فصلنامه دنیای میکروب ها
سال هشتم شماره 4 (پیاپی 25، زمستان 1394)

The grains are considered as the main food for poultries. Grains contain significant content of non- soluble starch polysaccharides (NSP). NSPs cause negative impact and incompatibility in birds. Digestion of NSP contents by β-glucanase enzyme supplement reduces the viscosity of polysaccharides in the digestive tracts, and increases their intestinal absorption. The aim of this study was to construction of glucanase gene of Bacillus subtilis in Escherichia coli.
In this experimental study, the glucanase gene of B. subtilis was amplified using specific primers and polymerase chain reaction. After purification of the bands, Bgl gene was cloned by T/A cloning technique in pGEM vector, and was transformed in E. coli. Afterward, the pGEM-bgl was transferred into E. coli Top-10 strain. The recombinant vectors were then transformed into E. coli competent cells, and the recombinants were plated on LB agar containing 100 µg/ml ampicillin. The recombinant plasmid transformed to E. coli Top10 cell and the colonies carrying plasmid were selected by PCR. The presence of glucanase construction was confirmed by enzymatic digestion.
The results showed that the glucanase gene was successfully cloned in E. coli. The results of cloning of 767 bp glucanase gene was confirmed by PCR assay.
The construction of B. subtilils glucanase gene and cloning of this gene in E. coli is a strong alternative for the production of probiotic used for poultry.

Hepatitis C virus infection initiates through binding of E2 glycoprotein to its specific human liver cell receptor. Therefore¡ this glycoprotein is considered as one of the important targets in drug and vaccine researches against this infection. This project was accomplished for the first time to transfer the full length of E2 gene by using recombinant pPICZAa-E2 (HCV-2a¡ JFH1) vector into Pichia pastoris yeast KM71H strain¡ and to evaluate the possibility of its expression in this expression system.
The E2 gene was amplified by using primers containing the EcoRI and XbaI restriction sites and was inserted into the (T-PTG 19) and (pPICZAa) vectors to be transferred into the E. coli. The recombinant pPICZAa-E2 vector was transferred into the yeast through electroporation¡ and it was evaluated by digestion and sequencing. The yeast was grown in YNB medium contained methanol for five days. Gene expression was studied by western blot.
Construction of a recombinant vector pPICZAa-(JFH1)E2¡ transforming the yeast and its expression in KM71H strain were successfully done.
In this study¡ the whole length of E2 Ag of HCV JFH1¡ as the protype strain of this virus¡ was successfully expressed. The result of this study can be used for further analysis of the structure and function of this protein.

There is a strong association between the presences of different types of human papillomaviruses to the development of genital lesions. HPV16 and HPV18 have been recognised as the most important etiologic agents for cervical dysplasia and carcinoma. The aim of this study was to investigate epidemiology of human papillomavirus (HPV) type 16 and 18 isolated from patients with cervical cancer admitted to Imam Khomeini Hospital in Tehran.
This case- control study was carried out on 100 patients with cervical cancer and 48 healthy people as control. DNA was extracted using phenol-chloroform method. Then, the relevant gene was amplified by PCR.
In this study, 79% of cases were HPV-positive, among them, 53% of samples was determined as HPV16 and 14% HPV18.
Since no association between these viruses and significantly detectable symptoms, and also because of the extreme prevalence of HPV in Iran, early detection of HPV can prevent cancer development as consequence of pre-cancer lesions. Also, molecular techniques such as PCR can contribute effectively identification of HPV cases to diagnose in a proper time.

Lysinibacillus is an endophytic bacterium that protects plant against environmental and physiological condition. The purpose of this study was to isolation and molecular identification of Lysinibacillus macroides from wheat roots and to analyse their antifungal activity against Trichoderma spp.
Material & The extract of root and stem cultivated wheats in different areas of Mazandaran and Golestan were used for isolation of endophytic bacteria. Their antifungal activity was evaluated in a dual culture method on the Potato Dextros Agar (PDA) medium. After genomic DNA extraction, 16S rRNA gene was amplified using PCR. Then the PCR product was sequenced by BLAST.
The isolated bacteria were able to inhibit the growth of biocontrol fungi. Based on the 16srRNA sequence studies, this bacterium belonged to Lysinibacillus and showed 99% similarity to L. macroides 1w.
This study is the first report of isolation of Lysinibacillus from the endophytic plants. The endophytic bacteria isolated in this study can be used for extraction, identification and application of its inhibitory metabolites for design and the production of antifungal agents.

