ISOLATION OF ACTIVE INVERTASE AND EFFECT OF PH ON ENZYMATIC

ACTIVITY

R.P.S. Roque, D.L.M. Santos, D.R.J Santos, G.C. Sevilla, C.A.M. Solis
Group 8, 2A Biochemistry General Biochemistry Laboratory
Faculty of Pharmacy, University of Santo Tomas

ABSTRACT

Enzymes are biological catalysts which can alter or speed up a chemical reaction, without itself being
chemically changed at the end of the reaction. Not all enzymes are protein in nature thus they are called Ribozymes
which is a RNA molecule that has catalytic activity. Determination of the effect of PH on enzyme activity is the
primary objective of the experiment. Dinitrosalicyclic acid (DNS) Assay method is utilized to monitor the enzymatic
activity of invertase. Invertase was subjected to different PH of buffer solution and was observed under 540 nm
absorbance using spectrophotometer. After observation and analysis, a peak was observed by plotting absorbance
versus ph and was known as optimum PH. Optimum PH is said to be the most favorable PH value or the point where
the enzyme is most active. And that invertase exhibits high activity over a broad PH range of 3.5 – 5.5 with optimum
PH near 4.5. But due to human errors, the acquired data was incorrect giving an unreliable basis for the
determination of the effect of PH on enzymatic activity.

INTRODUCTION served as the denatured enzyme stock

Invertase is a yeast derived enzyme
which is classified as a hydrolase. Its official
name is β-fructofuranosidase (EC 3.2.1.26)
and is classified as an hydrolase. Generally,
invertase can break peptide bonds and
specifically hydrolyzes sucrose to glucose and
fructose. Dinitrosalicyclic acid (DNS) Assay is
the method utilized to monitor its enzymatic
activity. Furthermore, PH is discussed in the
experiment and specifically tackled on the
effect of PH on enzymatic activity. PH is
defined as the measure of the concentration
of hydrogen ions in a solution.
Spectrophotometer was used to determine
the absorbance of the solution and was
plotted against the PH levels used on each
solutions.
The experiment tends to explain the
different principles involved from isolation of
active invertase to the principles behind
Dinitrosalicyclic acid (DNS) Assay and the
principle behind the relationship of PH to the
enzymatic activity of invertase. Further
discussions on the topics could be found on
the results and discussions section of this
report.
MATERIALS AND METHOD
For the preparation of denatured
invertase stock solution 100 ml of enzyme
stock solution was incubated in a boiling
water bath for about 10 minutes. The solution
was allowed to cool down. The solution was
solution.
7 test tubes including the blank test
tube were prepared for sucrose assay using
dinitrosalicylic colorimetric method.
Designated ml of sucrose standard solution
and ml of distilled water were placed in each
test tubes. 3 drops of concentrated HCl was
added to each of the test tubes and were
incubated in a 90°C water bath for 5 minutes.
0.15 ml of 0.5 KOH was placed to the test
tubes for the neutralization of the solutions.
2.80 ml of 0.1 M buffer PH 5 was placed on
the test tubes. 3 ml of DNS reagent was
added to the test tubes and were immersed
in a 95°C water bath for 10 minutes or until
the red-brown color developed. The solutions
were allowed to cool down. Absorbance at
540 nm was measured using
spectrophotometer. Hydrolyzed-sucrose
standard curve was constructed by plotting
A540 against concentration.
6 test tubes were prepared for the
determination of the effect of PH on
enzymatic activity, 2.90 ml of 0.1 M buffer
solution with different ph was added to each
of the 6 test tubes and were added as
follows:
Tube 1 2 3 4 5 6
PH 2 3 5 7 8 11
0.10 ml of enzyme stock solution was
added to each test tubes and were incubated
in 60°C water bath for 5 minutes. 1.50 ml of
sucrose solution was added to each test tubes
and were incubated again in 60°C water bath
for 5 minutes. 3 ml of DNS reagent was
added and test tubes were immersed in 95°C
water bath for 10 minute or until the solutions
becomes red-brown in color. The test tubes
were allowed to cool down. For the
preparation of blank solutions the steps above
were repeated but denatured enzyme were
placed instead of enzyme stock solution.
Absorbance at 540 nm was observed.
RESULTS AND DISCUSSIONS
As mentioned, invertase having the
official name of β-fructofuranosidase (EC
3.2.1.26) is classified as an hydrolase and are
catalyzing hydrolysis of the terminal
nonreducing β-fructofuranoside residues.
Generally, they are able to break peptide
bonds and can split sucrose to glucose and
fructose (Figure 1). In the presence of
invertase.
With the use of Spectrophotometer,
absorbance at 540 nm was observed. And
arrived at the conclusion that:
Absorbance ≈ Color intensity of solution ≈
Amount of DNS reduced ≈ Amount of reaction
products ≈ Amount of substrate consumed ≈
Rate of reaction
Different reagents were used for DNS
assay and each reagent plays important role.
Concentrated HCl was used for the complete
hydrolysis of glycosidic bonds. 0.5 N KOH was
used to neutralized excess acids. 1% DNS
reagent is composed of Dinitrosalicylic acid
will act as the oxidizing agent, Na2SO3 which
stabilizes the red color, NaOH which increases
the reactivity of sugars and changes the PH of
the reaction vessel along; with the ANS
production; halting the invertase reaction.

