METHODOLOGY

A. Extraction of Invertase from Yeast

A bakers yeast of 10 g was prepared and was dissolved in 30 mL of 0.1 M NaHCO 3.

The solution was let to withstand for 24 hours at room temperature. The
supernatant was diluted to 1:100 using distilled water. The resultant supernatant
was used as the enzyme solution in the next experiments.

B. Preparation of Denatured Invertase Stock Solution

The 25 mL enzyme stock solution was incubated in a boiling water bath for 10
minutes and was cooled after. The resultant supernatant was used as the denatured
enzyme stock solution in the next experiments.

C. Reducing Sugars Assay Using the Dinitrosalicylic (DNS) Colorimetric Method

A series of 6 test tubes were prepared. For each test tube, 0.50 mL of the 0.05 M
acetate buffer solution, with a pH of 4.7, and 2 mL of DNS reagent were added and
mixed. The test tubes were immersed in a boiling water bath for 10 minutes to
develop a red-brown color. The tubes were covered with marbles to prevent the
solvent from evaporating. The test tubes were cooled in an ice water bath and 5 mL
of distilled water was added and mixed to each. The absorbance was measured to
540 nm using a UV-Vis spectrophotometer.

D. Effect of pH on Invertase Activity

A set of six numbered test tubes were prepared. For each test tube, 0.50 mL of 0.1
M buffer solution was added (different pH numbers were assigned to each test
tube). To each test tube, 0.10 mL of the enzyme stock solution and 1.4 mL of
distilled water were added and mixed. The tubes were incubated in a 60 oC water
bath for 5 minutes. Then, 1 mL of 0.03 M sucrose was added and the reaction
mixtures were incubated in a 60 oC water bath for 5 minutes. DNS reagent was
added. The test tubes were immersed in a boiling water bath for 10 minutes to
develop a red-brown color. The tubes were covered with marbles to prevent the
solvent from evaporating. The test tubes were cooled in an ice water bath and 5 mL
of distilled water was added and mixed to each.

Another set of six test tubes were prepared for the reagent blank, each containing
0.10 mL of the denatured enzyme, 1.4 mL of distilled water and 0.5 mL of the
corresponding buffer. The mixtures were mixed thoroughly. The tubes were
incubated in a 60 oC water bath for 5 minutes. The next steps were repeated.

The absorbance was measured to 540 nm using a UV-Vis spectrophotometer.

E. Effect of Temperature on Invertase Activity

Water baths with 20 oC, 30 oC, 50 oC, 60 oC, 70 oC and 90 oC were set up. A set of six
test tubes were prepared. Each test tube contained 1 mL of 0.03 M sucrose, 1.4 mL
of distilled water and 0.50 mL of 0.05 M acetate buffer solution, pH 4.7. The tubes
were incubated separately (according to their assigned temperature) for 5 minutes
in a water bath. Then, 0.1 mL of the enzyme solution was added and incubated
again for 5 minutes. DNS reagent was added. The test tubes were immersed in a 95
o
C water bath for 10 minutes to develop a red-brown color. The tubes were covered
with marbles to prevent the solvent from evaporating. The test tubes were cooled in
an ice water bath and 5 mL of distilled water was added and mixed to each.

Another set of six test tubes were prepared for the reagent blank, each containing
0.10 mL of the denatured enzyme, 1 mL of 0.03 M sucrose, 1.4 mL distilled water
and 0.50 mL of 0.05 M acetate buffer solution, pH 4.7. The next steps were
repeated.

The absorbance was measured to 540 nm using a UV-Vis spectrophotometer.