Bacillus subtilis strains produce a wide variety of antimicrobial substances, such as iturin family lipopeptides, which are effective in biological control of many plant pathogens. The aim of present study was to investigation the antifungal activity of indigenous strains Bacillus subtilis against Fusarium moniliforme and Verticillium dahliae.
The forest soil samples were collected from seven different parks at Tehran. The isolates were screened by antifungal activity. Two best strains with greater inhibition zone were identified by PCR. Then, nutrient broth media were optimized to produce of large volumes of the antifungal metabolites from the selected native strains. Following 4 day incubation, the bacterial metabolites were purified, and the presence of iturin was confirmed by chromatography method.
Totally, 23 isolated strains were confirmed as B. subtilis. In subsequent experiments, two strains 36 and 78 showed the greatest activity against the Fusarium moniliforme and Verticillium dahliae respectively. 16srRNA sequence analyses for selected isolates confirmed 100% similarity to B. subtilis. The nutrient broth with glucose, yeast extract, neutral pH and 30 ⁰C incubation temperature were optimized for the best production condition. The HPLC results showed that both the ability of these strains to produce iturin A in a specific period was as the same as standard iturin.
These indigenous strains showed the ability to produce antifungal metabolites. Therefore, these strains can be used as good candidates for the biological control of plant pathogenic fungi and as an alternative for chemical fungicides.

Design of the production of single cell proteins depends on definition of the growth template of the producer microorganisms. This study was aimed to evaluate the production of Aspergillus niger single cell protein and simulation of cell biomass production based on several un-structured models.
In this experimental study, the fermentative process of single cell protein production was conducted in batch submerged culture with optimum culture medium formulation at 250C, pH 6 and 300 rpm for 200 h in an incubator shaker. At the end of process, the content of shaker flasks was used to analysis glucose concentration, cell dry weight and protein content in cell biomass.
The result simulation by Monod, Moser and Logistic models showed 92% 63% and 83% similarity, respectively. Increase in the pH from 3.5 to 6 caused 71% enhancement in protein content in cell biomass. However, pH more than 6 led to decrease in the cell biomass protein content and this values reached to 29% a pH 7. Increases in the initial glucose concentration from 10 to 50 g. L-1 did not show considerable effects on the cell biomass protein content. Cell biomass protein content of the media containing 50 g. L-1 initial glucose was only 5.67% more than the medium contained of 10 g. L-1 initial glucose.
The Monod kinetic model was proposed as a suitable model to simulate A. niger behaviour. Furthermore, pH of the media affects cell biomass protein content. However, initial glucose concentration in the media did not show significant effects on the cell biomass protein content.

Sorbitol is a 6- carbon polyol, which is used in food industries as a precursor for production of many products due to its sweetness and high solubility. The application of microbial process for sorbitol production has recently been becoming of point of interest because of requirement for suitable fermentative conditions, being cost effective and less environmental pollutions. The purpose of this study was to produce sorbitol from sucrose by Debaryomyces hansenii isolated from the nature.
The sorbitol producing yeast strain was isolated in a medium containing 40% glucose and 1% yeast extract. Following identification of yeast using ITS gene sequencing, we focused on the process of fermentation and production of sorbitol from sucrose. The quality and quantity of sorbitol production was analyzed by thin layer chromatography, colorimetric method and Megazime kit.
The sorbitol producing yeast strain was isolated from pollen of Malva sylvestris flower, and was identified as D. hansenii. This strain produced 5.23 g l-1 sorbitol after 5 days in medium containing of 150 g l-1 sucrose.
In this study sorbitol was produced by a D. hansenii isolated from the nature. Due to high efficiency of sorbitol production from sucrose by yeast, isolation of sorbitol producing native yeasts and optimization of the production conditions can be helpful for increases in quality and quantity of sorbitol production.

Mycotoxins are the secondary metabolites produced by Penicillium, Fusarium, and Aspergillus species. These metabolites are mainly produced by Aspergillus species. This study was aimed to isolation and characterization of the toxicogenic fungi strains from mostly consumed cereals (wheat, corn) used in Kerman city. This empirical basic study was carried out on 83 samples of wheat and corn collected from formal preservation units. Screening was conducted using sabouraud dextrose agar, sabouraud dextrose agar with chloramphenicol and PDA mediums. The isolated strains were identified according to morphologic characters. The presence of mycotoxins of pathogenic fungi was determined by HPLC. Overall, 37% of samples were infected with Aspergillus and A. flavus (14%) was the most frequent fungi. Also aflatoxins B1, B2 and G1 were isolated from Aspergillus using HPLC. Our results indicated the presence of toxigenic fungi on the mostly consumed cereals (wheat and corn). Therefore, a program must be planned to control the fungi on the cereals.