Research Journal of Biotechnology

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Research Journal of Biotechnology

Molecular Cloning and Characterization of a Novel 4-Aminobutyrate Transaminase cDNA from the Monascus ruber Mr-5

Ren Hao, Zhang Ting, Qi Yuping and Jiang Donghua

A full-length cDNA encoding 4-aminobutyrate transaminase (designated as MrGABA-T) which catalyzes the conversion of γ-aminobutyric acid (GABA) to succinic semialdehyde, was isolated from the GABA-rich Monascus ruber Mr-5. The full-length cDNA of MrGABA-T has a 1563 bp open reading frame (ORF) encoding a protein of 520 amino acid residues. The deduced protein has an isoelectric point (pI) of 9.12 and a calculated molecular weight of 57.5 kDa. Phylogenetic tree analysis reveals that MrGABA-T is more closely related to GABA-Ts from Aspergillus than to those of other fungi. In this study, the function of MrGABA-T, a gene regulating GABA production, was also analyzed by the disruption of MrGABA-T in M. ruber Mr-5. The results revealed that the GABA maximum production of MrGABA-T-deleted strain (△MrGABA-T) was about 2.8-fold as that of M. ruber Mr-5 (2.67g/L). This work will make a contribution to enhance GABA production at the molecular level in M. ruber.

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Isolation and characterization of citrate synthase gene CaCTS in pepper cytoplasmic male sterility

Fan Shi and Deng Ming-hua

In the present study, pepper citrate synthase (CaCTS) gene was cloned and characterized. This novel gene was then deposited into NCBI database and assigned to accession number JN885193. The results of cDNA sequences of the pepper CaCTS gene demonstrated that it is a gene with a complete open reading frame of 1413 bp encoding a CaCTS protein of 470 amino acids with a molecular weight of 52.466 kDa and pI of 7.26. Similarly comparisons for amino acid sequences reveal that the pepper CaCTS protein has high homology with the CTS proteins of six other species. The phylogenetic tree analysis revealed that the pepper CaCTS protein has a closer genetic relationship with the CTS protein of Solanum lycopersicum and Nicotiana tabacum than with those of Cucumis sativus, Theobroma cacao, Rhododendron micranthum and Populus trichocarpa. Real-time PCR analysis shows that pepper CaCTS gene is expressed in various tissues but at different levels. CaCTS mRNA was highly expressed in flower and seed, moderately expressed in placenta and pericarp, weakly expressed in root, stem and leaf. Transcriptional analysis of the CaCTS gene by qRT-PCR revealed that the gene expression in anthers of the CMS line was clearly lower than in the maintainer at the stages 2 and 3 but no significant differences in the stages 1 and 4.

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Heterologous transcription of a polyketide synthase gene from the lichen forming fungi Usnea longissima

Wang Yi, Zhao Neng, Yuan Xiaolong Hua Mei, Hur Jae-Seoun, Yang Yuming and Wang Juan

Usnea longissima is traditional medicine in Asia and several polyketide synthase (PKS) genes responding bioactive compound were isolated from U. longissima. However, the intron splicing of PKS gene from U. longissima is not clear in Aspergilli. In this study, a PKS of U. longissima (UlPKS6) was ligated into vector pNQ-ArgB which contained the A. nidulans niiA promoter, the Neurospora crassaqa4 terminator, took the ampicillin resistance gene and arginine as a selectable marker, first and then UlPKS6 was transferred into Aspergillus nidulans by polyethylene glycol mediated protoplasts transformation method. The integrity and copy number of UlPKS6 in transformants were detected by long and accurate polymerase chain reaction (LA-PCR) and Southern blot respectively; Reverse transcription-polymerase chain reaction (RT-PCR) and sequence alignment were used to detect gene expression and intron splicing of UlPKS6 in transformants. Nine transformants were obtained with single full UlPKS6 gene DNA from 106 protoplast. Intron splicing of UlPKS6 in transformants is same with in lichen forming fungi. UlPKS6 was transferred into A. nidulans and expressed in A. nidluans successfully. The DNA from U. longissima or other lichen can be used in heterologous expression in A. nidulans directly.

