Research Journal of Biotechnology

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Vol. 9(7) July 2014

Molecular Cloning and Expression of Alpha Amylase Gene from Thermophilic Bacillus subtilis in Escherichia coli

Fadime Kiran, Tugba Simsek and Ozlem Osmanagaoglu

The aim of this experiment was to isolate thermostable alpha-amylase producing bacteria and to clone the gene (AmyE) into Escherichia coli DHα for obtaining a low-cost source of a thermostable enzyme. Fourteen potential strains of α-amylase producer were isolated from water samples of different natural thermal springs in Turkey. A strain exhibiting the highest α-amylase activity (1.8 U/L) was selected for the molecular cloning experiments and was identified as Bacillus subtilis by 16S rDNA sequence analysis. The gene encoding an extracellular thermostable -amylase was amplified by Polymerase Chain Reaction (PCR). Gel purified PCR product and the vector pBR322 partially double digested with restriction enzymes and then in vitro ligations were performed. The resulting recombinant plasmid was called as pOZ322, was introduced into competent cells by transformation using CaCl2. Recombinant cells were selected via the ampicillin resistance gene acquired from the plasmid vector pOZ322 which allowed the E.coli cells to grow in the presence of ampicillin medium. -amylase gene was expressed successfully in the genetically engineered E.coli DHα and its enzymatic activity was closely found to be higher (2.2 U/L, p>0.05) than the activity of the donor strain.

Changes in Icariin contents and antioxidant activities of Epimedium koreanum Nakai leaf by far infrared irradiation

Cheng-Wu Jin, Yan-Lin Sun, Won-Woo Kim and Dong-Ha Cho

The objective of this research was to explore the suitable temperature to obtain a maximum total polyphenol content and optimum conversion of icariin using far infrared irradiation (FIR) as a thermal source of Epimedium koreanum Nakai leaf. The changes were monitored and quantified by HPLC and UV-spectrophotometry. Further, the alteration in antioxidant activity due to FIR at different temperatures was also evaluated. The increase in the temperature in FIR caused a gradual increase in icariin production. The maximum increase in icariin was achieved with 5.49 mg g-1 DW at 1000C. However, an over-exposure (1200C) decreased the content. The total polyphenol content was also altered with the increase in the temperature and reached a plateau (147.10 mg GAE g-1 DW extract) at 1000C which was 1.28 fold higher than that of the control. In an antioxidant assay, the DPPH free radical inhibition activity, reducing power and total antioxidant capacity were also increased with the temperature in a dose-dependent manner in the same trend of the total polyphenol content. Overall, the FIR thermal treatment could enhance the bioactivity (antioxidant) in Epidemidium koreanum leaf by increasing total polyphenol and icariin content.

Nanotechnology: Emerging tool in food sector

Take Ajaykumar M.

Food nanotechnology is an area of emerging interest and opens up a whole universe of new possibilities for the food industry. The principal areas in the food sector where nanotechnology has potential for use are in encapsulation and emulsion formation, in food contact materials and sensor development and some applications of the technology are close to utilization. Nano food packaging materials may extend food life, may improve food safety, may alert consumers that food is contaminated or spoiled, repair tears in packaging and even may release preservatives to extend the life of the food in the package. Nanotechnology applications in the food industry can be utilized to detect bacteria in packaging or produce stronger flavors and color quality and safety by increasing the barrier properties. Nanotechnology holds great promise to provide benefits not just within food products but also around food products. In fact, nanotechnology introduces new chances for innovation in the food industry at immense speed but uncertainty and health concerns are also emerging.

Effect of Clostridium thermocellum activity on enzymatic hydrolysis of alkaline peroxide pretreated wheat straw

Karagoz Pinar and Ozkan Melek

In this study, the microbial activity of Clostridium thermocellum on the hydrolysis of raw and pretreated wheat straw (WS) was investigated. The microbial activity was compared with the activity of enzyme mixture. It was found that alkaline peroxide pretreatment improves both microbial and the enzymatic digestibility of WS. Activity of C. thermocellum on the pretreated biomass is almost equal to the activity of 35 µl/g enzyme which is equal to 5% of enzyme usage for complete saccharification of pretreated biomass. It was observed that enzymatic saccharification after C. thermocellum activity increased sugar release by 6% as compared to the sole enzymatic digestion.

