The crude enzyme was precipitated at 70% saturation of ammonium sulfate, and the protein was collected by centrifugation (10,000×g for 10 min). It was suspended in minimal volume of double-distilled water. The precipitate was dialyzed and used for enzyme purification using column chromatography. The dialyzed sample was loaded on DEAE-cellulose column, which was equilibrated previously with the buffer A (50 mM sodium phosphate buffer, pH 7.4). The column was washed with the five bed volumes of buffer A, and the enzyme was eluted with buffer A
Abstract In this experiment, the reaction kinetics of the hydrolysis of t-butyl chloride, (CH3)3CCl, was studied. The experiment was to determine the rate constant of the reaction, as well as the effects of solvent composition on the rate of reaction. A 50/50 V/V isopropanol/water solvent mixture was prepared and 1cm3 of (CH3)3CCl was added. At specific instances, aliquots of the reaction mixture were withdrawn and quenched with acetone. In addition, phenolphthalein was added as an indicator.
This experiment is carried out to determine the percentage of calcium carbonate, CaCO3 in the toothpaste provided with the experimental technique known as back titration. A back titration is also known as indirect titration. A known mass of toothpaste is neutralised with a known concentration and volume of hydrochloric acid, HCl. The mixture is then further neutralised by a known concentration and volume of sodium hydroxide solution, NaOH to determine the number of mole of HCl that reacted with CaCO3 in the toothpaste. As the number of mole of CaCO3 is found through the mole ratio thus the mass of CaCO3 is known and the percentage of CaCO3 can be calculated.
3.1 Preliminary optimization studies 3.1.1. Effect of reaction time: Figure.3 represents the time progression for the enzymatic esterification of ethanol and hexanoic acid with 1:1 substrate ratio by Novozyme 435 (2 %) at 50 ˚C. It was observed that percentage conversion of ethyl hexanoate reached up to 73.6% in the initial 120 min. However, as the reaction proceeds further, a marginal change in conversion was observed after 120 min because of equilibrium of the reaction. This is attributed to the reversible nature of the esterification reaction.
Germinating peas and maize respire at faster rates than fresh and dried peas and maize at room temperature and 400C(graph no). This could be because pea seeds have two cotyledons as compared to the single one of maize so oxygen usage is also more. These results support my hypothesis stated earlier. Respiration is an enzymatic process where enzymes are required at every step in the breakdown of glucose. At 600C for both peas and maize the respiration rate went down as the enzymes denatured.
CH3 175 83.06% 287-289ºC 4. -OCH3 191 86.03% 275-277ºC 5. 204 78.78% 295ºC Step-3 Synthesis of 2-Methyl benzoxazin -4(3H)-one53 (4) Anthranilic acid (0.1M, 18g) was taken in acetic anhydride and refluxed under anhydrous conditions for 4 hrs. Excess of acetic anhydride was then distilled off under reduced pressure. Obtained product was immediately used for next step.
Catalase Test, in this test the microbial culture from Nutrient Agar plates were used. This test determines the production of catalase by the microorganisms. Catalase is an enzyme which decomposes hydrogen peroxide to water and oxygen gas thereby, protecting the microorganisms from the lethal effect of hydrogen peroxide which is accumulated as an end product of aerobic carbohydrate metabolism. (Bahrami-Hessari et. al.
2.3 Determination of radical scavenging activity (RSA) Determination of RSA was carried out according to Murthy, Jayaprakasha, & Singh (2002) using DPPH as stable free radical and butylatedhydroxyanisole (BHA) as standard. Different aliquots of extract and BHA were taken in 100 μl fixed volume. 0.1 mM methanolic solution of DPPH (5 ml) was added and shaken vigorously. The tubes were kept at room temperature in dark for 20 min. MeOH was used for baseline correction.
Laccase activity will be estimated by measuring oxidation of 2,6-DMP in 50 mM malonate buffer (pH 4.5) containing 1.0 mM 2,6-DMP at 469 nm. The filter paper activity will be determined by release of reducing sugars produced in 60 min from a mixture of 125 µL of appropriate clued enzyme, 250 µL of 50 mM acetate buffer (pH 5.0), and 20 mg of Watman No. 1 filter paper (1.5x1.0 cm) incubated at 50°C. One filter paper unit (FPU) will be defined as amount of enzyme that released 1 µmol glucose/min. Xylanase activity will also be determined by release of reducing sugars produced in 30 min from a mixture of 50 µL of appropriate clued enzyme and 200 µL of 50 mM acetate buffer (pH 5.0) containing 1% (w/v) birchwood xylan incubated at 50°C.
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.