1.

0 OBJECTIVES
1.1

To develop a suitable standard curve for enzyme assay

1.2

To analyse the effect of temperature on the activity of enzyme

1.3

To determine optimum temperature for enzyme in enzyme kinetics plots.

2.0 INRODUCTION
Enzyme are protein molecules that act as biological catalysts by increasing the rate of
reactions without changing the overall process. Enzyme can be used to catalyse the forming or
breaking of the chemical bond, without themselves undergoing permanent chemical changes.
Enzyme can also be used to lower down the activation energy needed for a chemical reaction.
The reactants of enzyme catalysed reactions are termed as substrates. Enzyme kinetics is the
study of the chemical reactions that are catalysed by enzymes. In enzyme kinetics, the reaction
rate is measured and the effects of varying the conditions such as substrate concentration,
enzyme concentration, pH and temperature of the reaction are investigated. Enzyme amylase is
used to catalyse starch hydrolysis.
For the experiment of the effect of temperature on enzyme activity, the substrate used is
starch solution whereas the enzyme used is

-amylase solution. During the process of

hydrolysis, amylase breaks down the internal

-1,4-glycosidic bond present in starch with

the production of reducing sugars, maltose. In this experiment, activity of amylase breaking
down starch are monitored by changing of the temperature of reaction between amylase and
starch. Enzyme activity is used to measure of how readily an enzyme interacts with the
substrate to generate the product of the reaction. Furthermore, colour reagent solution is used to
measure the concentration of reducing sugars produced in the enzyme-substrate reaction. At
540nm, the value of absorbance for the resultant solution is obtained. The intensity of colour
depends on the concentration of reducing sugars produced meaning that increased
concentration of reducing sugars produced can increase the intensity of colour following by the
increase of absorbance.

Figure 1.1 : Effect of incubation temperature on -amylase activity

One of the important parameters affecting the rate of a reaction catalysed by an enzyme is
the temperature. Like most chemical reactions, the rate of an enzyme-catalyzed reaction
increases as the temperature is raised. A ten degree

rise in temperature will increase the

activity of most enzymes by 50 to 100%. Variations in reaction temperature as small as 1 or 2

degrees may introduce changes of 10 to 20% in the results. In the case of enzymatic reactions,
this is complicated by the fact that many enzymes are adversely affected by high temperatures.
As shown in Figure 1.1, the reaction rate increases with temperature to a maximum level
(optimum temperature), then abruptly declines with further increase of temperature. Because
most enzymes rapidly become denatured at temperatures above 40C, most enzyme
determinations are carried out somewhat below that temperature.
Over a period of time, enzymes will be deactivated at even moderate temperatures. Storage
of enzymes at 5C or below is generally the most suitable. Some enzymes lose their activity
when frozen.

4.0 MATERIALS AND EQUIPMENTS

4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
4.10
4.11
4.12
4.13
4.14

1.0% (w/v) soluble starch solution

0.2% (w/v) maltose monohydrate solution
-amylase solution
Colour reagent solution
Distilled water
Test tubes
Water bath
Pipettes
Beakers
Hot plate and magnetic stirrer
Spectrophotometer
Measuring cylinder
Ice cubes
Thermometer

5.0 PROCEDURES
5.1

Development of maltose standard curve

5.1.1 Make different concentration of maltose solution (total volume of 1mL) by dilution with
distilled water in clean test tube
5.1.2 For each test tube, add 0.5 ml colour reagent solution and mix well.
5.1.3 Place in boiling water bath for exactly 5 minutes, then cool in ice to room temperature
and add 4.5 ml distilled water for each concentration and mix well.
5.1.4 Take sufficient amount from each test tube into different cuvettes and place each cuvette
in a colorimeter and record the intensity of dark orange red colour at 540 nm as the
'absorbance' or OD.
5.1.5 Plot a graph with the amount of maltose on X axis versus absorbance at 540nm on Y
axis.
5.2

Effect of temperature on enzyme activity

5.2.1 Pre-incubate the 0.5ml starch solution for 5 minutes in water bath at 10C.
5.2.2 Add 0.5 ml of enzyme solution in each test tube, mix by vortex, and incubate in water
bath at 10C for 3 min.
5.2.3 After incubation, immediately add 0.5 ml colour reagent solution to all test tubes, mix
well using vortex.

5.2.4 Repeat steps 5.1.3 to 5.1.4 at water bath at 20C, 30C, 40C and 50C to analyse the
reaction.
6.0 RESULT
6.1

Maltose standard curve

6.1.1 Determine the concentration of maltose solution and amount of maltose for each tube as
in the Table 1.1:

To find maltose concentration (%):

For V1 = 0.1ml,
M1V1 = M2V2
0.2% (0.1ml) = M2 (1.0ml)
M2 = 0.02 %

Where,
M1 = Concentration of maltose monohydrate solution 0.2%
V1 = Maltose solution (ml)
M2 = Maltose concentration (%)
V2 = final volume

To find amount of maltose in moles:

Molecular formula of Maltose = C12H22O11
Molecular weight of maltose = 12(12g/mole) + 22(1 g/mole) + 11(16 g/mole)
= 342 g/ mole
For maltose solution = 0.1 ml
Mass =

0.2 g x 0.1 ml
= 2 x 10-4g
100 ml

Amount of Maltose (moles) =

mass
Molecular weight

2 x 104 g
342 g/mole

= 0.5848 x

10-6 moles

Table 1.1

Std

Maltose
Concentration
(%)

Amount of maltose
(moles)

Maltose
Solution
(ml)

Distilled
Water
(ml)

0.02

0.5848

0.10

0.90

0.04

1.1696

0.20

0.80

0.06

1.7544

0.30

0.70

0.08

2.3392

0.40

0.60

0.10

2.9240

0.50

0.50

0.20

5.8480

1.00

Blank

1.00

Absorbance
at 540nm

6.1.2 Use the data of absorbance to develop a linear plot of OD (540nm) versus amount of
maltose (moles) using graph paper or Excel software and determine the regression
value.

6.2 Effect of substrate concentration on enzyme activity

6.2.1 Complete Table 1.2 by using the data of absorbance and maltose standard curve to
determine the amount of maltose and calculate the velocity/rate of reaction.
Linear equation obtained from graph above:

y = mx + c
Where y = abs at 540nm
x = amount of maltose (moles)
x=

yc
m

Velocity, V =

amount of maltose ( moles ) x dilution factor (ml)

time ( min ) x volume ofenzyme (ml)

The subsequent velocity at others temperature is shown in table below:

Table 1.2:

Temperature (C)

Abs at 540 nm

Amount of maltose
(moles)

Velocity, [V]
(moles/min)

10
20
30
40
50

6.2.2 Use the data of above to develop a plot of velocity versus temperature.
Expected Result is shown as Figure 1.1 below:
Figure 1.2

Reference
Enzymes - Factors affecting enzyme actions. (2010, January 31). Retrieved on November 12,
2014 from Dr Parry's Website http://drparry.co.uk/index.php?option=com_content&
view=article&id=57%3Aenzymes&catid=42%3Aenzymes&Itemid=54&limitstart=1