EFFECTS OF TEMPERATURE IN INVERTASE ACTIVITY

Redentor D.L. Laureta III, Bea Czarina T. Loque, Lara Mae S. Lorenzo,
Jeanette T. Lusung and Tobias Manuel Y. Maningat
Group 5 2H Medical Technology Biochemistry Laboratory

ABSTRACT
Several factors affect enzyme reaction rates, one of which is temperature. The aim of the experiment is to find the
relationship between temperature and enzyme reaction rate, with the enzyme invertase. Invertase is a hydrolase
which reacts with sucrose to form glucose and fructose. Several test tubes with equal amounts of sucrose and
invertase when subjected to varying temperatures, and then DNS reagent was added. Each test tube was subjected to
95°C to form the ANS, characterized by a red-brown color, then analyzed with the spectrophotometer at 540 nm.

INTRODUCTION distilled water until a volume of 250 mL was

Enzymes are protein catalysts found in nature
that speed up biological functions and processes
that would otherwise take a long time to
completely finish. The efficiency of enzymes is
affected by a variety of factors, most notably the
temperature of the surrounding environment.
Like pH, an extremely high or low temperature
will also reduce the enzyme activity. The enzyme
that will be used in this experiment is invertase,
an enzyme found in plants and acts as a catalyst
for the hydrolysis of sucrose. Invertase will be
obtained through extraction from baker’s yeast
which will be done in the first part of the
experiment.
The objective of the experiment is to observe
and take note the optimum temperature in which
enzymes react and process products, in this case,
the optimum temperature of invertase and its
reaction with sucrose, compared with a blank
control in the form of denatured enzymes.
This experiment also involves using
spectrophotometry to view the results of enzyme
activity in different temperatures.
EXPERIMENTAL
A. Materials and Compounds Used
A. Materials and Compounds Used
For extraction of invertase from yeast: 0.25g
Baker’s yeast, distilled water
For the effect of temperature on invertase
activity: 100mg/L Sucrose standard solution,
concentrated hydrochloric acid, 0.5M Potassium
Hydroxide, Dinitrosalicylic acid (DNS)
reagent,10g/L Sucrose solution, 0.1M buffer
solutions(pH 5), test tubes, pipettes, beakers,
volumetric flasks, paraffin film, hot plate, UV-
Vis spectrophotometer.
B. Procedure
1. Extraction of Invertase from Yeast
In the act of drawing out the invertase, 0.25g
of Baker’s yeast was weighed and added with
achieved. The solution was allowed to stand for
20 minutes at room temperature and the
collection of the supernatant was followed upon
the occurrence of sedimentation. The
supernatant was then used as the enzyme
stock solution for the other experiments.
2. Preparation of Denatured
Invertase Stock Solution
One hundred mL of enzyme stock solution
was incubated in a boiling water bath for 10
minutes. The solution was cooled and the
supernatant was collected. The collected
supernatant served as the denatured enzyme
stock solution.
3. Determining the Effect of
Temperature on Invertase Activity
A 20, 30, 50, 60, 70 and 90 degrees Celsius
water baths was prepared. Six test tubes with
each containing 1.5 mL sucrose solution was
prepared and incubated separately for 5
minutes in each water bath. In another test
tube, 0.80 mL enzyme stock solution was
mixed with 19.20 mL 0.1M buffer solution with
a pH of 5. Afterwards, 3 mL of dilute enzyme
solution was added to all test tubes and were
all incubated for another 5 minutes. DNS
reagent with amount of 3 mL was also added to
all test tubes. The test tubes were then
immersed in 95°C water bath for 10 minutes
for the development of the characteristic red-
brown color. The solutions were cooled and
covered to prevent evaporation. The same
procedures were used in the case of denatured
enzyme. The absorbance was measured at 540
nm. Finally, the amount of sucrose hydrolyzed
using sucrose standard curve was determined.
The construction of sucrose standard curve was
constructed in the dinitrosalicylic colorimetric
method.
RESULTS AND DISCUSSION
1
 After spectrophotometry, the results were Campbell, M.K., and Farrell, S.O. (2006).
noted down, (Table 1.) and graphed (Fig.4.1) Biochemistry, Fifth Edition. California, USA:
Thomson Brooks/Cole.
Table 1. Effects of Temperature on Invertase
Activity Delvin, T.M., (2002). Textbook of Biochemistry
with Clinical Correlations. New York, USA: Wiley-
Test Tube Temperature Absorbance Liss.
1 20 0.123
2 30 0.234 From the Internet
3 50 0.456 Li, T.S. Effect of temperature on soluble invertase
4 60 1.234 activity, and glucose, fructose and sucrose status
5 70 0.890 of onion bulbs (Allium cepa) in store. (2004)
6 90 0.432 Retrieved from
http://informahealthcare.com/doi/abs/10.1080/0
Fig.4.1 Graphical Presentation of Invertase 9637480412331290512. 2/3/2011
Activity Versus Temperature.
Effects of pH (Introduction to Enzymes). (n.d.)
Retrieved from http://www.worthington-
biochem.com/introBiochem/tempEffects.html.
2/3/2011

Effect of High Temperature on Sucrose Content

and Sucrose Cleaving Enzyme Activity in Rice
Grain During the Filling Stage. (2006) Retrieved
from
http://www.ricescience.org/qikan/manage/wenzh
ang/E060309.pdf. 2/3/2011

The Effect Of pH On Invertase Activity. (2003)

Retrieved from http://www.coursework.info/
AS_and_A_Level/Biology/Molecules__Cells/The_E
ffect_Of_pH_On_Invertase_Activity_L107500.ht
ml

As noticed in the graph shown at Fig4.1, there

is peak absorbance at 60°C. This point is the
optimum temperature, where in the enzyme is
most active. As the temperature increases, the
rate of reaction also increases, similar to
inorganic reactions. But as seen in Fig.4.1, at a
certain temperature, the rate of reaction drops
drastically, represented by the bell-shaped curve
apparent in the graph. This phenomenon can be
explained by the nature of the enzymes which
are proteins. All proteins with a tertiary structure,
like invertase, denature when exposed to enough
high temperatures. Denatured proteins do not
react as much as the normal proteins, therefore
lessening the reaction rate of the entire system.

REFERENCES
From Books
Boyer, R. F. (2006). Concepts in Biochemistry
(3rd ed.). Hoboken, NJ: Wiley.