ENZYMES

EFFECT OF pH ON INVERTASE ACTIVITY

Bob Dylan S. Pascual, Rhuoni D.F. Pasion,

Febie Anne C. Pelias, Christoper Alexander S. Payba, Katrina M. Piczon
Group 7 2H Medical Technology Biochemistry Laboratory

ABSTRACT

Enzymes are biological catalysts, protein in nature and reaction specific. The objectives of the experiment are: to
extract invertase that breaks existing bond in sucrose to form glucose and fructose from Baker’s yeast and to
determine the effects of the change in pH on the reaction rates of an enzyme-catalyzed reaction. To achieve its
objectives the group prepared six numbered test tubes added with the buffer solution, after which, the enzyme stock
solution was poured on each test tube and has been incubated. DNS was added and the absorbance was read at 540
nm using visible spectrophotometry. At the end of the experiment, the group has therefore concluded that the pH is
dependent on the enzyme activity. Invertase exhibits high activity over a broad pH range 3.5-5.5 with optimum pH
near 4.5.

INTRODUCTION

Enzymes as above mentioned are natural
catalysts. Other experimental factors that affect
the enzyme activity includes: enzyme
concentration, pH of the reaction solution,
temperature, substrate concentration, enzyme
inhibitors/ activators and Cofactors/ Coenzyme.
The objectives of the experiment are: 1.) to
extract invertase that breaks existing bond in
sucrose to form glucose and fructose from
Baker’s yeast and 2.) to determine the effects of
the change in pH on the reaction rates of an
enzyme-catalyzed reaction.

The experiment focused on the effect of pH on

the enzyme (invertase) activity. Invertase is a
yeast derived enzyme. Its official name is: β-
fructofuranosidase (EC 3.2.1.26). It is also
classified as hydrolase – catalyzing hydrolysis of
the terminal nonreducing β-fructofuranosidase
residues.
In the preparation of stock solution, invertase
was denatured at 95°C to stop Activation Energy
which enables the group to determine the distinct
period. Denaturation is the complete loss of
organized structure in a protein, it is irreversible
and it also involves disruption of the secondary
and tertiary structure without breaking the
primary structure.
DNS or Dinitrosalicylic acid, an oxidizing agent,
was used as an assay to monitor enzyme activity.
In the DNS assay, the rate of reaction is
monitored colorimetrically by measuring the
amount of reaction products that reacts with DNS
reagent. DNS reagent contains dinitrosalicylic
acid, Na2SO3 which stabilizes the red color,
NaOH that increases the reactivity of sugars and
changes the pH of the reaction vessel halting the
invertase reaction. The absorbance was read at
540 nm.
EXPERIMENTAL
A. Compounds tested (or samples used)
For extraction of Invertase from yeast: 0.25 g
Baker’s yeast, 250 mL distilled water.
For preparation of denatured invertase stock
solution: 100 mL enzyme stock solution, boiling
water bath
For Sucrose Assay Using Dinitrosalicylic
Colorimetric Method: 6 test tubes with one blank
tube: Test tube 1 with 0.25 mL sucrose std.
solution, test tube 2 with 0.50 mL, test tube 3
with 0.75 mL, test tube 4 with 1.00 mL, test tube
5 with 1.25 mL and test tube 6 with 1.50 mL
sucrose std. solution mixed with certain amounts
of distilled water. UV-Vis Spectrophotometer.
For Effect of pH on Invertase activity: 6
numbered test tubes, 2.90 mL 0.1M appropriate
buffer solution, enzyme stock solution, 60°c and
95°C water bath, 3 mL DNS reagent, 1.50mL
sucrose solution and UV-Vis Spectrophotometer.
B. Procedure The red-brown characteristic was developed
after adding 3 mL of DNS reagent and after
1. Extraction of Invertase from Yeast immersing the test tubes in 95°C water bath for
15 minutes. The solutions were then cooled.
The extraction of invertase was prepared by Blank solutions were prepared by following steps
dissolving 0.25 grams of Baker’s yeast in distilled aforementioned, after which enzyme stock
water to make a 250-mL solution. The mixture solution was added and the absorbance was
was allowed to stand for 20 minutes before measured at 540 nm. With the values obtained a
collecting the supernatant which served as the hydrolyzed-standard curve was constructed using
enzyme stock solution. the dinitrosalicylic colorimetric method.
2. Preparation of Denatured Invertase
Stock Solution
RESULTS AND DISCUSSIONS
Denatured Invertase stock solution was
prepared by incubating 100 mL of enzyme stock
solution in a boiling water bath for 10 minutes
then cooled. The supernatant was then collected
to serve as the denatured enzyme stock solution Figure 1. Hydrolyzed Sucrose Standard Curve
that will be used for the succeeding experiments.
Table 1. Sucrose Assay using DNS Colorimetric
3. Sucrose Assay Using Dinitrosalicylic
Method
Colorimetric Method
mL
To construct the hydrolyzed-sucrose curve at test Absorbanc
sucrose mL H2O
A540 against concentration (mg/ml), six test tube # e
standard
tubes were prepared with specific amounts of blank - 1.50 0.000
sucrose standard solution and distilled water
1 0.25 1.25 0.001
covered with marble or parafilm to prevent
evaporation of solvent. Afterwards, 3 drops 2 0.50 1.00 0.002
(~0.05 ml) of concentrated HCl was added to 3 0.75 0.75 0.003
each tube. After which they were incubated at
4 1.00 0.50 0.005
90°C water bath for 5 minutes to hydrolyze
sucrose. The solution was neutralized by adding 5 1.25 0.25 0.012
0.15 mL of 0.5M KOH. A 2.80 mL 0.1M buffer 6 1.50 - 0.011
solution was added and was mixed using the
Vortex mixer. Then, 3 mL of DNS reagent was
added. The test tubes were then immersed in a
Dinitrosalicylic acid (DNS) Assay was used to
95°C water bath for 10 minutes; doing so will
monitor enzyme activity. The principle involved in
develop the characteristic red-brown color. Allow
DNS assay, is that DNS (3,5-dinitrosalicylic acid,
the mixture to be cooled. After cooling, the
IUPAC name 2-hydroxy-3,5-dinitrobenzoic acid)
absorbance was then measured at 540 nm and
reacts with reducing sugars (eg. Glucose and
the hydrolyzed-sucrose curve was constructed by
fructose) to form 3-amino-5-nitosaliclylic acid
plotting A540 against concentration.
(ANS). DNS does not react with sucrose because
4 . Effect of pH on Invertase Activity it is a non reducing sugar. Accordingly, the rate
of reaction (enzyme activity) is monitored
For the determination of the effect of pH on (colorimetrically) by measuring the amount of
Invertase Activity, six test tubes containing a reaction products (reducing sugars- equimolar
total of 2.90 mL of the appropriate buffer solution mixture of glucose and fructose) that react with
were again prepared and labeled. After the DNS reagent.
preparation, 0.10 mL enzyme stock solution was The absobance shows the color intensity of the
added to each test tube. It was mixed thoroughly solution which is the amount of DNS reduced that
and then were all incubated in a 60°C water bath affects the amount of reaction products and the
for 5 minutes. 1.50 mL of sucrose solution was amount of substrate consumed thereby affecting
added and then incubated the reaction mixture or altering
Sucrose the rate α-D-glucose
of reaction. Glucose is an
+ β-D-fructose
again in 60°C water bath for 5 minutes. aldose because of itsaldonic
aldehyde
acid functional group.
Incubation allows the solution to undergo
enzymatic hydrolysis.

