2015, Volume 9, Issue 1 (Volume 9, Issue 1, January-December 2015)

Production, purification and characterisation of thermostable metallo-protease from newly isolated Bacillus sp. KG5
Nazenin Ahmetoglu, Fatma Matpan Bekler, Omer Acer, Reyhan Gul Güven, Kemal Güven

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Abstract

Background: Due to the importance of microbial proteases in biotechnological applications, a number of microorganisms are being explored. The production, purification and characterisation of extracellular metallo-proteases by producing Bacillus sp. KG5 was studied.
Material and Methods: Bacterial strain KG5 was isolated from Kös (Bingöl) hot spring. The strain KG5 was identified by morphological, physiological, biochemical and 16S rRNA gene sequencing. The effects of various parameters on protease production, such as time, temperature, pH, carbon and nitrogen sources and CaCU were studied. The enzyme was purified by ammonium sulphate precipitation and Sephadex G-75 gel permeation chromatography. Molecular weight was calculated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymographic analysis. The effects of some metal ions, chelators and inhibitors on enzyme activity were determined.
Results: The optimum temperature, pH and incubation period for protease production were 40-45°C, 7.0 and 24 h, respectively. It was determined that the best nitrogen sources were yeast extract and urea, while the best carbon sources were lactose and galactose. However, glucose as a source of carbon was found to inhibit the production of the enzyme. The maximum enzyme production was increased in the presence of CaCU. The molecular weight of purified enzyme was found to be approximately 48 kDa. It was found that the enzyme was fully stable in the presence of 2 mM CaCU at 50°C after 120 min. Purified protease was significantly activated by Ca2 + and Mg2 + , while it was greatly inhibited by Cu2 + , Zn2 + , Hg2 + and SDS as well as by the metal ion chelators ethylenediaminetetraacetic (EDTA) and 1,10-phenanthroline. Phenylmethylsulfonyl fluoride (PMSF) had a little effect on the enzyme.
Conclusions: Our findings suggest the potential of this isolate for protease production and that this enzyme may be suitable for biotechnological applications.

Keywords: bacillus sp. KG5, biotechnology, protease production and characterisation

Citation: Ahmetoglu N, Bekler FM, Acer O, Güven RG, Güven K. Production, purification and characterisation of thermostable metallo-protease from newly isolated Bacillus sp. KG5. Eurasia J Biosci. 2015;9(1):1-11. https://doi.org/10.5053/ejobios.2015.9.0.l

Isolation and characterization of plasma membrane Na7H+ antiporter (SOS1) gene during salinity stress in kallar grass {Leptochloa fusca)
Banafsheh Taherinia, Hamid Reza Kavousi, Sara Dehghan

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Abstract

Background: Leptochloa fusca is a halophyte plant which is highly tolerant to saline and sodic soils and water. Moreover, L fusca is an attractive model plant to study the mechanism of salt tolerance mainly due to its characteristics as a typical euhalophyte, having both accumulating and excreting salt properties. Soil salinity adversely affects plant growth, development and disturbs intracellular ion homeostasis resulting cellular toxicity. The Salt Overly Sensitive 1 (SOS1) gene encodes a plasma membrane NaVH+ antiporter that plays an important role in imparting salt stress tolerance of plants.
Material and Methods: Using conserved sequences of S O S 1 , the coding sequence of plasma membrane NaVH+ antiporter (SOS1) in kallar grass was partially isolated and its expression profile during salinity stress was investigated.
Results: The aa (amino acid) sequence of the isolated region of /./SOS1 possesses the maximum identity up to 96% of its orthologue in Distichlis spicata. The results of semi-quantitative RT-PCR revealed that salinization was affected SOS1 transcript level positively. The expression of i./50S1 in leaves of kallar grass progressively increased under all salinity levels compared to control.
Conclusions: The results suggest that i./SOS1 may play an essential role in the salt tolerance of L fusca and may be useful for improving salt tolerance in other crop species.

