Research Journal of Biotechnology

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Research Journal of Biotechnology





Comparing LC50 and Bioaccumulation rate of Nano Zinc Oxide and Zinc Oxide by Eisenia fetida in vermicompost

Soodabeh Nadiri, Ghasemali Omrani, Mina Makki Ale Agha, Mozhgan Emtyazjoo and Hojat Zakeri

In recent years, instead of using metal oxides, nanoparticles have received much attention due to their unique properties. Apart from being useful, nanoparticles can cause risks to the environment from the beginning of the production to disposal. Therefore, environmental risk assessment of nanoparticles is necessary. Earthworms are indicators of environmental pollution, therefore in this research, Eisenia fetida was selected to determine its capability towards up-taking nano-Zno and ZnO. The toxic effects of the metals were examined as well. In order to determine the absorption rate, the earthworms Eisenia fetida were exposed to the concentrations 0, 2.5, 5, 10, 20 and 40 g/kg dw of nano-Zno and ZnO for 28 days. Afterwards, the earthworms were digested and measured with atomic absorption device. To determine LC50, the earthworms were exposed for 42 days at different concentrations of nano-Zno and ZnO including 20, 40, 80, 160, 320 and 640 g/kg dw in the substrates of natural soil as well as soil and cow manure separately. The obtained results indicated that the earthworms can absorb nanoparticles just like the zinc heavy metal so that with increasing the concentration of ZnO and nano-Zno in soil, their accumulation rate was increased in the earthworm tissue as well. Highest values of bioaccumulation factor were assigned to the samples containing ZnO concentration of 5 g/kg dw and the samples containing nano-Zno concentration of 40 g/kg dw. The LC50 obtained for Zno and nano-Zno were respectively equal to 73.525 g/kg dw and 66.651 g/kg dw in natural soil and 249.183 g/kg dw and 216.745 g/kg dw in vermicompost. The organic matter, nitrogen and C/N ratio were measured at the beginning and end of the experiment. After 28 days, the amount of organic matter decreased from 32.3% down to 24.6%.

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Effects of Microwave irradiation and conventional heating in controlling microbial Loads in selected Tropical Fruit pulps

Kanimoli S. and Alagusundaram K.

Food industry in the recent years has witnessed the emergence of microwave oven as a substitute to thermal oven for a number of food manufacturing processes and products. Application of microwave energy for various industrial food-processing operations is found to be economical and inevitable. In this study, attempts were made to determine the effects of microwave and conventional heating in reducing the microbial loads (Bacteria and Fungi) in mango, papaya, sapota and jackfruit pulps. A thermostat controlled water bath was used for conventional heating. The fruit pulps were heated to 60, 70 and 800C for 15, 30 and 60 s. A domestic microwave oven was used for the microwave irradiation studies. The fruit pulps were irradiated for 15 s at 40, 70 at 100% power levels (at 100% level the microwave oven will supply 900 W of power). Number of survivors, decimal reduction time (D value), the process sterilizing value (for a 12 D cook) and the temperature change required for a 10 fold change in D value (Z value) were presented. Microwave heating resulted in an excellent killing of bacteria and fungi compared to conventional heating. In conventional heating the D values ranged between 1 min 3 s to 1 min 52 s for the four fruit pulps studied. The Z value varied from 330 C to 970 C. A phenomenal saving in energy was observed when fruit pulps were sterilized using microwaves instead of conventional heating. The quantity of heat required for one log cycle reduction in population ranged from 145.8 kJ / kg to 253.5 kJ/ kg for the fruit pulps exposed for conventional heating. The energy absorbed during microwave sterilization ranged from only 1.39 kJ / kg to 3.7 kJ / kg for the fruit pulps.

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Identification of Resistance Gene Analogs (RGAS) from Highly Wilt Resistant Castor (Ricinus Communis L.) Genotype

Kapadia C. V., Mahatma M. K., Parekh M. J., Patel Nafisa and Tomar R. S.