DNS ANS
(Yellow solution) (Red-brown solution)
 function. Also, pH dependence of enzyme activity
is a consequence of either acid-base behavior or
changing degree of ionization of groups in the
enzyme, in the substrate, or in both. Invertase
exhibits high activity over a broad pH range 3.5-
5.5 with optimum pH near 4.5 as discussed
above.
Figure 2. Hydrolysis of Sucrose to yield glucose
and fructose

REFERENCES

From books:

Boyer, R. (2006). Concepts in Biochemistry. 3rd

ed. New York: John Wiley & Sons, Inc.

Crisostomo,A.C., Daya,M.L., de Guia,R.M.,

Farrow,F.L., Gabona,M.G., Liu,M.I.D., Pena,G.T.,
Pena,L.L., Santiago,L.A., Santiago,M.R.,
Sarile,A.S., Torres,P.C., Vargas,A.G., Ysrael,M.C.
(2010). Laboratory Manual for General
Biochemistry, Quezon City, Philippines: C&E
Publishing, Inc.
Figure 3. Effect of pH on Enzymatic Activity
Voet, D., Voet, J.G. (2004). Biochemistry:
Table 2. Effect of pH on Invertase Activity Biomolecules, Mechanisms of Enzyme Action, and
Metabolism. 3rd ed. New York: John Wiley & Sons,
Inc.
Test
pH Absorbance540
Tube From the Internet
1 2 0.023
2 3 0.145 Effect of pH (Introduction to Enzymes).
3 5 0.987 http://www.worthington-
biochem.com/introBiochem/effectspH.html
4 7 0.632
02/04/11
5 8 0.430
6 11 0.081 pH effects on Enzymes
http://www.buzzle.com/articles/ph-effect-on-
enzymes.html 02/04/11

According to the graph as well as the table From Journals

above, the reaction of the hydrolyzed sucrose
and the activation energy is directly proportional. Thomas, P.G., Russell, A.J., Fersht A.R.
The curve is bell-shaped. The peak of the curve Tailoring the pH dependence of enzyme catalysis
represent the optimum pH (~pH 4.2- 4.5) or the using protein engineering 318 (1985) 375-376
best pH condition wherein the EA is highest. The
optimum pH also shows the best conformation in Wang,J., Araki, T., Matsuoka, M., Ogawa, T.
which enzyme activity is the highest, meaning A graphical method of analyzing pH dependence
the residues of the enzyme is properly of enzyme activity 524 (1999)
protonated/ deprotonated. The phenomenon of
optimum pH explains that the slight alteration of
pH affects the structure thereby altering its