Preparation of CMCS The carboxymethylation process of CS into water soluble CMCS was carried out according to the method reported previously (Qian et al., 1996; Chen, et al., 2004). Briefly, CS powder (10 g) was suspended in 100 ml of isopropyl alcohol and the resulting slurry was stirred in a 500 ml flask at room temperature. 25 ml of 10 N aqueous NaOH solution, divided into five equal portions, was then added to the stirred slurry over a period of 25 min. The alkaline slurry was stirred for additional 30 min. Subsequently, monochloroacetic acid (60 g) was added, in five equal portions, at 1 min intervals.
One unit (U) of glucoamylase is defined as the amount that liberates 1 µmol of reducing sugar as glucose/ml/min under the assay condition. One ml of the diluted enzyme extract was added to 1.0 ml of 5% soluble starch solution prepared in acetate buffer (pH 4.8). The enzyme substrate mixture was incubated at 60 0 C for one hour. Then 2 ml of Dinitrosalicylic acid reagent (DNS) was added to each test tube. The test tubes were placed in boiling water for 5 minutes and cooled to room temperature.
The DEAE CL-6b gel was washed twice with 0.5m Hcl, twice with 0.5m Naoh and twice with PB ph6.0 before utilization. For DEAE 1ml gel every 1ml serum was utilized. In the wake of blending for 1 hour at 200c the suspension was centrifuged at 4500g for 25 minutes. The supernatant was thought by Ammonium sulfate precipitation and pellet was broken up in refined water and the ensuing arrangement
coli transformants was isolated by alkaline lysis method (Sambrook et. al., 1989). Clones obtained on plates were inoculated in 5ml LB broth containing appropriate antibiotic and incubated overnight at 37°C under shaking conditions. The cells were harvested by centrifugation at 7000rpm for 5min and resuspended in 200 μl of GTE (Glucose-tris-EDTA) buffer. For lysis two volumes i.e.
The residual biomass was separated by filtration and washed with distilled water. For alginate extraction, the acidified algal biomass was suspended in 3% Na2CO3 solution at different alkali: alga ratio (20, 40, and 60 mL/g). The different extraction temperatures ranged from 25 to 45º C, and lasted for 1 to 3 h. For each experimental run, sodium alginate was collected by filtration and precipitated with absolute ethanol (1:2 v/v). The mixture was maintained at 4º C overnight. The precipitate was collected by vacuum filtration and allowed to dry at room temperature.
188.8.131.52-Extraction method No.2: Fifty grams of powdered aerial parts of portulaca oleracea extracted by soxhlet with 500ml 0f 70% ethanol for 8 hr. then the extract cool at room temperature ,filter and evaporate to dryness by rotatory evaporator at 60ºC .The dried extract hydrolyzed by reflux with 250 ml of 2N of hydrochloric acid for 3 hr. The final extract cool at room temperature ,filter then partitioning with ethyl acetate three times each one with 50 ml of ethyl acetate ,combine the three lower layer and evaporate to dryness by rotatory evaporator to give the crude extract(12.765 gm) as shown in scheme
The mixtures was filtered with Whatman #1 filter paper and the filtrate was used to repeat the extraction for another two times. Then, the methanol solvent in mixtures was removed using the Rotary Evaporator (EYELA, N1100) at 140° hPa; 60°C; speed 5. The distilled water and excessive methanol were removed with Freeze Dryer (GENEVAC LTO, EZ 2.3 ELITE) for a week. The crude obtained were weighed and stored at -20°C until use. 1.2 Partitioning method
Each isolates was inoculated into 50ml cellulase enzyme production broth medium with inoculum size of 2 x 106 spore concentration. 3.1.10 Preparation of crude enzyme After a desired period of incubation, the cellulase enzyme production broth was poured into a sterile Falcon tube and centrifuged at 10 000 rpm at temperature of 5oC to separate the cell and the broth. The supernatant was kept as a crudes enzyme sources for enzyme assay. 3.1.11 Preparation of sodium citrate buffer 3.2 Analysis techniques 3.2.1 Reducing Sugar Assay
* At the same time, 33 mg of fullerene was dissolved in 5 ml of Orthodichloro benzoic acid. And it was stirred for about 3 hours in a separate conical flask. * With the help of a dropper the dissolved fullerene was added into the RB which had the hydrazone. * Once the addition was complete the temperature was increased to 80 degrees and it was kept on refluxing for overnight duration. * The progress of the reaction was checked from time to time by checking the TLC in the solvent of the column, that is
The reaction mixture was poured into ice cold water acidify with 1% HCl and precipitates were collected, filtered and dried and recrystalized with ethanol. 4.2.2 General procedure for synthesis of flavones: 50mg of chalcone was taken in round bottom flask. After that 15-20 ml of DMSO was added and mixed properly, 5-6 granules of iodine were added and reflux the reaction mixture up to 3-6 hours and kept for overnight. Then reaction mixture was poured into ice water and the precipitates were neutralized with sodium thiosulphate, to remove the unreacted iodine, washed with water, filtered, dried and recrystalized with