Journal of Applied Biotechnology Reports
Volume:4 Issue: 4, Autumn 2017

Laccase is a polyphenol oxidase, highly glycosylated that mainly presents as monomeric proteins with varying mass of 50-90 kDa. This enzyme oxidizes lignin using molecular oxygen which produces water as the only by-product but it shows specificity to broad range of substrates such as phenols including ortho- and para-diphenols, amino phenols, methoxy phenols, polyphenols, polyamines, aryl diamines and ascorbate. Laccase can be found in fungi, plants, insects and bacteria. Laccases are involved in a wide range of biological functions including pigment formation in fungi, ectomycorrhizal symbiosis, metabolism of proanthocyanidins, virulence of pathogen fungi and sexual development. Regarding its unique function it is getting more attention for novel applications in biosensors, microfuel and bioelectrocatalysis. In addition, it is used in food, pharmaceutical and cosmetic, pulp and paper and textile industries. It has especial potential to be used in bioremediation to remove water and soil pollutions resulted from different industries. This has made researchers to produce transgenic plants containing heterologous laccases to be able phytoremediate polluted soil and water resources with chemicals including different organophosphorus pesticides and nerve agents. Additionally, hydroponic culture of these transgenic plants can be considered as an inexpensive approach for commercial production of laccase exploiting rhizosecretion strategy.

Anthrax, a common disease of human and cattle, is caused by Bacillus anthracis infection. Protective antigen (PA) from Bacillus anthracis is a potent immunogen, which has been of interest in the development of new candidate vaccines against the disease. In this study, the toxicity effects of this antigen on prokaryotic (Escherichia coli and Staphylococcus aureus) and eukaryotic (MCF-7) cells were investigated. Antibacterial effects of the recombinant PA were analyzed using MTT and MIC (Minimum Inhibitory Concentration) assays. Cytotoxicity effect of the recombinant protein (in concentrations ranging from 0.5 to 2 µg/ml) on MCF-7 cell line was analyzed using MTT, Neutral red uptake, and comet assays. MCF-7 cells' oxidative stress following the treatment with PA (0.5-2 μg/ml) was analyzed by NO assay, reduced glutathione assay (GSH), and catalase activity assay. MTT and MIC assays showed that PA has a low inhibitory effect on Escherichia coli and no inhibitory effect on Staphylococcus aureus. Cell cytotoxicity assays indicated that the antigen significantly inhibits the growth of MCF-7 cells. Comet assay also showed that the antigen induces apoptosis in MCF-7 cells. According to nitrite oxide, reduced glutathione, and catalase activity assays, PA has not a significant effect on MCF-7 cells in comparison to the control (P<0.001). Protective antigen has no significant inhibitory effect on the growth of bacterial cells. However, it significantly inhibits the growth of the breast cancer cells (P<0.001). The effect of PA on breast cancer cells is pharmacologically important so that the antigen can be considered as a candidate anticancer molecule.

L-ascorbate acid is the scientific and common name for vitamin C. This vitamin is derived from L-threo-hex-2-enono-1,4-lactone. GME enzyme can modify GDP-Dmannose via epimerase effect and turns it to GDP-l-galactose. Thus, it creates interaction and relation between the synthetic pathway of vitamin C and the synthetic pathway of cell wall polysaccharides. Also, GEM enzyme produces GDP-l-glucose via another epimerase effect on GDP-l-galactose which is recognized as a new intermediate in vitamin C pathway of plants. In the biosynthesis pathway of vitamin C, GME has the most amount of protein protection. In this research, the GME gene of Actinidia deliciosa cultivar Hayward was cloned into the pTG19 plasmid. Sequencing analysis of the GME gene showed that this fragment contains 1161 bp. Results of blast showed that our sequence had high similarity (1973 score) with Actinidia deliciosa cultivar Qinmei and lowest similarity (1002 score) with Musa acuminate. According to the results of this study both phylogenic trees (DNA and protein) were divided into 7 separate groups. Also, Chlamydomonas reinhardtii and Oryza sativa Japonica in this dendrogram were placed in a separate group. Based on the results, Vitis vinifera was placed in two distinct groups in DNA and protein phylogeny trees. In contrast to DNA phylogenic tree in the protein phylogenic tree, all Solanums plants are grouped in one group that in dictate, although they are different in DNA sequencing, they are very similar in protein sequences.

Noble metal nanoparticles have a great potential for biological study, especially the use of gold nanoparticles is popular. In this work gold nanoparticles (GNPs), silver nanoparticles (SNPs) and gold-silver hybrid nanoparticles (HNPs) synthesized and used as a carrier for electrochemical investigation of redox protein. Optical characterization of these nanoparticles was performed by UV-Vis spectroscopy. The optical absorption spectra of HNPs solution shows only one plasmon absorption, it is concluded that mixing of gold and silver leads to a homogeneous formation of alloy nanoparticles. LCR meter study shows the HNPs is best conductance in compare of GNPs and SNPs. Therefore, the electron transfer of the homogenous glucose oxidase (GOx), horse radish peroxides (HRP) and hemoglobin (Hb) was investigates by electrochemical method in presence of HNPs. They demonstrated quasi-reversible cyclic voltammograms with a formal potential of -479, -178 and,168 mV in 50 mM phosphate buffer solution at pH 7.4 respectively.