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Simultaneous Determination of Glucuronic Acid, Malic Acid, Taurine and Potassium Sorbate in Functional Drinks by Ion Chromatography

Sha Ruyi and Cai Chenggang

A method for simultaneous determination of four acidic ingredients of glucuronic acid, malic acid, taurine and potassium sorbate in functional drinks was developed by ion chromatography. The mobile phase of 20 mmol·L-1 KOH in 1.0 mL·min-1 was run for 20 min through the anion separation column at 35 °C. The anion suppressor current and the cell temperature were set at 50 mA and 30°C respectively. This method is able to separate and analyze four components of glucuronic acid, malic acid, taurine and potassium sorbate at the same time with their limits of detection of 0.72, 0.64, 2.58 and 1.5μg·mL-1 and the corresponding correlation coefficient of 0.999, 0.996, 0.989 and 0.958 respectively. The average recovery range was of 94–104% with the relative standard deviations of 0.77-1.08% (n=6). The established ion chromatography method can be used in the acid drinks analysis and quality control during the processing and storage of functional drinks.

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Efficacy of plant growth hormones for shoot induction and regeneration in Sesame (Sesamum Indicum L.)

Pusadkar Pratik, Kokiladevi E. and Bonde Shilpa

In vitro regeneration protocol was standardized for two varieties of Sesamum, namely TMV 7 and SVPR 1. Deembryonated cotyledons isolated from whole seeds were used as an explant. The explant cultures were raised on Murashinge and Skoog’s (MS medium) supplemented with different concentrations of BAP (0.5 -2.5 mg/l) in combination with IAA (0.5 mg/l) + ABA (0.5 mg/l) + AgNO3 (0.25 mg/l) with 6 % sucrose. Responded explants resulted into maximum percentage of shoot initiation 67 % and 65.33 % on a medium fortified with BAP (2 mg/l) + IAA (0.5 mg/l) + ABA (0.5 mg/l) + AgNO3 (0.25 mg/l) with 6 % sucrose in TMV 7 and SVPR 1 respectively. Subsequent culturing of these responded explants for shoot elongation on MS medium containing different concentration of GA3 was studied. Highest elongated shoots 47 % and 37 % were recovered on MS medium containing GA3 (0.3 mg/l) in SVPR 1 and TMV 7 respectively. Proliferated and well developed elongated shoots were then rooted in a medium having different concentrations of NAA. This work is an attempt for standardization of regeneration protocol in Sesame and it helps for further genetic improvement by transformation studies.

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Optimal on Bioconversion of Phytosteril into Androst-4-ene-3, 17-dione by Mycobacterium SP-UC-8

Yang Ying, Li Shun, Zheng Zhi and Li Xingjiang

Androst-4-ene-3,17-dione(AD)is an intermediate product in sterol hormones synthesis without any substitutions. It shows an important regulative effect in organisms, thus giving it wide market potential. To improve the yield of AD in the bioconversion process, single factorial experiments and response surface method were carried out to identify the optimal culture medium and fermentation conditions. Results showed that the optimal medium composition for AD production was as follows: molasses 58.5 mL/L, sunflower oil 211.6 mL/L, phytosteril 6.0 g/L, NH4NO3 3 g/L, (NH4)2HPO4 0.8 g/L and HgCl2 0.055 g/L. The production of AD was found to be 2.55 g/L, and the bioconversion rate of AD was 85.8%. The optimum fermentation conditions were identified as temperature of 300C, rotational speed of 220 rpm/min and an initial pH of 8.2 which resulted in AD yield of 2.71 g/L and bioconversion rate of 88.31%. The bioconversion experiment was scaled up to a 5 L stirrer tank, and the optimized conditions were: Volume of medium, 3 L; initial pH, 8.2; stirring speed, 400 r/min; ventilation rate, 3.0 L/min; inoculum ratio, 12% and cultivation temperature, 300C; the production of AD reached 4.42 g/L, and the conversion rate was 92.1%. Using the optimal medium and fermentation conditions, the yield of AD increased significantly along with the bioconversion rate of AD from phytosteril with Mycobacterium SP-UC-8.

