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.
After which the digestions were examined by gel electrophoresis. The samples were run on a 50 mL 0.9% (w/v) agarose gel in 1X TAE buffer at 100 V until the leading track dye traveled 2/3 the distance of the gel. The gel was then soaked in GelRed for 20 minutes and examined under UV light. To prepare the digestions 10 μL of each digestion was mixed with 2 μL of 6X track dye in a micro centrifuge tube. 12 μL of 1 kb DNA ladder and each digestion was run on the gel.
The incubation mixture contained 2.5 ml of 1.2% (w/v) fibrin, 2.5 ml of 100 mM Tris–HCl buffer, 10 mM CaCl2 (pH 7.8), and 20 µg of enzyme. The incubation was carried out at 37°C for 30 min, and the reaction was stopped by adding 5 ml of 110 mM trichloroacetic acid containing 220 mM sodium acetate and 330 mM acetic acid. This reaction mixture was centrifuged at 3,000×g for 5 min, and the absorbance of the trichloroacetic acid (50 mM) soluble product was determined at 275 nm. One unit of fibrinolytic enzyme activity was defined as the amount of enzyme required to liberate 1 µg of L-tyrosine per minute at 37°C. The total protein determination was performed as described by Lowry et al.
We inoculated them with our stain and incubated them on shaker for 24 hours. After 24 hours incubation, we took O.D at 600nm and plotted a graph. To check effect of different temperatures on rhizospheric nitrogen bacterial growth: We took 6 flasks (100ml), and added 20ml L-broth in each flask and autoclaved it. We inoculated them with our stain. We selected different temperatures 4°C, 15°C, 37°C.
Nutrient agar medium (peptone 10.0 g, beef extract 3.0 g, sodium chloride 5.0 g, agar 20.0 g per liter and final pH 7.0) was prepared and autoclaved at 121°C for 30 min. Isolation was made using pour plate method and the Plates were incubated at 37°C for 48 h. (Three plates
The aqueous extract was prepared by dissolving 1g of dry extract with 20 ml of sterilized distilled water, so the final concentration of extract would be 0.05 g/ml, from this solution other concentration were prepared (0.1-0.2) g/ml. the solutions were shaken for 30 min. The extract was centrifuged (30,000 rpm; 15 min) and the supernatant was Separated. To hydroalcoholic extract, 80 g of the powder was extracted with aqueous methanol (75%). The other two concentrations were prepared from soaking sixfold aqueous methanol (75%) with different amounts of powder.
All reaction tubes, enzyme stock, and the two tubes with water were warmed in a 37 °C water bath for ten minutes. After heating, the reactions were started by adding 250 µL of the enzyme solution into each of the A and B tubes and 250 µL of the deionized water to the C tube. The tubes were kept in the water bath for 15 minutes. After the 15 minutes, 750 µL of 0.20 M NaOH was added to each tube to stop the
The dried mushroom samples were further heated at 105 ºC overnight until constant weight obtained for moisture content determination. The samples were incinerated at 550 ºC for 24 h and reweighed to determine ash content. MicroKjeldahl method was employed for the crude protein content (N × 4.38). The crude fat was determined by extracting a known weight of sample in Soxhlet apparatus using petroleum ether as a solvent. Total carbohydrates calculated by the difference.
The sample was transferred to a 250 ml conical flask kept in water bath for alkali treatment. 75 ml of 17.5% caustic soda was measured using a measuring cylinder at 20°C. 15 ml of 17.5% NaOH was added and fibres were macerated gently with a flattened glass rod for 1 minute. 10 ml more NaOH was added and the solution was mixed for 45 seconds. 10 ml NaOH was again added and mixed for 15 seconds to make lump free slurry.
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