Molecular Characterization of Leptin Exon 3 gene in sheep of Saudi Arabia

Mahmoud A. H., Saleh A. A., Almealamah N. A., Abou-tarboush F. M., Aljumaah R. S., Shafey T. M. and Abouheif M. A.

Leptin hormone plays an important role in many biological processes in animals and humans such as appetite, immune system and reproduction. We evaluated the genetic variability in the leptin (LEP) exon 3 gene in Saudi indigenous Najdi (n=41) and Naeimi (n=40) sheep. A 1041-1058 bp fragment was amplified, sequenced and three non-synonymous single nucleotide polymorphisms (two transition and one-transversion) were found at positions 170, 286 and 332 of the exon 3 coding region. In addition, two insertion-deletion (indel) mutations were detected in the 3' UTR of LEP exon 3; the first indel was 16 nucleotides in length and the second indel was only a single nucleotide. Four out of the five detected mutations have been found in other international sheep breeds. The most frequent LEP genotypes (01 and 02) in Najdi and Naeimi sheep were also found to be the most common genotypes in GenBank database. However, the data revealed that the single base indel in the 3' UTR was unique to the Najdi breed. On the other hand, 42% of the total studied individuals were heterozygous at LEP locus.

Pharmacognostical and preliminary phytochemical investigations in stem and root of Cajanus cajan (L.) millsp.

Mahitha Banala, Archana Pamulaparthi, Srikanth Kagithoju and Ramaswamy Nanna

The present study was aimed to investigate the pharmacognostical studies and also to screen various phytochemicals present in the stem and root extracts of pigeon pea. Pharmacognostic studies which include macroscopic, microscopic, phytochemical prospection was performed using aqueous, acetone, benzene, chloroform and methanol extracts of stem and root, fluorescence analysis and physicochemical parameters of pigeon pea by standard procedures. The macroscopic characters of the plant were observed and anatomical investigation of stem and root showed epidermis, trichomes, cortex, cambium, pith, secondary xylem and phloem. Root showed the presence of secretory ducts containing tannin like material. Phytochemical screening showed the presence of bioconstituents like alkaloids, glycosides, tannins and flavonoids in stem and root extracts in varying quantities. Fluorescence analysis showed characteristic colouration when treated with different solvents and reagents. Physicochemical parameters of root powder showed varied ash values such as total ash 7.4%, acid insoluble ash 1.5% and water soluble ash 2.24% respectively and varied extractive values such as aqueous 8.1, acetone 7.3, benzene 5.7, chloroform 6.2 and methanol 5.0 respectively. The various pharmacognostical studies and phytochemical prospectation of root and stem of C.cajan can be used as a diagnostic tool for the identification and standardization of the plant drug.

Synergistic use of Nisin with Cassia alata extract as a biopreservative in traditional Ayurvedic medicines

Leon Ittiachen, Gothandam K. M. and Karthikeyan S.

Ayurvedic medicine, a widely used traditional medicinal system in India, seeks to alleviate disease with natural herbal remedies. Due to a burgeoning global market, Ayurvedic formulations are now being bottled with the addition of chemical preservatives instead of being freshly prepared when prescribed. Our study took samples of contaminated bottles of manjistada kashayam obtained directly from Ayurvedic manufacturing units which had been returned from the market due to spoilage. These were then screened and six contaminants were isolated; three fungal strains, Aspergillus flavus, Haplosporangium parvum, Paecilomyces lilacinus and three bacterial strains Bacillus cereus, Escherichia coli and Bacillus subtillus . The shrub Cassia alata was selected as a possible bio preservative. Extractions were done with 75% dry weight of flowers and 25% seeds using ethanol, water and ultrasound. MBC and MFC values were obtained for these against the above isolated contaminants. High activity was seen against the fungal contaminants but action against two of the gram positive bacterial contaminants was low. Further studies involving the conjoint use of the extract with the natural probiotic Nisin, was attempted. The proposed biopreservative using hurdle technology was seen to be an effective way to combat contamination in Ayurvedic formulations.