invertase
Sucrose ------------------- glucose + fructose

FIGURE 1. REDUCTION OF SUCROSE

FIGURE 3. SUCROSE STANDARD CURVE

Figure 3 served as the basis of the

FIGURE 2. REDUCTION OF GLUCOSE AND experiment. Concentration of the solution
FRUCTOSE depend on the absorbance in 540 nm.

Furthermore, figure 2 explains the pH dependence of enzyme activity is a

reduction of glucose and fructose using the
Dinitrosalicyclic acid (DNS) Assay method.
This method is utilized to monitor the
enzymatic activity of invertase. DNS; 3,5-
dinitrosalicylic acid whose IUPAC name is 2-
hydroxy-3,5-dinitrobenzoic acid, reacts with
reducing sugars such as glucose and fructose
to form 3-amino-5-nitrosalicylic acid also
known as ANS. DNS strictly does not react
with sucrose which is a non-reducing sugar.
consequence of acid-base behavior or
changing degree of ionization of groups in the
enzyme, in the substrate, or in both.
Hypothetically, Invertase exhibits high
activity over a broad pH range 3.5-5.5 with
optimum pH near 4.5.

In DNS assay, rate of reaction of

enzyme activity is monitored colorimetrically
by measuring the amount of reaction
products which are reducing sugars –
equimolar mixture of glucose and fructose
that react with DNS reagent.
 PH ABSORBANCE540
Blank 0
2 0.025 REFERENCES
3 -0.009
5 0.005 FROM BOOKS:
7 0.009
8 0.002  Voet D.J. & et. Al. (2008). Principle of
11 0.054 Biochemistry (3rd edition). United
FIGURE 4. ACQUIRED DATA: PH VERSUS States: Aptara Inc. page 332.
ABSORBANCE
 Chang R. (2009). General Chemistry:
The Essential Concepts (5th edition).
Technically, the acquired data (Figure
United States: The McGraw-Hill
4) is incorrect maybe because of human error
Companies. Pages 484-486.
such as following the methods or such. If PH
levels versus absorbance at 540 nm were to  Crisostomo A.C. & et. Al. (2010).
graph, it should form a bell shape figure. As Laboratory Manual in General
seen in figure 5 below. Biochemistry. Philippines: C & E
Publishing Inc. pages 41-43.

FROM INTERNET:

 http://www.worthington-
biochem.com/introbiochem/effectsph.h
tml
TAKEN 15-16 JANUARY 2011
 http://www.123helpme.com/view.asp?
id=121162
TAKEN 15-16 JANUARY 2011
 http://www.scribd.com/doc/7247472/
Chapter-4-Enzymes
TAKEN 15-16 JANUARY 2011
FIGURE 5. EFFECT OF PH ON ENZYMATIC  http://www.scribd.com/doc/8303966/
ACTIVITY 43-Enzyme-Activity
TAKEN 15-16 JANUARY 2011
Figure 5 illustrates the effect of PH on
the reaction rate of invertase. The optimum
PH can be observed in the graph. Optimum
PH is defined as the most favorable pH value
- the point where the enzyme is most active
or simply it the PH value in which your
enzyme works at best. In other sources it is
defined as The pH at which an enzyme
catalyses a reaction at the maximum rate.

Furthermore, changes in PH lead to

the breaking of ionic bonds that holds the
tertiary structure of the enzyme on place. The
enzyme begins to lose its functional shape,
particularly the shape of the active site, such
that the substrate will no longer fit into it, the
enzyme is said to be denatured. Also changes
in PH affect the charges on the amino acids
within the active site such that the enzyme
will not be able to form an enzyme-substrate
complex.