Effect of pH on Invertase Activity

ABSTRACT saccharase is a type of enzyme, a natural catalytic agent for biochemical reactions, can be obtained in Bakers Yeast. finish of the effect of pH on invertase activity is the primary objective of the experiment. Dinitrosalicyclic acid (DNS) Assay rule is utilized to monitor the enzymatic activity of invertase. Invertase was subjected to different pH (3.87, 4.0, 5.5, 7.3 and 10.55) of mince rootage and was find under 540 nm absorbance using spectrophotometer. After 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 demand for starting any type of chemical reaction. With a low energy requirement for activation, the reaction takes coif faster. The overall performance of an enzyme depends on various factors, such as temperature, pH, cofactors, activators and inhibitors. Invertase is an enzyme which is usually found in plants. It acts as a catalyst for the hydrolysis of sucrose.saccharose is a disaccharide composed of glucose and fructose linked by a glycosidic attach. 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 important product of photosynthesis. Invertase is also used in the confectionery industry 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 generally direct in loss of activity for most enzymes. Furthermore, there is a most favorable pH for enzyme the shoot for where the enzyme is most active. This point is known as the optimal pH. The aim of this experiment is to find out the feed of pH which invertase is effective. The objectives of this experimentare to extract invertase from Bakers Yeast and to receive 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 wing resolvings (pH 1, 3, 5, 7, 9, 11), ucrose solution (10 g/L), footrace organ pipes, pipets, beakers, volumetric flasks, paraffin 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 pee to make a 250-mL solution. When the solution is lively (complete dissolvation of Bakers Yeast) it is thusly allowed to stand for 20 minutes at room temperature. Provided that the sediments form, the supernatant essential be collected as it bequeath be used as the enzyme stock solution that will be used in the succeeding experiment. Sucrose Assay utilize Dinitrosalicylic colorimetrical MethodIn preparation of this fragmentise of the experiment, a series of test tubes were prepared as foll ows 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 body of 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 test tube. Noted that the tubes were confused well and then incubated after at a 90 degrees Celsius water clean for 5 minutes. After the incubation, 0.15 mL of 0.5 M KOH was added to neutralize the solution. another(prenominal) 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 earlier the test tubes were immersed in a water bath at 95 degree Celsius for 10 minutes to develop the characteristics of a red-brown colour solution. After cooling, the solution were subjected into spectrophotometry to measuring rod the absorbance at 540 nm. Effect of pH on Invertase ActivityIn finding the effect of pH on invertase activity, six numbered test tubes were prepared with 2.90 mL appropriate 0.1 M buf fer solution as shown at a lower place 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 time was right, another 1.50 mL of sucrose was added. The solution was then incubated again and treated 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 quest steps 1-4 again, but instead of using the enzyme stock solution, alter enzyme was added. All the test tubes containing the solution were then subjected to spectrophotometry to measure the absorbance at 540 nm.EXPERIMENTALSucrose Assay Using Dinitrosalicylic Colorimetric MethodA. Materials used S ucrose Standard Solution, Distilled Water, Concentrated HCl, 0.5 M KOH, 0.1 M Buffer 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 take Solution, 1.50 Sucrose Solution, 3 mL DNS Reagent, interrogation Tubes, UV-Vis Spectrophotometry.B. ProcedureAfter preparing the required test tubes, they were introduced with 0.10 mL enzyme stock solution before being incubated for 5 minutes in a water bath at 600C. Then, 1.5 0 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 asked to plot the hydrolized-sucrose standard curve by plotting Absorbance against engrossment (mg/mL)Chart 1. Standard Curve of Absorbance against Concentration.Effect of pH on Invertase Activity The following table shows the results from the UV-Vis Spectrophotometer in esteem 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.