Keywords: Leptochloa fusca, NaVhT antiporters, SOS pathway, salinity tolerance, semi-quantitative RT-PCR

Citation: Taherinia B, Kavousi HR, Dehghan S. Isolation and characterization of plasma membrane Na7H+ antiporter (SOS1) gene during salinity stress in kallar grass {Leptochloa fusca). Eurasia J Biosci. 2015;9(1):12-0. https://doi.org/10.5053/ejobios.2015.9.0.2

Morpho-physiological and phytochemical traits of gazania (Gazania rigens) affected by foliar application of bio-stimulants
Amir Foroutan Nia, Sanaz Bahman, Hassanali Naghdi Badi, Ali Mehrafarin, Mohammadreza Labbafi

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Abstract

Background: Gazania (Gazania rigens), a member of the Asteraceae family, is an ornamental plant that is cultivated on a large scale in gardens and landscapes.
Material and Methods: : This experiment was carried out to evaluate the effects of bio-stimulants compounds on morpho-physiological and phytochemical traits of gazania on the basis of randomized complete blocks designed (RCBD) with three replications. The treatments included biostimulants of aminolforte, kadostim, fosnutren, humiforte (each of them at 2 and 4 L.ha-1), and control treatment (foliar spray with distillated water).
Results: The effects of treatments were significant (pN0.01) on all of the traits except for the number of leaves and capitula per plant, and capitula diameter. The maximum amount of the plant height and total dry weight was obtained in the treatment of kadostim at 2 L.ha-1. The largest collar
diameter of the stem and total fresh weight was observed in the treatment of aminolforte at 4 L.ha-1. The greatest diameter of the flower and stem was observed in the treatment of aminolforte and fosnutren at 2 L.ha-1, respectively. Also, the highest quantity of SPAD value, chlorophylls a and b, and total chlorophyll, carotenoids, and nitrogen content were related to the foliar application of aminolforte at 4 L.ha-1, but the highest percentage of potassium content was attained in humiforte treatment at 4 L.ha-1. In comparison, the least amount of all traits was observed in control
treatment.
Conclusions: The findings showed that the foliar applications of bio-stimulants based on bio-active amino acid compounds with a mixture of nutrients have positive effects on the growth, morphophysiological, and phytochemical traits of the gazania.

Keywords: amino acids, bio-stimulants, Gazania rigens, morpho-physiological and phytochemical traits

Citation: Nia AF, Bahman S, Badi HN, Mehrafarin A, Labbafi M. Morpho-physiological and phytochemical traits of gazania (Gazania rigens) affected by foliar application of bio-stimulants. Eurasia J Biosci. 2015;9(1):21-8. https://doi.org/10.5053/ejobios.2015.9.0.3

Physiological effect of phenanthrene on Triticum aestivum, He Ha nth us annus and Medicago sativa
Seyed Yahya Salehi-Lisar, Somayeh Deljoo

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Abstract

Background: Polycyclic aromatic hydrocarbons (PAHs) are widespread organic pollutants released into the environment by both natural and anthropogenic processes. PAHs can negatively affect different aspects of plant growth and development. However, the mechanisms of PAHs effects and physiological response of plants to PAHs have not been adequately studied. Accordingly, the aim of this study was evaluation of the germination, growth and physiological responses of wheat, sunflower and alfalfa to phenanthrene toxicity.
Material and Methods: This experiment was conducted as a pot culture of plants using a completely randomised design (CRD) with four replications. Plants were cultivated in soil containing 50 and 100 mg kg1 of phenanthrene for 14 days under greenhouse conditions. All biochemical assays were performed spectrophotometrically after the determination of growth parameters.
Results: Soil contamination with phenanthrene differently decreased seed germination and the subsequent seedling growth of plants. Alfalfa showed the highest resistance at both the seed germination and seedling growth phases. Wheat and sunflower were the most sensitive species at the seed germination stage and seedling growth phase, respectively. Phenanthrene contamination induced oxidative stress in plants and POD was determined to be the important enzyme involved in ROS detoxification.
Conclusions: Phenanthrene effects on seed germination, seedling growth and physiological parameters of plants are species-dependent. The induction of oxidative stress and decrease in photosynthetic pigments content are two of the reasons for lower plant growth in phenanthrenecontaminated soil, and POD was an important enzyme in the detoxification of ROS. At least in some species, the higher resistance at seed germination could be followed by the higher resistance of seedlings to PAHs.