Castor (Ricinus communis) is highly susceptible to bacterial and fungal pathogens. The identification of resistance gene analogs holds great promise for development of resistant castor cultivars. Primer selection was performed based on different combinations of motifs conserved in R-gene classes. Degenerate primers designed based on known resistance genes (R-genes) were used in combinations to elucidate resistance gene analogs from Castor. Only seven primer combinations gave amplicons with expected sizes of 450-900 bp. The nucleotide sequence of these amplicons was obtained through sequencing; their predicted amino acid sequences compared to each other. Only two sequences, namely p6.4 and 23 had signifi¬cant sequence identity with known R-genes sequences of other plants. The findings of conserved domains, viz., kinase-1a, kinase-2 and hydrophobic motif, provided evidence that the sequences belong to the NBS-LRR class gene family. Homology and % identity searches of the isolated RGA sequences with the NCBI GenBank database showed that p6.4 had 89% identity to the disease resistance like proteins in castor and 23 had 100% identity with I2C disease resistant proteins in tomato. Phylogenetic analysis indicated that the RGAs clustered with R genes found in Arabidopsis and Tomato. The findings will be useful for studying of resistance mechanism in castor to Fusarium oxysporium.

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Design and fabrication of core-shell gold coated Fe3O4 magnetic nanoparticles as Cancer Cells Targeting agent

Karamipour Shahnaz, Farhadyar Nazanin and Seyed Sadjadi Mirabdullah

Core shell gold coated magnetite iron oxide nanoparticles can be used in biomedical applications such as magnetic bioseparation, bioimaging, drug delivery and cancer cell treatment. In this work, we synthesized gold coated magnetic nanoparticles in various molar ratios of Fe3O4 and gold using magnetite nanoparticles prepared in an average size of 5.6 nm diameter by a simple chemical co-precipitation method. Prepared nanostructure was then functionalized by 3- aminothiophenol (3-ATP) and folate-conjugated to prepare folate receptor targets on the tumoral cells surface. Characterization of gold coated magnetic nanoparticles was carried out by various techniques such as X-ray powder diffraction (XRD) analysis, Fourier transform infrared (FT-IR) and UV-visible spectroscopy, Field emission scanning electron microscopy (FESEM) and finally Vibrating Sample Magnetometer (VSM) analysis. The results indicated attachment of the functional agent, 3-ATP on the surface gold nanoparticle through thiol groups and its conjugation with folic acid via amine group of 3-aminothiophenol.

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Effect of Transcranial Magnetic Stimulation’s Repetition Pulse on Cox-2, IL-6 and cell death in ischemia induced rats ‏

Kim Whi-Young

The majority of strokes are caused by ischemia and result in brain tissue damage, leading to problems of the central nervous system including hemiparesis, dysfunction of language and consciousness and dysfunction of perception. This paper reports the development of an economic compact pulse type transcranial magnetic stimulation with a 1kHz pulse repetition rate. The high-voltage pulse powered device using a high-voltage high frequency pulse transformer and IGBT (Insulated Gate Bipolar Transistor), which is suitable for tens of kHz-switching, was engrafted to a transcranial magnetic stimulation and a new type of high-repeating transcranial magnetic stimulation was developed. To accurately control two of the IGBT that were used in power unit, an AVR One-Chip-Microprocessor was used for the controlling part and the stimulating coil was manufactured using a water-cooling method. An examination of the output of the stimulating coil according to the changes in the pulse repetition rate and control of the finished device showed that the maximum output of the transcranial magnetic stimulation (700 W) was reached at a pulse repetition rate of 80 Hz. After induction of brain ischemia, TMS was applied to acupuncture at ST 36, at 12, 24 and 48 hours. Protein expression was investigated using immuno-reactive cells which react to PARP antibodies in cerebral nerve cells and Western blotting. PARP expression after 24 hours significantly decreased (p<0.05) in the TMS group compared to the GI group. As a result, based on the results of this study, TMS can be an effective method of treating dysfunction and improving function of brain cells in brain damage caused by ischemia.

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Enhancement of in vitro shoot multiplication in banana cv. Rajapuri (AAB) using TDZ

Manjula R., Jholgiker Praveen, Venkata Subbaiah K., Swamy G .S. K. and Prabhuling G.

