Research Journal of Biotechnology

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Vol. 13(11) November 2018

Phylogenetic analysis of diverse uncultured eubacterial groups from saline-alkaline soil of basaltic terrain in western Maharashtra, India

Kumar Satish, Krishnani Kishore Kumar, Gramopadhye Sanchita, Bhushan Bharat and Vadivel Rajagopal

Page No: 1-11

Abstract: The microbial communities of saline-alkaline soil from basaltic terrain in western Maharashtra, India were investigated using culture independent and culture dependent approaches. The bulk community DNA of saline-alkaline soil was isolated and 16S rRNA gene libraries were created. The sequence analysis of the 18 phylotypes from 109 metagenomic clones resulted in sequences from five major soil phyla namely Proteobacteria, Firmicutes, Actinobacteria, Acidobacteria and Bacteriodetes.

In our analysis we could retrieve 16S rRNA gene sequences, displaying 16S rRNA gene sequence homology as low as 83% to any known sequence in the NCBI-GenBank database. The phylogenetic analysis of the metagenomic clones showed the presence of entirely novel microbial lineages in basaltic saline-alkaline soil not retrieved so far in any other study. In our culture dependent analysis of 236 cultured isolates, we could isolate microbes belonging to the 13 microbial genera. Our results suggest that the soil of basaltic region in western Maharashtra harbors novel microbial lineages and can have great biotechnological potential.

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Valorization of Black carrot marc: Antioxidant properties and enzyme assisted extraction of flavonoids

Kumar Manoj, Dahuja Anil, Sachdev Archana, Kaur Charanjit, Varghese Eldho, Saha Supradip and Sairam K.V.S.S.

Page No: 12-21

Abstract: The present investigation assesses the impact of green enzyme assisted processing (EAP) method for extraction of flavonoids with maximum antioxidant potential from black carrot waste employing pectinase. A Box-Behnken design with three-level, three-factor, under response surface methodology (RSM) was used to optimize the different concentrations of pectinase (0.1–0.3 %), incubation time (30–90 min) and extraction temperatures (40–60 ºC).

From response surface analysis of the data, a three-degree polynomial equation was developed which provided the following optimal extraction conditions: enzyme concentration = 0.277%, temperature = 56.66 °C and extraction time = 50.74 min. Under the optimal conditions, black carrot marc (BCM) extract obtained through EAP had Total Flavonoid Content (TFC) (1125.74 ± 35.46 mg QE/L) and antioxidant capacity (AOC) (20.79 ± 1.31). Results demonstrate that pectinase is a potential enzyme for enhancing the extraction of TFC with high AOC from BCM.

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Initial characterization and expression pattern analysis of common tobacco (Nicotiana tabacum) metal tolerance protein C4 and 11 genes

Yu Lei, Liu Yonggang and Kong Chuisi

Page No: 22-27

Abstract: The complete coding sequences of common tobacco metal tolerance protein C4 and 11 genes were amplified by RT-PCR. The tobacco metal tolerance protein C4 gene encodes a protein of 471 amino acids which shares high identity with the metal tolerance protein C4 of four species-wood tobacco (97%), nicotiana tomentosiformis (93%), potato (85%) and capsicum annuum (84%). The tobacco metal tolerance protein 11 gene encodes a protein of 399 amino acids which shares high identity with the metal tolerance protein 11 of six species-wood tobacco (99%), nicotiana tomentosiformis (99%), lycopersicon pennellii (92%), lycopersicon esculentum (92%), potato (92%) and capsicum annuum (92%). Prediction of transmembrane helices showed that metal tolerance protein C4 and 11 might be two transmembrane proteins.

Phylogenetic analysis revealed that the common tobacco metal tolerance protein C4 and 11 genes both have a closer genetic relationship with the wood tobacco metal tolerance protein C4 and 11 genes. Computer-assisted analysis showed that metal tolerance protein C4 gene is structured in 13 exons and 12 introns. Metal tolerance protein 11 gene is structured in 6 exons and 5 introns. The gene expression profile analysis indicated that the common tobacco metal tolerance protein C4 and 11 genes were differentially expressed in root, flower, leaf and stem. The expression of metal tolerance protein 11 gene was ubiquitously higher than that of metal tolerance protein C4 gene in all detected tissues.

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Statistical Optimization for Thermostable Xylanase Production by Geobacillus stearothermophilus NASA267 and access to Sugar Production from some Agricultural Waste

Soilman Nadia, Ali Safaa and Omar Sana

Page No: 28-37

Abstract: This study has been undertaken to: isolate thermophilic, xylanlytic bacteria, optimization of fermentation conditions and characterization of the xylanase enzyme produced by a potent isolate. The selected experimental potent strain (NASA267) was identified by sequencing the PCR amplified 16S rRNA gene. The isolate was found affiliated to the genus Geobacillus where it showed 99% similarity to Geobacillus stearothermophilus. The environmental growth conditions including incubation time, pH, temperature, carbon, nitrogen sources and additives, that affect the xylanase production were studied using the Plackett-Burman statistical experimental design. Geobacillus stearothermophilus NASA267 initially produced 89 Uml-1min-1 of xylanase. A pre-optimized medium based on this experiment yielded an enzyme activity of 115 Uml-1min-1.