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Effect of Storage Conditions on the Stability of the Extracts from Aronia melanocarpa Elliot

Lee Hyeon Yong

The stability of the ethanol extract from Aronia melanocarpa Elliot was studied by being associated with its antioxidant activities according to various storage conditions of temperature, periods and the forms of the extract such as powder and solution. It was interesting that the contents of a major anthocyanin, cyanidin-3-O-galactoside (cya-gal) in the powder were not much changed during the storage period at 250C while those in the solution were decreased after the 4th week of the storage under the same condition. Also, cya-gal contents decreased both in the forms of powder and solution at 400C. In addition, cya-gal contents were not changed for overall storage periods at 40C. The structural changes of cya-gal were also observed by showing significant decrease of UV spectrum at 460∼600 nm wave length during the storageat 400C which was well corresponding to the decrease of pH of the extracts. For the antioxidant activities, DPPH free radical scavenging activity and reducing power of the samples were not much changed at 40C for up to the 5th week of the storage while they were decreased at room temperature and the lowest at 400C by showing great decrease of its activities from the 2nd week of the storage. In this work, it is first reported that the optimal storage condition of Aronia extract was determined at 40C in the form of the power for up to the 6th week of the storage period by simultaneously considering the structural changes and biological activities of cya-gal.

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Screening of selected medicinal plants from Tamil Nadu, South India for antibacterial activity against selected fish pathogenic microbe

Bhuvaneswari Rajagopal, Manickam Narasimman and Bhavan Periyakali Saravana

The purpose of this study was to observe the antibacterial activity of aqueous methanolic extracts of 10 plants against fish pathogen gram negative Aeromonas hydrophila. The minimum inhibitory concentration (MIC) and minimal bacterial concentration were determined by agar well diffusion method and agar dilution method. The bacterium was susceptible to different plant extracts. Acorus calamus, Indigofera aspalathoides and Coleous aromaticaus showed antibacterial activity against all the tested bacteria. The extract of Acorus calamus showed maximum antibacterial activity of the 10 plant extracts used. It is clear from the results of the present studies that the plant extracts have great potential as antimicrobial compounds against bacteria.

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Immobilization of Cephalexin and Cefixime on Carboxylated Multi Walled Carbon Nanotubes (MWCNT) using poly ethylene glycol (PEG) as a cross linking agent

Kumar Vikram, Parecha Darshan Kumar, Choudhary Jignesh and Pathak Amrendra Nath

Multi-walled carbon nanotubes (MWCNTs) possess variable anti-microbial activity. They possess remarkable chemical, electronic, mechanical and optical properties. Being among the most promising materials in nanotechnology, they are also likely to revolutionize medicine. CNTs are hydrophobic in nature. But, after proper functionalization their hydrophobic property can be silenced. Carbon nanotubes can be transformed into sophisticated biosensing and biocompatible drug-delivery systems for specific targeting. In this study, we have demonstrated that the covalent immobilization of the antibiotics Cefalexin and Cefixime on MWCNT-COOH via poly ethylene glycol (PEG) as a linking-agent is possible. Immobilization was confirmed by scanning electron microscopy and Fourier Transform Infra-Red (FTIR) spectra. FESEM images obtained clearly indicate successful attachment of Poly Ethylene Glycol (PEG) via Esterification reaction and immobilization of Cephalexin and Cefixime drugs using Hexamethylene Diisocyanate. FTIR spectra show broad peak at ~3440 cm-1 in MWCNT-COOH and MWCNT-PEG samples which refer to the O-H stretch of the hydroxyl group which can be ascribed to the oscillation of carboxyl groups. But, in case of MWCNT-Cephalexin and MWCNT-Cefixime this peak is shifted to ~3345 cm-1 which refers to amine group of Cephalosporin derivatives. Given the easy, inexpensive immobilization procedure, the MWCNT-drug composite can be used as an effective and economical antibacterial material for biomedical applications in medical treatments of human race.

