**2.3. Chromosomal DNA and plasmid extraction**

Chromosomal DNA was prepared from overnight culture in LB,using AxyGEN Biosciences DNA extraction kit, according to manufacturer's instructions. Plasmid extraction was performed using Wizard mini prep. extraction kit (Promega) according to manufacturer's instructions with slight modification, where 50 L of lysozyme (200 mg/mL) were added to the resuspended buffer and incubated at 37 °C for 1 h then the protocol was carried on as described in the kit (O'Sullivan & Klaenhammer, 1993; Sambrook, et al., 1989).

## **2.4. PCR amplification for molecular identification**

To amplify the 16S rRNA gene, a primer pair hybridizing to two conserved regions in 16S rRNA genes from *Bacillus spp*. was used: (bac-F and bac-R) (Ash et al., 1991; Kwon et al., 2009). For the amplification of the 16–23S intergenic region, a primer pair was used: L516SF and L523SR. While for recA gene, a primer pair corresponding to conserved regions in recA genes from *Bacillus sp*. was used: recA-F and recA-R. Speciesspecific primer set for *B. subtilis* corresponding for ytcP gene was used: ytcP-F and ytcP-R. All polymerase chain reaction amplifications were performed with the Taq DNA polymerase kit (Promega). Reaction mixtures consisted of 20 mm Tris–HCl (pH 8.4), 50 mm KCl, 3 mm MgCl2, 50 mm of each of the four deoxynucleoside triphosphates (dNTP), 1 U Taq polymerase, 5 pmol of each primer and 1 L of template DNA in a final volume of 50 L. Samples were amplified in a GeneAmp polymerase chain reaction system 2700 (Applied Biosystems) programmed as follows: initial denaturation of DNA for 5 min at 94 °C, 30 cycles of 30 s at 94 °C, 30 s at 50 °C and 30 s at 72 °C. Polymerase chain reaction products were quantified by electrophoresis on a 1% (w/v) agarose gel containing ethidium bromide. Polymerase chain reaction products obtained from the selected isolates were purified, using QIA quick polymerase chain reaction purification KIT (Qiagen) and then sequenced commercially by Sigma-Egypt. The sequencings were performed and manually aligned, using DNAMAN software (version 4.0). Sequence homologies were examined by comparing the obtained sequence with those in the NCBI database and the NEB cutter V 2.0 database. NEB cutter V 2.0 is an on-line DNA sequence tool used to find large, nonoverlapping, open-reading frames and works for all restriction enzymes. It provides a website, which allows users to check nucleotide sequences for restriction enzyme sites. The sequences were submitted and AluI enzyme was chosen for digestion. Finally gel photograph using 2% agarose was viewed (Ash et al., 1991).

## **2.5. Bacterial strains and growth conditions**

Bacillus strains used in this work were cultivated in Luria-Bertani (LB) broth or agar at 37 °C. Defined medium was used for cellular production of levansucrase (Yanase et al., 1992). It had the following composition (g/L): yeast extract, 2.5; commercial sucrose, 80; MgSO4, 0.2 and K2HPO4, 5.5. The medium was completed by the addition of 1 L distilled water and the pH was adjusted to 7.0 before autoclaving. The parameters included initial incubation temperature (25–45 °C); different concentrations of sucrose (80–160 g); incubation time (16– 48 h); (50–150) rpm; pH (5-9) and NaCl (1–4% w/v) were studied. The sucrose was substituted with fructose, glucose, lactose (80 g/L) and beet molasses (equivalent to 80 g sucrose) to study their effects on enzyme production.

### **2.6. Cellular production**

Cultivation was carried out in 250 mL Erlenmeyer flasks. Each flask contained 50 mL production medium and was autoclaved for 15 min. The flasks were then inoculated with 2.0 ml inoculum and incubated for 24 h at 30 °C. The culture broth was then centrifuged in a cooling centrifuge (K70; Janektzki, Germany) at 10,397×g to separate the bacterial cells from the supernatant.

### **2.7. Assay of levansucrase**

Levansucrase assay was performed according to the method of Yanase et al. (1992) with some modification. 0.5 ml of culture filtrate was incubated with 1 ml 20% (w/v) sucrose and 0.5 mL 0.1 M acetate buffer at pH 5.2 and incubated at 37°C for min. The decreasing amounts of sugars produced were measured by glucose oxidase kits. One unit of enzyme activity was defined as the amount of enzyme that produced decreasing sugars equivalent to 1 \_mol of glucose/min.

### **2.8. Separation of levan polymer**

The levan producing organisms were cultivated on a defined medium as described above. After growth, the culture was centrifuged to remove bacterial cells; the levan was precipitated with two volumes of absolute ethanol. The precipitate was collected and dried under vacuum.

## **2.9. Chromatography**

198 The Complex World of Polysaccharides

**2.6. Cellular production** 

**2.7. Assay of levansucrase** 

to 1 \_mol of glucose/min.

under vacuum.