Genetic diversity and its climate influencing factors of the endangered Rhodiola sachalinensis using molecular markers

Wei Zhao, Wenting Zhao, Zhang Jiang and Xia Chen

Genetic structure in ecologically important features of species is a crucial element in biodiversity and conservation, yet these are not clearly studied in many endangered medicinal plants, especially in some species living in high altitude zone. R. sachalinensis is an important medicinal and endangered species distributed in Changbai Mountain China with fragments habitats. In this study, we applied a genomic scan approach using RAPD (Random amplified polymorphic DNA) makers to analyze genetic variation of different population of R. sachalinensis. In addition, we analyzed relationship among genetic diversity, climate factors (annual average temperature and annual precipitation) and altitude. The results showed that the population of R. sachalinensis distributed in different altitude had a high level of genetic diversity. At the species level, the percentage of polymorphic loci (PPL) and Shannon’s information index (I) were 100% and 0.5039, respectively. The coefficient of genetic differentiation (Gst =0.4847) and AMOVA analysis revealed that most genetic variation was distributed among populations (56.10%). The UPGMA clustering results using RAPD showed that all individuals from the same altitude were gathered together. Correlation analysis revealed close relationships among genetic diversity, annual average temperature and annual precipitation. These data highlight that environmental condition of alpine especially temperature and precipitation may play an important role in shaping the genetic diversity of R. sachalinensis.

Correlation between antioxidant activity and phenolic content of Curcuma angustifolia Roxb.

Dhal Yogamaya, Deo Bandita and Sahu Rajani Kanta

Curcuma angustifolia Roxb. is widely used as traditional medicine by the tribes of Odisha. This study aimed to work on antioxidant activity and phytochemical study on crude extracts of the plant in order to discover new natural sources for the treatment of various diseases. In order to estimate the correlations between phenolic contents and antioxidant mechanisms, different concentrations of C. angustifolia plant extracts were examined in this study. It showed antioxidant activities at all concentrations with respect to DPPH radical scavenging activity, hydroxyl radical-scavenging activity and total antioxidant capacity by phosphomolybdenum method. The phenolic content was found to be 0.74±0.05 mg/100g. A positive correlation was observed between antioxidant activity and phenol content of the analyzed extracts, however a negative correlation was found with the total antioxidant activity and the phenolic content. The results concluded that C. angustifolia Roxb. could be used as a potential source of natural antioxidant and a beneficial chemo preventive agent.

Relationship of Tree Height with Soil Properties, Soil and Needle Nutrients in Cedar (Cedrus libani A. Rich.) Plantations

Karatepe Yasin, Başaran M. Ali and Çoban H. Oğuz

This study was conducted in the cedar (Cedrus libani A. Rich.) plantation area located in the Mediterranean Region of Turkey (Burdur). The study aimed to assess the relation of tree height with certain soil properties (sand, silt and clay contents, pH, EC, lime and organic matter), soil nutrients (P, K, Ca, Mg, Na, Fe, Mn, Zn and Cu) at 3 different soil depths (0-30 cm, 30-60 cm and 60-90 cm), concentration of tree needle nutrients (N, P, K, Ca, Mg, Fe, Mn, Zn and Cu). The highest relationships between the tree height and soil properties were found at the depth of 0-30 cm. At this depth, the highest correlation (R2=0.5062) was found to be negative between the tree height and silt rate in soil while the relation was positive with clay and negative with pH and lime respectively by the order of significance. Mn, Cu and K that were the soil nutrients and concentrations of P, N, Cu and Mn that were the needle nutrients had a significantly positive correlation with the tree height.

Seven Potential Nucleic Chemicals enhance efficiency of Polymerase Chain Reaction

Zhong Xu, Hexiang Zhao, Fuming Sang, Xin Zhao and Zhizhou Zhang

It is a tedious task to search more and more PCR (polymerase chain reaction) enhancers. In this study, a simple but powerful approach to screen for novel PCR enhancers was reported which was based on a fact/hypothesis that natural DNA replication happens mostly in the nucleus so that some of the nucleic molecules shall be able to facilitate DNA replication/amplification. Through bioinformatics study under the help of CYGD (The Comprehensive Yeast Genome Database) database, over eight hundred of potential nucleic chemicals (PNCs) were collected and 96 such molecules were tested in several PCR models. Four amino acids (methinione, valine, leucine, isoleucine) plus uridine, uracil and inosine were found satisfactory in improving the amplification efficiency of different PCR reaction models.

