EXTRACTION OF INVERTASE FROM YEAST

First, weigh 10.0 g of the baker’s yeast. Dissolve it in 30 mL of the 0.1 M NaHCO3.
Second, let the solution stand for 24 hours at room temperature. Third, decant the
supernatant if sedimentation occurs. Dilute the supernatant to 1:100 using distilled
water. Lastly, the resultant supernatant will be used as the enzyme solution in the
succeeding experiments.

REDUCING SUGAR ASSAY USING DNS COLORIMETRIC METHOD

1. Prepare a series of test tubes as follows:

Test
Blank 1 2 3 4 5 6
Tube No.
Invert
Sugar
Standard 0 0.10 0.30 0.50 0.80 1.00 1.20
Solution,
mL
Distilled
2.50 2.40 2.20 2.00 1.70 1.50 1.30
H2O, mL

2. Add 0.50 mL of the 0.05 M acetate buffer solution, pH 4.7, to each. Mix well.
3. Add 2 mL of the DNS reagent. Immerse the test tubes in a boiling water bath for 10
minutes to develop a characteristic red-brown color.
Note: Cover all the tubes with marbles to prevent the solvent from evaporating.
4. Cool the test tubes in an ice water bath. Add 5 mL of distilled water to each tube. Mix
well.
5. Measure the absorbance at 540 nm.
6. Construct the invert sugar standard curve by plotting A540 against the concentration of
the invert sugar (mol/L).

EFFECT OF TEMPERATURE ON INVERTASE ACTIVITY (+GRAPH)

 1. Set up 20 °C, 30 °C, 50 °C, 60 °C, 70 °C, and 90 °C water baths.
2. Prepare six test tubes. Each test tube should contain 1 mL of the 0.03 M sucrose, 1.4
mL of distilled water, and 0.50 mL of the 0.05 M acetate buffer solution, pH 4.7. Incubate
the test tubes separately for 5 minutes in each of the water baths.
3. Add 0.1 mL of the enzyme solution to the tubes. Incubate the test tubes for another 5
minutes.
Note: Do not remove the test tubes from their respective water baths.
4. Add 2 mL of the DNS reagent. Immerse the test tubes in a 95 °C water bath for 10
minutes to develop a characteristic red-brown color.
Note: Cover the tubes with marbles to prevent the solvent from evaporating.
5. Cool the test tubes in an ice water bath. Add 5 mL of distilled water to each tube. Mix
well.
6. Prepare another test tube for the reagent blank containing 0.1 mL of the denatured
enzyme, 1 mL of the 0.03 M sucrose, 1.4 mL of distilled water, and 0.50 mL of the 0.05
M acetate buffer solution, pH 4.7. Incubate them for 5 minutes at room temperature.
7. Repeat steps 4-5
8. Measure the absorbance at 540 nm.
9. Determine the amount of invert sugar produced in the first experiment.
10. Plot the temperature against the amount of the invert sugar.

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