEX1 was confirmed by appropriate hydrogen bond interactions that favored the stable APEX1-inhibitor complex. The lead candidates also favored with drug likeness properties. In conclusion, the computational screening on human APEX1 showed the novel inhibitors N-(4-phenylthiazol-2-yl) oxamide and N,N'-bis (2-hydroxypropyl) oxamide with effective inhibition. Thus, these results can be further confirmed by experimental studies for future anti-glioma drugs.