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Rapid molecular authentication of Ashoka [Saraca asoca (Roxb.), de Wild], vulnerable medicinal plant species and its adulterant Polyalthia longifolia Benth by the development of SCAR markers and multiplex-PCR

Kumar Vinay

Definitive identification and authentication of genuine medicinal plant species is important for standardizing the herbal medicine. Ashoka [Saraca asoca (Roxb.) de Wild] a high valued medicinal plant is listed in the red data list of International Union for Conservation of Nature and Natural resources (IUCN) as ‘globally vulnerable’ species. Due to its critical status, herbal formulations and crude drugs made from S. asoca are often being adulterated with morphologically similar plant, Polyalthia longifolia. This type of substitution or adulteration adversely affects the therapeutic efficacy of the medicinal formulations. The present study was conducted to develop molecular markers for identification and differentiation of S. asoca and its widely used adulterant species P. longifolia using RAPD derived SCAR markers. Out of sixty RAPD primers used, OPQ-01 and OPQ-06 produced specific bands of 448 and 556 bp respectively in all S. asoca lines. Similarly, OPN-19 and OPF-01 produced specific band of 535 and 336 bp respectively in all P. longifolia lines. Species specific fragments were cloned and sequenced for development of SCAR markers. Saraca specific SCAR marker SQ6-1 produced an amplicon of 193 bp from Saraca while there was no amplification in Polyalthia. Similarly, Polyalthia specific SCAR marker PN19-1 production 395 bp fragments exclusively in Polyalthia. Furthermore multiplex-PCR, using these SCAR markers, was developed for the simultaneous discrimination of both the species. SCAR markers developed could be useful for identification and discrimination of Saraca from Polyalthia or vice versa, thus finding fruitful applications in quality control for preventing adulteration in medicinal formulations of Saraca asoca.

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Molecular cloning and expression analysis on a new putative antimicrobial peptide gene encoding a defensin like protein from Allium cepa L.

Wu Peng

Onion (Allium cepa L.) is a vegetable plant which has the potential to become medicinal plant due to its high level of antimicrobial peptide content. A full - length cDNA encoding defensin-like protein (designated as AcD) was isolated from onion (Allium cepa L.) by RT-PCR and RACE techniques. The full - length of the putative AcD was 509 bp containing 231 bp open reading frame (ORF) encoding a polypeptide of 76 amino acids. Comparative and bioinformatic analyses revealed that AcD showed extensive homology with defensin genes from other fungi species. The deduced 77 amino acid sequence showed a high homology with the defensin-like proteins, also known as antimicrobial peptides from the other plant species. Phylogenetic tree analysis indicated that AcD belonged to the fungi group and had close relationship with the defensin-like protein from Carnobacterium divergens. Tissue expression pattern analysis using the method of quantitative real-time PCR (qRT-PCR) indicated that the putative AcD was constitutively expressed in all the tested tissues and strongly in the leaves, weak in the roots. The putative AcD was found to be an elicitor-responsive gene which could be induced by exogenous elicitors including Staphylococcus aureus and Escherichia coli. The transcript levels of AcD were up-regulated after treated by Staphylococcus aureus and Escherichia coli in the leaves, but not in the roots. These results would help to understand the role of the putative antimicrobial peptide gene, AcD, in Allium cepa L.