The ‘Rajapuri’ (AAB) banana cultivar is very popular in Belgaum and Bagalkot districts of Karnataka. However, in recent years its cultivation decreased due to rapid spread of Sigatoka leaf spot disease through sucker propagation. Availability of tissue culture multiplied plant is very scarce as it is highly recalcitrant to tissue culture. The present investigation was undertaken to enhance shoot proliferation using TDZ. Shoot tip explants derived from sword suckers were inoculated on MS medium supplemented with different concentrations of Thidiazuron [TDZ (0.2 and 0.3 mg/L)], 6-Benzylaminopurine [BAP (2.0 and 5.0 mg/L)] and Naphthalene acetic acid [NAA (0.2 mg/L)] alone or in combinations. Cultures were incubated at 25±30C with a 16 hour photoperiod (2000 lux) provided by cool white florescent tubes. Among the all treatments TDZ 0.2 mg/L produced the maximum number of shoots (3.04) and number of adventitious buds (2.67) per explants as compared to BAP.

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Estimation of correlation and path analysis for yield and yield attributing traits in Blackgram [Vigna mungo (L.) Hepper]

Lenin kumar Y., Anuradha CH., Sokka Reddy S., Srinivas A. M. N. and Venkata Subbaiah K.

Correlation and path analysis studies were made for yield and its attributing traits in fifty blackgram genotypes. Based on correlation studies, it was observed that the number of branches per plant, number of clusters per plant, number of pods per plant, number of pods per cluster, pod length and number of seeds per pod showed highly significant positive correlation with seed yield per plant at genotypic level and improvement of these characters might contribute to high seed yields in blackgram. Path analysis revealed the highest positive direct effects on seed yield by number of seeds per plant followed by number of pods per plant, number of seeds per pod, pod length, number of branches per plant and 100 seed weight. Therefore these characters must be given priority in selecting for high yielding varieties in blackgram.

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Novel strategy for enhanced expression and purification of recombinant Bacteriorhodopsin in Escherichia coli

Jesu John Peter J., Chellaram C. and Ponmurugan P.

Purification of recombinant bacteriorhodopsin (BR) from E.coli was achieved by two step process that includes the expression of Bacterio-opsin (BO, apoprotein) and renaturation with retinol. However, the expression of BO in E.coli was hampered by the extensive cytoplasmic proteolytic degradation. The usage of N-terminal signal sequence particularly, the Mistic sequence was efficient enough to avoid proteolysis and yields a maximum quantity of BO through E.coli expression system. The present investigation was designed to maximize the final yield of BO by optimizing the codons of Mistic and BO sequence according to the codon usage in E.coli expression system. Codon optimized Mistic-BO expression construct was developed and successfully expressed in E.coli, efficiently purified and renatured to obtain a functional BR to a maximum yield of 200 mg/l. The observed yield is highest among the existing reports of recombinant BR produced by overexpression in E.coli system.

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Genotyping of Scented Rice Land Races of Odisha using SDS-PAGE of Total Seed Storage Protein

Tripathy Swapan K.

Odisha is the genetic paradise of short bold and medium grain local scented rice. More than 100 such land races are available in Odisha (India). Many of these land races are in verge of extinction owing to preference for large scale cultivation of high yielding varieties. Therefore, maintenance and genotyping of scented rice land races are indispensable. In the present pursuit, a set of fourty eight elite scented rice land races including two recently released genotypes (Nuakalajeera and Acharmati) were genotyped following SDS-PAGE of total seed storage protein.“Barikunja” was identified to have highest protein content (14.98%) followed by Deulabhog, Dhobaluchi, Badshabhog, Nuadhusura-2, Chatianaki and Kalajauban. The SDS-PAGE of total seed storage protein revealed altogether 23 scorable polypeptide bands with molecular weights ranging from 10.0 to 116.0kD. Polypeptide bands at 93.0, 67.3, 60.0, 57.0, 53.0, 47.0 and 37.0kD were found to be monomorphic and rest of the bands had shown polymorphism to the extent of 69.56% among the test genotypes. Basumati-1, Basmatibhog, Chatianaki and Kalikati-1had shown maximum 23 polypeptide bands while Kukudajata revealed as low as 13 bands only. In the present investigation, Kukudajata can be identified from rest of the test genotypes by absence of polypeptide band at 116.0kD and 70.0kD. Similarly, Heerakani had unique absence of 108.0kD band. Besides, 51.2kD and 39.0kD polypeptide bands were specifically absent in Ganjeikali and the low mol. wt. 13.0kD prolamin band was absent in Ganjeikali and Seetabhog as compared to other test genotypes. Such a genotype specific fingerprint is of immense value for varietal identification. Nuadhusura-2 and Kukudajata were identified to have medium amylose content based on the staining intensity of the protein marker at 60kD. Ganjeikali and Seetabhog were identified to be qualitatively superior for high lysine content owing to lack of the 13.0kD prolamin polypeptide band. The dendrogram constructed based on UPGMA analysis revealed seven genetic clusters at 76% phenon level. Altogether 38 protein types including seven common protein types were identified among the present set of 48 local scented rice genotypes, Kukudajata, Nuadhusura-1, Gangabali, Dhanaprasa, Dangar Basmati, Khosakani, Bishnubhog, Dhobaluchi and Ganjeikali have been identified as highly divergent genotypes for seed storage protein expression. Besides, the genotype-specific seed protein fingerprints revealed in this pursuit is of immense value for varietal identification.

