Effect of pH on Invertase Activity

ABSTRACT sucrase is a vitrine of enzyme, a natural catalytic agent for biochemical reactions, can be obtained in bakers Yeast. Determination of the re beginning of pH on invertase activity is the primary objective of the experiment. Dinitrosalicyclic sulphurous (DNS) Assay method is utilized to monitor the enzymatic activity of invertase. sucrase was subjected to different pH (3.87, 4.0, 5.5, 7.3 and 10.55) of buffer resultant and was observed under 540 nm absorbance use spectrophotometer. subsequently observation and analysis, a peak (optimum pH) was observed by plotting absorbance versus pH.INTRODUCTIONEnzymes be proteinaceous catalysts, which speed up the rate of a biochemical reaction. They reduce the activation energy that is essential for starting any type of chemical reaction. With a low energy requirement for activation, the reaction takes slur faster. The over both performance of an enzyme depends on various factors, such as temperature, pH, cofactors, activators a nd inhibitors. Invertase is an enzyme which is usually found in plants. It acts as a catalyst for the hydrolysis of sucrose. sucrose is a disaccharide composed of glucose and fructose linked by a glycosidic bond. When this bond is cleaved in a hydrolysis reaction, an equal amount of glucose and fructose. Invertase is a significant enzyme because glucose is an principal(prenominal) product of photosynthesis. Invertase is besides used in the confectionery diligence where fructose is preferred over sucrose because it is sweeter and does not crystallize easily.Enzymes are affected by changes in pH. Extreme pH values broadly result in loss of activity for most enzymes. Furthermore, there is a most favorable pH for enzyme the point where the enzyme is most active. This point is cognise as the optimal pH. The aim of this experiment is to find out the stove of pH which invertase is effective. The objectives of this experimentare to extract invertase from bread makers Yeast and to det ermine the effects of changes in pH on reaction rates of an enzyme-catalyzed reaction.MATERIALSThe materials used in this experiment are Bakers Yeast, Sucrose Standard Solution (100 mg/L), Concentrated HCl, 0.5 M KOH, DNS reagent, 0.1 M buffer solutions (pH 1, 3, 5, 7, 9, 11), ucrose solution (10 g/L), running tubes, pipets, beakers, volumetric flasks, alkane series film, hot plate and UV-Vis Spectrophotometer.METHODOLOGYExtraction of invertase from yeastTo extract the invertase from Bakers Yeast, 0.25 g of it was dissolved in distilled water to make a 250-mL solution. When the solution is lively (complete dissolvation of Bakers Yeast) it is then allowed to stand for 20 minutes at room temperature. Provided that the sediments form, the supernatant must be collected as it result be used as the enzyme stock solution that will be used in the succeeding experiment. Sucrose Assay exploitation Dinitrosalicylic colorimetric MethodIn preparation of this part of the experiment, a seri es of canvas tubes were prepared as follows Tube No. Blank 1 2 3 4 5 6 mL sucrose std. solution 0 0.25 0.50 0.75 1.00 1.25 1.50 mL distilled water 1.50 1.50 1.25 1.00 0.75 0.50 0.25After, 3 drops of concentrated HCl (0.05mL) were introduced to each analyze tube. note that the tubes were mixed well and then incubated after at a 90 degrees Celsius water tubful for 5 minutes. After the incubation, 0.15 mL of 0.5 M KOH was added to avoid the solution. Another 2.80 mL of 0.1 M buffer solution at pH 5 were added, then the solution was mixed well again. Then, 3 mL of DNS reagent was added forrader the test tubes were immersed in a water bath at 95 degree Celsius for 10 minutes to develop the characteristics of a red-brown polish solution. After alter, the solution were subjected into spectrophotometry to measure the absorbance at 540 nm. Effect of pH on Invertase ActivityIn finding the effect of pH on invertase activity, sixsome numbered test tubes were prepared with 2.90 mL appro priate 0.1 M buffer solution as shown below Tube No. 1 2 3 4 5 6 pH buffer solution 0.1 0.3 0.5 1.7 1.9 1.11Then, 0.10 mL enzyme stock solution was added to each test tube. After mixing thoroughly, all test tubes were incubated in 60 degrees Celsius water bath for 5 minutes. When the prison term was right, another 1.50 mL of sucrose was added. The solution was then incubated again and set to the same water bath for the same amount of time, 5 minutes. Then, 3 mL of DNS reagent was added before immersing the solution in a water bath (95 degrees Celsius) for 10 minutes until the solution turns into a red-brown colour solution. After cooling the first test tube, blank solutions were prepared by following stairs 1-4 again, unless instead of using the enzyme stock solution, denatured enzyme was added. All the test tubes containing the solution were then subjected to spectrophotometry to measure the absorbance at 540 nm.EXPERIMENTALSucrose Assay use Dinitrosalicylic colorimetric Metho dA. Materials used Sucrose Standard Solution, Distilled Water, Concentrated HCl, 0.5 M KOH, 0.1 M fan Solution, DNS Reagent, and UV-Vis Spectrophotometry. B. Procedure After collecting the supernatant from the enzyme stock solution, each test tube were introduced to 3 drops of conc. HCl before incubating at 90oC water bath for 5 minutes. 0.5 M KOH was then added to neutralize. Then, 2.80 mL of 0.1 M buffer solution was added before the solution was introduced to DNS reagent. The solution was in water bath at 950C for 10 minutes (until it is a red-brown solution). After cooling, it is subjected to spectrophotometry to measure absorbance at 540 nm. Effect of pH on Invertase ActivityA. Materials usedBuffer Solution, Enzyme Stock Solution, 1.50 Sucrose Solution, 3 mL DNS Reagent, strain Tubes, UV-Vis Spectrophotometry.B. ProcedureAfter preparing the required test tubes, they were introduced with 0.10 mL enzyme stock solution before existence incubated for 5 minutes in a water bath at 600C. Then, 1.50 mL sucrose solution was added before the solution was incubated again for 5 minutes in a water bath with the same temperature. After cooling, 3 mL DNS reagent was added before immersing the test tubesagain in a water bath at 950C until the red-brown color appears. Repeat steps 1-4 but this time, instead of adding the enzyme stock solution, add the denatured enzyme. After all the test tubes were prepared, they were sunjected to UV-Vis Spectrophotometry to measure absorbance at 540 nm.Image 1. The red-brown coloration after water bathRESULTSSucrose Assay Using Dinitrosalicylic Colorimetric Method The following table shows the results from the UV-Vis Spectrophotometer of Sucrose Assay using DNS Colorimetric MethodTest Tube No. Amount of Acid-Hydrolized Sucrose Absorbance Blank 0.0 0.000 A 1 0.56 0.335 A 2 1.11 -0.456 A 3 1.67 1.248 A 4 2.22 1.800 A 5 2.78 -0.238 A 6 3.33 -0.319 A Table 1. Results of Sucrose Assay using DNS Colorimetric Method The students were also as ked to plot the hydrolized-sucrose standard curve by plotting Absorbance against Concentration (mg/mL)Chart 1. Standard meander of Absorbance against Concentration.Effect of pH on Invertase Activity The following table shows the results from the UV-Vis Spectrophotometer in respect to the Effect of pH on Invertase ActivitypH Amount of Acid-Hydrolized Sucrose Absorbance Blank 0.0 0.000 A 3.87 2.02 0.162 A 4.0 9.12 0.78 A 5.5 12.6 0.975 A 7.3 1.883 0.151 A 10.55 9.33 0.748 A Table 2. Results of the Effect of pH using Colorimetric Method.