Enzymes

Extraction of Invertase from Yeast

Preparation of Denatured Invertase Stock Solution

Sucrose Assay using Dinitrosalicylic Colorimetric Method

Effect of pH on Invertase Activity

Effect of Temperature on Invertase Activity

Weigh 0.25 g Bakers yeast and dissolve in distilled water to make 250-mL solution.

Incubate 100 mL enzyme stock solution in a boiling water bath for 10 minutes.

Prepare a series of test tubes

Prepare 6 numbered test tubes. Add 2.90 mL appropriate 0.1 M buffer solution.

Set up 20, 30, 50, 60, 70, and 90 C water baths.

Allow to stand for 20 minutes at room temperature.

Allow the solution to cool.

Add 3 drops concentrated HCl to each test tube. Mix well. Incubate at 90 water bath for 5 minutes.

Collect the supernatant (if sedimentation occurs). The supernatant serves as the enzyme stock solution that will be used for following experiments.

Collect only the supernatant if frothing occurs. This serves as the denatured enzyme stock solution that will be used for succeeding experiments.

Add 0.15 mL 0.5 M KOH to neutralize the solution.

Add 0.10 mL enzyme stock solution to each test tube. Mix thoroughly. Incubate all tubes in 60C water bath for 5 minutes.

Prepare 6 test tubes with each test containing 1.5 mL sucrose solution. Incubate the test tubes separately for 5 minutes in each water bath.

Add 2.80 mL 0.1 M buffer solution, pH 5. Mix well.

Add 1.50 mL of sucrose solution and incubate reaction mixture in 60Cwater bath for 5 minutes.

In another test tube, mix 0.80 mL enzyme stock solution with 19.20 mL 0.1 M buffer solution, pH 5.

Add 3 mL of DNS reagent. Immerse the test tubes in 95 C water bath for 10 minutes to develop the characteristic red brown color.

Add 3 mL of DNS reagent. Immerse the test tubes in 95 C water bath for 10 minutes to develop the characteristic red-brown color. Allow the solutions to cool.

Add 3 mL of dilute enzyme solution to all tubes. Incubate for another 5 minutes.

Add 3 mL of DNS reagent. Immerse the test tubes in 95 C water bath for 10 minutes to develop the characteristic red-brown color. Allow the solutions to cool.

After cooling, measure the absorbance of 540 nm.

Prepare blank solutions by following steps 1-4. Add denatured enzyme instead of enzyme stock solution.

Construct the hydrolyzed sucrose standard curve by plotting A540 against concentration (mg/mL).

Prepare blank solutions by following steps 2-5. Add denatured enzyme instead of enzyme stock solution.

Measure the absorbance of 540 nm. Measure the absorbance at 540 nm. Determine the amount of sucrose hydrolyzed using hydrolyzedsucrose standard curve.

Determine the amount of sucrose hydrolyzed using hydrolyzedsucrose standard curve.