Construction of MiR-145 Eukaryotic Expression Vector and its Tumor-suppressive Activities in Human Colon Cancer Cells

Gao Feng Liang, Aihua Jing, Junchao Xu, Xiangfeng He and Baoan Chen

Recent research indicates that miR-145 is involved in various biological processes including cell cycle, cell differentiation, apoptosis and colorectal cancer. In this study, we report the construction of recombinant plasmid miR-145 expression vector and its tumor-suppressive activities in transfected human colon cancer cells (HCT-116). The double strands oligo encoding the miR-145 was designed and synthesized to generate the mature miR-145 expression plasmid. PHsa-miR-145, the new constructed plasmid vector and its controls were transfected to HCT-116 cells. Fluorescence detection displayed that fluorescence intensity of GFP was highest during 48 and 72 hours post-transfection and qRT-PCR showed a significantly increase in miR-145 expression in the vector transfected cells compared with the expression in its controls, then its tumor-suppressive activities were further investigated. Cell proliferation and cell cycle assay indicate that miR-145 treated HCT-116 cells pronouncedly enhance tumor-suppressive effect by inducing cell cycle G1 arrest. Our results indicate that transduction of miR-145 offers a feasible approach to signiﬁcantly inhibit HCT-116 cells proliferation and increase the apoptosis index. Thus, targeted delivery of miR-145 into HCT-116 cells may be useful for the treatment of colon cancer.

Improvement of a High Frequency Regeneration System for in vitro Cultured Cotyledons of Chinese cabbage (Brassica rapa L. ssp. pekinensis)

Qian Meng and Hui Feng

The current study aims at establishing a high frequency regeneration system in Chinese cabbage (Brassica rapa L. ssp. pekinensis). The effects of genotype, medium conditions, seedling age and types of cellophane wraps were studied for the regeneration system using four inbred lines of Chinese cabbage. The results showed that the regeneration is highly genotype depend, the line 11A078 was easier to induce adventitious bud than other three lines. The highest shoot regeneration was 89.33% achieved with line 11A078 on MS medium containing 5mg/L 6-BA and 0.5mg/L NAA. 4 day old seedlings could obtain highest shoot regeneration rate (89.10±1.38a) for line 11A078. It was observed that a culture medium supplemented with 10g/L agar and 2 g/L Phytagel™ for cotyledons of 11A078 resulted in better shoots regeneration rate (83.82±2.64a). Rooting frequency as well as plantlet survival reached their maximum frequency (100%) in MS medium containing 0.1 mg/L NAA and 0.1 mg/L IBA. High survival rate (95.44±0.83%) was achieved with line 11A078 when breathable membrane was used for cellophane wrap.

Alternative Low Cost Technology Implications in Plant Tissue Culture

Shalini S. and Deepa Sankar P.

Tissue culture is a widely accepted technique utilised for micro-propagation of plants, obtaining disease free plants, synthesising artificial seeds, secondary metabolites, carrying out gene transfers, protecting endangered species, maintaining genetic diversity etc. The search for alternative low-cost media constituents for plant tissue culture technology is a continuing process. Plant breeders in developing countries have undertaken research work to bring down the cost of micropropagules produced through plant tissue culture to serve the farming community. Alternative sources for gelling agents, carbon source, macro and micronutrients, reduction of laboratory construction cost and equipments have been reviewed in this paper.

Methods for Identification, Quantification and Charaterization of PolyhydroxyAlkanoates- A Review

Godbole Suchitra

Polyhydroxyalkanoates (PHAs) are the polymers of hydroxyalkanoates that accumulate as a carbon/energy storage material in various microorganisms. PHAs have been attracting considerable attention as biodegradable substitutes for conventional polymers, because of their similar material properties to conventional plastics. A number of review articles on the general features of PHAs, the physiology, genetics and molecular biology, the development of PHAs having novel monomer constituents, production processes, biodegradation of PHAs are available. Recently much effort has been devoted to develop a process for the economical production of PHAs. The isolation, analysis and characterization of PHAs are important factors for any process development. A number of methods have been developed for the analysis of PHAs. This paper is an effort to compile the methods available for the identification, quantitative estimation and characterization of PHAs.