Many of the anti-cancer compounds which are currently used have an origin in natural sources including plants. Aloe vera is one of the plants that has been used in traditional medicine for centuries to treat a variety of diseases and cancer prevention. In this study, the cytotoxic effect of Aloe vera crude extract on tumor cell lines including HL60 human acute myeloid leukemia and MCF-7 breast cancer cells was studied by cell viability assay, changes in cell morphology, and apoptosis analysis. According to the results, the treated cells with Aloe vera extract in comparison to the untreated cells exhibited significant decline in viability in a time and dose dependent manner. MTT assay showed that IC50 value of Aloe vera extract on MCF-7 cells was 0.5 mg/ml during the first 24 hours, while in this time IC50 of extract on HL 60 cells was 1 mg/ml. Also, morphological characteristics of treated MCF-7 & HeLa cells showed typical features of cell death at the morphological level such as rounding off of cells, cell shrinkage and detachment from the substrate, thus indicating that Aloe vera extract induces cell death by apoptosis. Cell death mediated by through the apoptotic pathway was also confirmed by TUNEL assay. Interestingly, Aloe vera extract did not have any significant cytotoxicity towards normal cells. Therefore, the difference in sensitivity to the Aloe vera extract between MCF-7 and HL60 cancer cells and normal cells suggested that Aloe vera extract can be used as a chemotherapeutic drug for treatment of human acute myeloid leukemia and breast cancer.

Human infection by Escherichia coli enterohemorhagic (EHEC) can lead to watery diarrhea, blood flow, or hemolytic uremic syndrome (HUS). This syndrome occurs in 5 to 10 % of patients with E. coli O157: H7 infection. Children under the age of 5 years old and the elderly and people with immune deficiency are the most prone to severe complications caused by this pathogen. The entry of this bacteria that has the ability to produce Stx-like toxin causes gastrointestinal symptoms including diarrhea and intestinal mucus. This toxin is a hexamer protein with a molecular weight of 70.5 kDa and is composed of A and B units. The purpose of this study is to purify the Shiga-like toxin, which can be used to provide a diagnostic kit, antibody production and vaccine studies. First, E. coli O157: H7 was confirmed by PCR technique and cultured in LB medium. After centrifugation, the cell wall of the bacteria was destroyed by a sonication. Since the toxin is secreted both in the medium and intra-cellular, to increase the concentration of toxin, the precipitate and supernatant were mixed together then the mixture was precipitated with ammonium sulfate salt, it was dialyzed against the salt in a PBS buffer. The presence of toxin was confirmed by SDS-PAGE and Western Blot techniques. In order to confirm the toxicity of protein, supernatant, lysed sediment and a mixture of both were injected into mice groups. In this experiment, the yield of toxin production was 650 μg/ml and the final purity was 90%. Our results demonstrate that Shiga-like toxin (Stx) can be purified without chromatographic methods whit an acceptable purity and yield.

The soil is the main habitat of saprophytic and pathogenic fungi. Heat, rainfall (humidity), soil ingredients are important factors in the growth of fungi. Soil-borne fungi are a major cause for different degrees of allergy or another fungal disease in human and animals. This study was carried out with the aim of isolation and identification of non-pathogenic and pathogenic fungi from the soil of Greater Tunb, Abu-Musa and Sirri islands, Persian Gulf, Iran. In this study, a total of 60 soil samples were collected from the three islands of Greater Tunb, Abu-Musa, and sirri. The soil suspensions were prepared by sterile physiologic saline (0.9% NaCl) and then antibiotics of penicillin and streptomycin were added and 0.2 ml of the suspension was added to Sabouraud’s dextrose agar medium containing chloramphenicol with and without cycloheximide and incubated at 27°C for 2-3 weeks. The fungal isolates were examined macroscopically and microscopically. A total of 483 fungal isolates including 30 genera were isolated as follows: Aspergillus spp. (22.99%), Mycelia sterilia (16.15%), Penicillium spp. (8.9%), Chrysosporium spp. (6.83%), Cladosporium spp. (5.6%), Fusarium spp. (4.97%), Alternaria spp. (4.76%), Acremonium spp. (3.73%) and other fungi (26.07%). In the current study, a fungus of Sporothrix schenckeii isolated from the soil of Greater Tunb and AbuMusa. The results of this study contribute towards a better understanding of the incidence pattern of soil-borne fungi, Given that no study has investigated this issue, the findings of the present study can be beneficial for the management of public health surveillance, epidemiologists as well as physicians.

Integrons are mobile genetic elements which carry effective genetic factors in antibiotic resistance. These elements have several classes and play an important role in the development of antibiotic resistance in Gram-negative bacteria. Klebsiella pneumonia as Gram-negative bacteria caused a variety infection and increasing the antibiotic resistance of this bacterium leads to a majority of problems in its treatments. The present study was conducted to investigation of class I and II integrons among Klebsiella pneumoniae with focus on association with antibiotic resistance. In this cross sectional study, a total of 100 Klebsiella pneumoniae isolates were collected from hospitals of Semnan were identified by biochemical tests. Detection of antibiotic susceptibility was performed by disk diffusion method. For detection of class I and II integrons, PCR by integrase genes, intI and intII, were performed. A p value of <0.05 was considered statistically significant. The highest rate of resistance was observed for trimethoprim/sulfamethoxazole (49%), ceftriaxone (41%) and ceftazidime (40%) while only 10% of isolates showed resistance to imipenem. PCR for intI were positive in all resistance isolates (46%) and intII was positive in lower rate (40%). Overall a significant association was observed between the prevalence of integrons and resistance to antibiotics (p<0.05). This study demonstrated that integrons are widely prevalent and play an important role in multidrug resistance in Klebsiella pneumoniae isolates in this region.