**2.8. Separation of levan polymer** 

the supernatant.

aligned, using DNAMAN software (version 4.0). Sequence homologies were examined by comparing the obtained sequence with those in the NCBI database and the NEB cutter V 2.0 database. NEB cutter V 2.0 is an on-line DNA sequence tool used to find large, nonoverlapping, open-reading frames and works for all restriction enzymes. It provides a website, which allows users to check nucleotide sequences for restriction enzyme sites. The sequences were submitted and AluI enzyme was chosen for digestion. Finally gel

Bacillus strains used in this work were cultivated in Luria-Bertani (LB) broth or agar at 37 °C. Defined medium was used for cellular production of levansucrase (Yanase et al., 1992). It had the following composition (g/L): yeast extract, 2.5; commercial sucrose, 80; MgSO4, 0.2 and K2HPO4, 5.5. The medium was completed by the addition of 1 L distilled water and the pH was adjusted to 7.0 before autoclaving. The parameters included initial incubation temperature (25–45 °C); different concentrations of sucrose (80–160 g); incubation time (16– 48 h); (50–150) rpm; pH (5-9) and NaCl (1–4% w/v) were studied. The sucrose was substituted with fructose, glucose, lactose (80 g/L) and beet molasses (equivalent to 80 g

Cultivation was carried out in 250 mL Erlenmeyer flasks. Each flask contained 50 mL production medium and was autoclaved for 15 min. The flasks were then inoculated with 2.0 ml inoculum and incubated for 24 h at 30 °C. The culture broth was then centrifuged in a cooling centrifuge (K70; Janektzki, Germany) at 10,397×g to separate the bacterial cells from

Levansucrase assay was performed according to the method of Yanase et al. (1992) with some modification. 0.5 ml of culture filtrate was incubated with 1 ml 20% (w/v) sucrose and 0.5 mL 0.1 M acetate buffer at pH 5.2 and incubated at 37°C for min. The decreasing amounts of sugars produced were measured by glucose oxidase kits. One unit of enzyme activity was defined as the amount of enzyme that produced decreasing sugars equivalent

The levan producing organisms were cultivated on a defined medium as described above. After growth, the culture was centrifuged to remove bacterial cells; the levan was precipitated with two volumes of absolute ethanol. The precipitate was collected and dried

photograph using 2% agarose was viewed (Ash et al., 1991).

**2.5. Bacterial strains and growth conditions** 

sucrose) to study their effects on enzyme production.

Paper chromatography was performed according to Block et al. (1995). Hydrolysate of products of levan were analyzed by either paper chromatography on whatman No. 1. The mixtures at the end of incubation time were boiled for 3 min to stop the reaction. Chromatographic development was carried out with a solvent system of nbutanol:acetone:water (4:5:1) and detected by spraying with aniline hydrogen phthalate. The acid hydrolysate of the polysaccharide produced by the six isolates was analyzed using high-pressure liquid chromatography (HPLC). A 7.8 mm ×300 mm PL-HI-PLEXPB column was linked to a differential refractometer. The column temperature was maintained at 80 °C. The aqueous mobile phase was delivered at a flow rate of 0.6 ml/min.

## **2.10. Determination of molecular weight**

Different concentrations of levan and oligosaccharide were prepared and the flow time of equal volume for each concentration at 30°C was determined in a U-shaped Ostwald viscometer. Flow time of the same volume of distilled water was also determined as control. Thus, specific viscosity/C (gsp) was estimated. A plot of levan and oligosaccharide concentration (C) against intrinsic viscosity (C) (gsp/C) therefore yielded a straight line.

### **2.11. Antivirus detection**

Two types of viruses were used, highly pathogenic avian influenza H5N1 virus Egyptian isolate, was used at titre of 106 EID50/mL (embryo infective dose per mL) and adenovirus type 40 with different doses 201×104, 1× 105, and 1×106 infectious particles/mL obtained from the Holding Company for Biological Products & Vaccines (VACSERA).

### **2.12. Specific pathogen free (SPF) eggs**

SPF embryonated chicken eggs were used at nine days old and inoculated via the allantoic sac route. SPF eggs (Brown et al., 2007) were obtained from Nile SPF Eggs, Koomoshiem, Fayoum, Egypt.

### **2.13. Cytotoxicity test**

It was done according to Simoes et al., (1999) and Walum et al., (1990). Briefly, All samples (100 mg) were dissolved in 500 \_L of water or ethanol. Samples A, E, M, and K were dissolved in ethanol while samples C and G were dissolved in water. Decontamination of samples was done by adding 12 \_L of 100x of antibiotic–antimycotic mixture to 500 \_L of each sample. Then, bi-fold dilutions were done to 100 \_L of original dissolved samples and 100 \_L of each dilutions were inoculated in Hep-2 cell line (obtained from the Holding Company for Biological Products & Vaccines VACSERA, Egypt) previously cultured in 96 multi well plates (Greiner-Bio one, Germany) to estimate the non toxic dose of the tested samples. Cytotoxicity assay was done using cell morphology evaluation by inverted light microscope and cell viability test applying trypan blue dye exclusion method.