For further optimization, thirteen trials’ multifactorial Box–Behnken experimental design was applied to find out the optimum level of each of the significant variables. The optimal levels of the three components were founded to be (g/ L): CMC (Carboxy Methyl Cellulose), 15; Y.E (Yeast-Extract), 7.5; and Glycerol, 7.1; with a predicted activity of approximately 183.6 Uml-1min-1. This study also described the usage of the these xylanolytic enzymes in hydrolysis of the pretreated lignocellulosic agricultural materials as raw materials for ethanol production.

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Genetic diversity and population structure of Sinomenium acutum (Menispermaceae) based on nuclear marker

Ying He, Chun Guo, Jin-jing Teng, Xi-yao Zeng, Xing-yao Xiong and Hua Yang

Page No: 38-44

Abstract: Sinomenium acutum (Menispermaceae) is a traditional Chinese medicine. In recent years, extensive harvesting for medicinal purposes has resulted in a sharp decline in its population. To investigate its genetic diversity and population structure, we studied its nuclear ribosomal internal transcribed spacer 2 (ITS2) sequence. Five haplotypes (from H1 to H5) were identified in a survey of 121 samples from 7 populations. The most common haplotype was H1 which was shared by all the populations. The other four haplotypes were unique. HT / HS test described a low level of genetic differentiation within and among populations.

The occurrence of significant genetic structure was not supported by GST / NST test. In addition, mismatch distribution, Tajima’s D and Fu’s FS tests confirm fails are to undergo a recent demographic expansion in Sinomenium acutum. Analysis of molecular variance (AMOVA) indicates that the majority of variation appeared within population. Low genetic differentiation between populations noted a large gene flow in the distribution regions, which may be due to a strong adaptability of Sinomenium acutum to the environment, huge quantity of insects to promote pollen spread during the long flowering stage and bird-accelerated seed transmission. These results provide an important information for future to define the appropriate conservation strategies in the area.

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Does rhizospheric bacterium influence root exudation of sucrose? A preliminary investigation using Bacillus subtilis RR4

Kandaswamy Rekha, Dongal D. Sarbesh and Balasundaram Usha

Page No: 45-49

Abstract: Root exudation, the secretion of phytochemicals through roots, plays a critical role in regulating the microbial community in the plant rhizosphere. Sugars, particularly sucrose, comprise a major portion among the root exudates. Our aim was to demonstrate the effect of a rice rhizosphere isolate, Bacillus subtilis RR4, on the gene expression of sucrose transporters (OsSUT1-5) and root exudation of sucrose. Aseptically grown rice plants were treated with RR4 in a hydroponic setup for 48 h.

Semi-quantitative reverse transcription-PCR was performed using RR4-treated rice roots to analyze the gene expression profile of the sucrose transporters (OsSUT1-5). HPLC analysis of the collected root exudates was performed to analyze the levels of sucrose exuded by the RR4-treated and untreated rice plants. Semi-quantitative RT-PCR analysis of sucrose transporter genes (OsSUT1-5) and HPLC analysis of root exudates revealed that B. subtilis RR4 did not influence the exudation of sucrose.

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Genetic variation and phylogenetic relationships of some distinguished edible cabbages

Sun Yan-Lin, Bai Xin-Fu, Zheng Shi-Lin and Hong Soon-Kwan

Page No: 50-63

Abstract:The genus Brassica, known as oil crops or vegetables, is widely cultivated in Canada, Australia, Asian and European countries. As Brassica species have high yields and good environmental adaptation, abundant germplasm and hybrids with various genetic diversity and elite characteristics were bred out. However, it brought difficulties on the understanding of their genetic diversity and phylogenetic relationships. In this study, we collected 19 distinguished edible cabbages including Brassica campestris L., B. oleracea L. and Salvia plebeian R. and evaluated their genetic variation based on nrDNA ITS region sequences.

S. plebeian R., called savoy cabbage in the folk and considered as one of allied Brassica vegatables, was just selected as the outgroup. Seen from the phylogenetic tree, B. oleracea L. was completely separated out of the Brassica group, but the other two B. oleracea L. accessions were positioned inside. Due to hybridization, B2, B15 and B18 showing three different types of leaf profile were exactly divided into one group. Involved in the subgenus genetic relationship, B. oleracea showed higher genetic diversity than B. campestris. This report provided more evidences for evaluating the genetic variation of Brassica species. Our results would conduct functional plant breeding and trait improvement of Brassica.

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Nonlinear response of Gold nanoparticles pertaining to immunotoxicity in chicken lymphocytes culture system

Ambwani Sonu, Kakade Datta P., Arora Sandeep and Ambwani Tanuj Kumar

Page No: 64-73

Abstract: Nanotechnology is undoubtedly one of the most significant industrial innovations of the 21st century. Nanomaterials are being used in various industrial applications and products as well as for numerous biomedical purposes. Since the manufacture and use of nanoparticles are increasing exponentially, humans and animals are more likely to be exposed occupationally via consumer products or through environment. In particular, gold nanoparticles (AuNPs) have attracted substantial attention and have been used for various biomedical applications. AuNPs are considered to be non-toxic, similar to that of bulk gold; however, numerous in vitro and in vivo studies have been performed to establish AuNPs toxicity which have revealed conflicting results.