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Bioremediation and biomass production through mixotrophic cultivation of novel cyanobacteria, Desertifilum tharense MSAK01: A statistical study

Khemka Ankita and Saraf Meenu

The stratagem undertaken for increment of algal biomass with nutrient uptake is through incorporation of nutrients in the coupling methods of biofuel production and dairy wastewater treatment. In present statistical study, Plackett–Burman (PB) design was employed to deduce the functionality of physical and chemical factors, and response surface methodology (RSM) was utilized for their optimization. The concentration of total phosphorus (TP) and total nitrogen (TN) present in the cultivation medium was 183.4 mg L-1 and 160.0 mg L- respectively. The results for subsequent biomass production and TN and TP removal before and after optimization were 2.65 g L-1, 80.2% and 72%, and 3.6 g L-1, >96% and >93% respectively. Gas chromatography and mass spectrometry analysis (GC-MS) of the fatty acid methyl esters (FAME) extracted from optimized biomass showed enhancement in the saturated fatty acid after optimization (C16:0 and C18:0; ~ 63%) compared to the original fatty acid profile (~ 60.7%). Thus, the present study strongly confirmed Desertifilum tharense MSAK01 as potential and valuable feedstock for bioremediation of dairy wastewater.

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Optimization of sample preparation for Two-Dimensional Gel Electrophoresis analysis of Gluconobacter oxydans membrane proteins

Lulu Shi, Jinping Lin and Dongzhi Wei

Gluconobacter oxydans is widely used in industrial biotechnology. To optimize the treatment methods of two-dimensional electrophoresis (2-DE) and establish the 2-DE profiles of membrane proteins of Gluconobacter oxydans, we emphasized on evaluation and comparison of three different solubilization buffers and optimized 2-DE conditions for membrane proteins of Gluconobacter oxydans. Efficient solubilization was obtained with 2-DE buffer composition of 5 M urea, 2 M thiourea, 2% (w/v) CHAPS, 0.5% (v/v) Triton X-100, 2% (w/v) SB 3-10, 1% (w/v) DTT, 40 mM Tris-base, 0.5% (v/v) pharmalyte 3-10. More protein spots were detected when the cells were broken by high-pressure homogenization. The optimal protocol for isoelecric focusing (IEF) was accumulated voltage of 100,000 Vh. A better distribution of the protein spots on the map from pI 5 to 7 was observed while2-DE was carried out with non-linear pH gradient IPG strips (24 cm, pH 3-10). Using optimized conditions, we used 2-DE analysis of type II NADH dehydrogenase overexpressing G. oxydans toidentify 8 differentially-expressed protein spots byMALDI-mass spectrometry.In conclusion, we have successfully optimized the conditions for Gluconobacteroxydans membrane proteins analysis by 2-DE. These optimized conditions will lay the basis for further profound studies in the field of proteomics.

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Nanoencapsulated Tea Polyphenols as Anticancer Agent

Karikalan Kulandaivelu and Mandal Abul Kalam Azad

Polyphenols are important anticancer compounds present in tea. Among the constituents of tea polyphenols (TPP), catechins constitute about 30-42% of the dry weight. TPPs target many signaling pathways in cancer such as initiation, promotion, metastases and apoptosis. Various studies revealed that treatment with EGCG (major fraction of catechins) reduces the incidence of tumor genesis and multiplicity in different organ sites like stomach, liver, breast, prostate skin and colon. TPP is an effective therapeutic agent but the bottlenecks to use TPP as a therapeutic agent are its chemical instability, potentially low bioavailability and short half-life. To overcome these limitations, approaches like nanoencapsulation have been attempted. Nanoencapsulation of TPP in gelatin, solid lipid nanoparticles (SLN), poly lactic acid (PLA), poly lactic glutamic acid (PLGA) and other type of nanoparticles can be a novel way to protect TPP from chemical instability and enhance bioavailability. In this review we discussed the TPP as anticancer agent, its problem to be used as therapeutic agent and use of nanotechnology to overcome these problems.

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