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Enhanced 2,3-butanediol production by Serratia marcescens H30 with over-expression of 2,3-butanediol dehydrogenase

Zhang Liaoyuan

2,3-Butanediol dehydrogenase (BDH) from S. marcescens H30 is responsible for converting acetoin (AC) into 2,3-butanediol (2,3-BD) during sugar fermentation. Our previous studies indicated that the amount of BDH in S. marcescens H30 limited the conversion of AC into 2,3-BD, resulting in AC accumulation. To improve 2,3-BD production, in this study, the budC gene encoding BDH enzyme was over-expressed in S. marcescens H30. Batch fermentation in 5-l bioreactor showed that excess BDH could significantly decrease the AC accumulation by 86.1% and 2,3-BD formation was enhanced by 78.5% accordingly. Meanwhile, the main byproducts of ethanol, lactate and succinate by recombinant strain were reduced by 45.3%, 27.2% and 51.9 %, relative to the wild strain. BDH activity assays indicated that the activity of BDH in the recombinant strain was over 21-fold higher than that of wild strain which resulted in a lower level of NADH pool and a higher level of NAD+ when compared with the wild strain. These results suggested that a high yield of 2,3-BD could be obtained by S. marcescens H30 with over-expression of BDH.

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Study of the reconstruction of haplotypes and recombination events in some regions of sheep genome

Raed Al-Atiyat M.

The main objective of this study was the investigation of haplotyping structure and recombination observed for the three intervals on each chromosome 2 and 6 of male sheep genome. The haplotype information was utilized for studying recombination events in sires using MERLIN software. The comparison of two-locus and three-locus non-recombinant and recombinant haplotypes showed that the most frequent haplotypes have allele 152. Only one two-locus haplotype (201_220 of CSRD2015-OARHH30) was found ten times as non-recombinant and paternal and three times as maternal haplotype. Allele 152 of this locus possibly has a positive interaction relationship with allele 222 of locus CSRD2105 and allele 118 of CSRD240. It is possibly a positive interaction of allele 152 with allele 118 that forms more of two-locus haplotypes had been expected. This combination of the alleles in the two-locus haplotype (152_118) could be caused by better fitness of such haplotype that for instance may cause higher fertilizing capacity of the sperm cells carrying this haplotype. These observations need more investigation as it is possible that such haplotypes had a paternal origin and they were passed to the dams from grandparents, great-grandparents or even many generations before. Lastly, the results provided additional data which add to the common knowledge that sheep haplotype structure and dynamics are affected by evolutionary forces such as gene flow, selection and many other random factors.

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The effect of casein genotypes selection on the genetic structure of Romanian Spotted, Holstein Friesian and Montbéliarde cattle populations and the genetic variability of kappa-casein and beta-lactoglobulin in Romanian Grey Steppe

Grădinaru A.C., Ilie Daniela Elena and Creangă Şt.