The immune system is considered as a sensitive indicator against exposure of such foreign materials. Keeping in view the above scenario, present study was carried out to evaluate immunomodulatory effect of AuNPs in chicken lymphocytes cell culture system. The study revealed maximum non-cytotoxic and minimal cytotoxic dose to be 50µg/ml and 60µg/ml in chicken lymphocytes cell culture system. At minimal cytotoxic dose, immuno-toxic effects were observed through lymphocytes proliferation assay. However, at lower doses of AuNPs, there was increase in lymphocytes proliferation and maximum proliferative dose was determined to be 20µg/ml at which immuno-potentiating effects were observed. Thus, it could be inferred that AuNPs displayed non-linear response in chicken lymphocytes culture system in relation to immunomodulatory effects.

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Optimization of the Production Conditions of Gamma Aminobutyric Acid by Absidia Fermentation

Wang Zhichao and Wang Zhifei

Page No: 74-78

Abstract: Gamma aminobutyric acid (GABA) is also known as 4- aminobutyric acid. Its molecular formula is C4H9NO2, with a relative molecular weight of 103.2. It widely exists in the cells of microorganism, animals and plants1. As a kind of neuro-inhibitory transmitter in mammals, a variety of physiological and metabolic activities are involved. It is greatly demanded by the market for the special physiological function and medicinal value with its unique molecular structure: it has the function of lowering blood pressure, treating epilepsy, enhancing memory, anti-sleeping and delaying senescence with higher physiological activity2.

We used Absidia as the fermentation strain and found out the best fermentation conditions: temperature:310C; time: 70 hrs; pH value: 4.9; inoculation age:18 h, inoculation amount: 8%, rotation speed :170 r/min. When the fermentation test was performed according to the conditions, the molar conversion rate of GABA can reach 32.03%, this can provide theoretical support for large-scale industrial production in the future.

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Coordinate effects of betaine in reducing intra and extracellular erythropoietin aggregation

Mortazavi M., Shokrgozar M.A., Sardari S., Azadmanesh K., Mahdian R., Kaghazian H., Hosseini S.N. and Moazzami R.

Page No: 79-84

Abstract: Recombinant protein production involves a secretory bottleneck that induces intracellular protein aggregation. Intra and extracellular protein aggregations which reduce the yield and quality of recombinant protein production, can be overcome using chemical chaperones as protein stabilizers.

The aim of the present investigation was to study the effect of betaine on erythropoietin aggregation in recombinant CHO cells expressing erythropoietin and their culture supernatant. The effect of betaine on subcellular localization of erythropoietin was assessed through confocal microscopy, in which betaine treated cells showed reduced levels of intracellular erythropoietin retention and increased erythropoietin secretion. Extracellular aggregation in supernatant was examined through western blot analysis under nonreducing conditions. Betaine treatment reduced erythropoietin aggregation in the media.

The results of western blot analysis were indicative of the reduction in protein aggregation in the supernatant of betaine treated cells. This finding suggests that a low concentration of betaine can specifically reduce both intra and extracellular aggregation. Therefore, betaine can be used as a specific ligand to reduce erythropoietin aggregation via stabilizing it and increasing its secretion.

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Glutamate dehydrogenase activity, protein and total nitrogen content in seedlings of maize at varying growth temperatures

Sengar Rakesh Singh, Sengar Kalpana and Garg Sanjay Kumar

Page No: 85-93

Abstract:Seedlings of Zea mays L. cv. Ganga Safed-2 were raised with ½ strength Hoagland ‘s solution containing 5 mM NH4NO3 as sole nitrogen source at 12 h photoperiod (70 Wm-2) and at varying temperature of 10-40 °C, to examine the effect of temperature on l-glutamate dehydrogenase activity nitrogen status of roots and shoots and growth of roots and shoots. Total NADH-glutamate dehydrogenase activity in both roots and shoots increased with the increase in temperature up to 30 ºC and then it declined sharply during a temperature range between 30-40 ºC. The protein and Kjeldahl nitrogen contents of the shoots also showed almost a similar trend. However, the root protein content remained almost unchanged at a temperature range between 15-30 ºC and declined slightly at higher temperature. The optimum temperature for maximum Kjeldahl nitrogen content in the roots was 25 ºC whereas that for the shoots was 35 ºC, although the growth of the either organ, as measured by their fresh or dry weights increased up to 40 ºC.

The in vitro stability of NADH-GDH from root tissue was fairly high up to 5 h a low temperature (5-20 ºC) whereas the same from the shoot tissue was stable significantly up to 10 ºC only. A phase separation temperature was observed in in vitro stability of both, root-as well as shoot-enzyme. The experiment however, demonstrated some differences in the NADH-GDH activity and nitrogen productivity of root and shoots tissues at different growth temperatures and indicates a possible involvement of this enzyme in ammonia assimilation in the plants growing at a wide temperature range.

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