Keywords: helianthus annus, Medicago sativa, oxidative stress, PAHs, Triticum aestivum

Citation: Salehi-Lisar SY, Deljoo S. Physiological effect of phenanthrene on Triticum aestivum, He Ha nth us annus and Medicago sativa. Eurasia J Biosci. 2015;9(1):29-37. https://doi.org/10.5053/ejobios.2015.9.0.4

Optimization of an in vitro protocol for the production of ascorbic acid in Hibiscus cannabinus leaf-derived normal root cultures
Sandopu Sravan Kumar, Prabhakaran Manoj, Parvatam Giridhar

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Abstract

Background: Hibiscus cannabinus (kenaf) is cultivated in many parts of the world for its bast fibers that find extensive industrial applications. In the southern states of India, kenaf is a popular leafy
vegetable. The leaves are rich in ascorbic acid.
Material and Methods: The present study reports an efficiently optimized protocol for in vitro normal root cultures for the biomass establishment of kenaf and the production of substantial amounts of ascorbic acid. The explants prepared from cotyledonary leaves of 15 day old in vitro seedlings of kenaf were inoculated onto Murashige and Skoog (MS) basal medium supplemented with 0.5-3 mg/L indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) or naphthalene acetic acid (NAA) individually for root initiation.
Results: MS medium containing 2 mg/L NAA provided the best response (100% of explants) with a root length of 12-14 cm. A light greenish friable callus was also obtained in most of the explants. Efficient root biomass and ascorbic acid production was evident upon sub-culturing ~1 cm length root segments onto MS liquid medium containing the same growth regulators regimen used for the initiation of roots. Root cultures showed maximum biomass by the eighth week (5.51 g/100 g fresh weight). Ascorbic acid production was maximal in the seventh week (48.43 mg/100 g fresh weight).
Conclusions: This protocol will facilitate scale up studies for obtaining higher yields of ascorbic acid in vitro using kenaf root cultures on a commercial scale; it could also be used to investigate the kinetics of the ascorbic acid biosynthetic pathway.

Keywords: ascorbic acid, biomass, Hibiscus cannabinus, root culture

Citation: Kumar SS, Manoj P, Giridhar P. Optimization of an in vitro protocol for the production of ascorbic acid in Hibiscus cannabinus leaf-derived normal root cultures. Eurasia J Biosci. 2015;9(1):38-45. https://doi.org/10.5053/ejobios.2015.9.0.5

Effect of BAP on total hypericin production in shoot cultures of Hypericum scabroides: An endemic species in the Eastern Anatolia Region of Turkey
Hilal Surmus Asan, Hasan Cetin Ozen, Ahmet Onay, Nurettin Asan

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Abstract

Background: Due to the therapeutic importance of hypericin, a number of Hypericum species are being investigated. The aim of this study was to determine the effect of various BAP (6- benzylaminopurine) concentrations on seed germination, shoot proliferation, and total hypericin in a tissue culture of Hypericum scabroides which is endemic to the Eastern Anatolia region, Turkey.
Material and Methods: Hypericum scabroides specimens were collected from Eastern Anatolia (Elazig, Turkey). The effects of various BAP concentrations (0.0 (control), 0.5, 1.0, and 2.0 mg/L) on seed germination, shoot multiplication, and the accumulation of total hypericin were determined using tissue cultures of H. scabroides. Murashige and Skoog (MS) medium was used for germination and shoot cultures. Measurements of total hypericin were taken using a UV spectrophotometer.
Results: The best germination rate (59.2%) was obtained using hormone-free MS medium (control group). Apical tips of freshly germinated seedlings were proliferated on the MS medium supplemented with various BAP concentrations (0.5, 1.0, and 2.0 mg/L) and the control group (without BAP). The highest number of shoots (42.7 shoot/explant) and longest shoot length (2.50 cm) were obtained on MS medium supplemented with 2.0 mg/L BAP. Total hypericin was found in trace amounts and it was found that the total hypericin was not affected by the concentration of BAP.
Conclusions: Our results showed that increasing concentrations of BAP stimulated shoot multiplication but did not affect seed germination rates or total hypericin in in vitro cultures of H. scabroides.