This scientific paper aims to provide a theoretical selection model in Romanian Spotted, Holstein Friesian and Montbéliarde cattle populations reared in the Romanian farms, considering the results from the scientific reports regarding the main casein alleles and their genotypes influence on cheese production. Furthermore, the genotype and allele frequencies at κ-CN and β-LG loci in Romanian Grey Steppe, one of the oldest Romanian cattle breed, were calculated. The alleles frequencies changing rate due to the selection applied (Δp, Δq, Δr) was established as a result of the difference between the allele frequencies after the selection and before that: Δp=p’A-pA; Δq= q’B-qB and Δr=r’C-rC. As a result of assumed selection, it occurred in Romanian Spotted investigated population a β-CN A1, B and C alleles increased frequencies by 8.88%, 2.36% and 0.36% respectively and a κ-CN B allele increased frequency by 8.36%. In Holstein Friesian investigated population, the expected results on β-CN locus were achieved, the A2allele frequency decreased by 11.19% and the A1 and B allele frequencies compensatory increased by 8.22% and 2.96%, respectively. As a result of the total selection applied for the κ-CN AE, BE and EE genotypes, the E allele was completely removed from the investigated Holstein Friesian population, the B favourable for cheese production allele frequency increased by 9.97% and that for A allele decreased by 2.87%. A new selection model for Montbéliarde investigated population was unnecessary, the genetic structure of these individuals being obviously directed towards obtaining milk for cheese production. Considering the κ-CN and β-LG genotypes found in Romanian Grey Steppe investigated population, the frequencies for the A and B identified alleles were: 0.464 and 0.536 respectively for κ-CN and 0.409 and 0.591 for β-LG. The AB genotype was the most prevalent both for κ-CN and β-LG proteins (0.491 and 0.564 respectively) whereas the AA genotype was with the least frequencies (0.218 and 0.127 respectively) for the same proteins.

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Preparationand characterization of immobilized xylanase from Streptomyces rameus L2001 and its advantageous characteristics in xylo oligosaccharide production

Zhu Yunping and Li Xiuting

The xylo oligosaccharide producing xylanase from Streptomyces rameus L2001 was immobilized on Eudragit S 100 using response surface methodology (RSM) based on single-factor experiments. A 99.45% activity yield was obtained. The optimum pH of the immobilized xylanase increased from 5.3 to 5.8 while its optimum temperature increased from 60°C to 65°C. The immobilized enzyme also exhibited higher thermal stability than the free xylanase at temperatures ranging from 60°C to 80°C. The half-life of the immobilized xylanase at 80°C was over 30 min while only 37% of the activity of the free xylanase was retained. The primary products from the hydrolysis of water-insoluble corncob xylan using the immobilized enzyme were xylobiose (X2) and xylotriose (X3) while the xylose was below detectable levels. The immobilized enzyme could be repetitively used over 5 cycles while retaining more than 88% of its original activity. Therefore, the immobilized xylanase from Streptomyces rameus L2001 exhibits potential for production of xylo-oligosaccharides from the hydrolysis of corncob.

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Study on production of itaconic acid with Aspergillus terreus utilizing glucose

Sun Ting and Li Xingjiang

Itaconic acid is an important biochemical product and widely used in chemical intermediate. In order to improve the yield of itaconic acid in the fermentation process, we optimized the culture medium and fermentation conditions by using single factor experiment and response surface method (Box-Behnken). The results showed that the optimal medium compositions for itaconic acid production were as follows: glucose, NH4NO3, MgSO4, KH2PO4, FeSO4, ZnSO4, corn steep liquor were 120 g/L, 2.5 g/L, 1.9 g/L,0.2 g/L, 0.2 g/L, 0.15 g/L and 2 g/L respectively. The optimum fermentation conditions through single factor experiments were found to be a temperature of 320C, initial pH of 2.5, rotation speed of 200 r/min and shake flask liquid volume of 60mL/250mL leading to the itaconic acid yield of 42.71±0.24 g/L. Then fermented in the stirred tank with a ventilation volume of 4.0 L/m, rotation rate of 500 r/min, fermentation period was 144 hours leading to the itaconic acid yield of 53.63±0.21 g/L. Thus, by fermentation processing optimization, the yield of itaconic acid was raised effectively.

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