Keywords: hypericum scabroides, hypericin, shoot culture, BAP

Citation: Asan HS, Ozen HC, Onay A, Asan N. Effect of BAP on total hypericin production in shoot cultures of Hypericum scabroides: An endemic species in the Eastern Anatolia Region of Turkey. Eurasia J Biosci. 2015;9(1):46-51. https://doi.org/10.5053/ejobios.2015.9.0.6

Characterization of cellulase enzyme produced by Chaetomium sp. isolated from books and archives
Moza Mohammed AL-Kharousi, Nallusamy Sivakumar, Abdulkadir Elshafie

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Abstract

Background: Cellulase is an important industrial enzyme used to degrade cellulosic biomass. The demand for cellulase enzyme is continuously increasing because of its applications in various industries. Hence, screening of cellulase producing microorganisms from different sources has gained significant importance.
Material and Methods: In this study, fungi isolated from books and archives were screened for their cellulase producing abilities. Four different fungi were isolated from books and archives using potato dextrose agar. Screening of these isolates for cellulase production was carried out using
carboxymethyl cellulose broth. The most efficient fungus was subjected to cellulase fermentation and enzymes produced were purified and partially characterized.
Results: Four different fungi, Chaetomium sp., Aspergillus niger, Aspergillus nidulans and Penicillium sp., were isolated from books and archives. All the isolates were tested for their ability to produce cellulase enzyme. During the primary screening Chaetomium sp. showed good growth and higher cellulase activity (155.3±25.6 U/mL) in carboxymethyl cellulose medium than the other fungi. The cellulase fermentation study was conducted with Chaetomium sp. using carboxymethyl cellulose as a substrate. During the stationary phase (144 h) of the growth, the cellulase activity of Chaetomium sp. was significantly high. The maximum mycelial weight of this fungi was obtained at 168 h. Viscosity of the Chaetomium sp. inoculated fermentation medium continuously decreased until 144 h because of the degradation of carboxymethyl cellulose. During cellulase fermentation, pH increased from the initial neutral pH to 8.5. Purified cellulase showed a specific activity of 7.3 U/mg. It exhibited maximum activity at 20°C and was stable between pH 5 and 9.
Conclusions: Books and archives could be a good source for the isolation of cellulase producing fungi.

Keywords: archives, books, cellulase, Chaetomium sp., viscosity

Citation: AL-Kharousi MM, Sivakumar N, Elshafie A. Characterization of cellulase enzyme produced by Chaetomium sp. isolated from books and archives. Eurasia J Biosci. 2015;9(1):52-60. https://doi.org/10.5053/ejobios.2015.9.0.7

The impact of hydroelectric project development on the ethnobotany of the Alaknanda river basin of Western Himalaya, India
Khilendra Singh Kanwal, Hema Joshi

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Abstract

Background: This study focuses on the ethnoflora used by local communities in the Alaknanda river basin of Uttarakhand state in Western Himalaya, India. The objectives of the study were to collect ethnobotanical information, to assess the impact of hydropower projects on ethnoflora and to suggest conservation and management measures for the protection of ethnoflora.
Material and Methods: A well-designed questionnaire based survey was conducted in the ten villages of the study area to collect ethnobotanical information. The conservation status of plants was also evaluated following the IUCN Red list, the Red Data Book of Indian Plants and the CITES criteria.
Results: A total of 136 plant species belonging to 61 families and 112 genera were used by local communities for various ethnobotanical purposes. The majority of plant species were used for medicinal purposes (96 spp.), followed by fodder (46 spp.), wild edibles (31 spp.), fuel (29 spp.),
timber (17 spp.), fish poison (9 spp.), agriculture implements (6 spp.), fibre (6 spp.), religious use (6 spp.) and handicraft (1 sp.). For the preparation of herbal medicine, rural people of the region use different parts of medicinal plants such as the whole plant (20%) followed by roots/rhizomes/tubers (20%), leaf (18%), fruit (10%), seed (9%), bark (9%), stem (6%), flowers (6%) and resin (2%).
Conclusions: Development of hydropower projects will influence the diversity and distribution of ethnoflora in the region. Therefore, for the conservation of the ethnoflora of the area, conservation and management measures have been suggested.

Keywords: biodiversity conservation, ethnobotany, Himalayas, hydropower project, sustainable development

Citation: Kanwal KS, Joshi H. The impact of hydroelectric project development on the ethnobotany of the Alaknanda river basin of Western Himalaya, India. Eurasia J Biosci. 2015;9(1):61-77. https://doi.org/10.5053/ejobios.2015.9.0.8