The effect of temperatures on the activity of enzyme amylase.

Introduction:
Biotechnology [1] is growing day by day and many new applications are seen for enzymes [2]. We
produce amylases [3] from microorganisms, plants and humans. For industrial purpose we use
microbial enzymes. We produced amylolytic enzyme from filamentous fungi. Our main focus in
industry is α-amylase [4] enzyme as it is employed in starch for hydrolysis of polysaccharides [5].
This enzyme is economical on industrial scale. We also use amylase for production of glucose.
Amylase can be bacterial enzymes or fungal originated. The purpose of studying the
temperature effect on the enzyme activity is that it affect the growth of the organism. Most of the
enzymes are studied between the ranges of 25-37 ºC. The optimum temperature for starch is 37
C.
As we discussed above that the starch industry is our main focus of amylases. At the initial
phase of starch hydrolysis, the starch granules are kept at high temperature to obtain the better
performance of the enzymes in starch. The World main focus now a days is Aspergillus niger [6]
that is used for amylases production and studies on fungal amylases is getting more interest.
Key Words: Biotechnology, Amylases, Temperature, Polysaccharides, Aspergillus niger.
Materials:

 Water bath
 Test tubes (2 for each temperature for starch and amylase solution)
 Syringe for pouring solution.
 Stop Watch
 Glass rod for mixing purpose.
 Thermometer to measure the temperature.
 Iodide solution
 5 ml solution of starch
 5 ml solution of amylase
Methods:
To perform the experiment successfully we followed the mention steps.
1) We set the thermostatic bath at 40 oC temperature.
2) We heat the water bath to 60 oC and 80 oC. We can speed up our experiment by getting
hot water from sink.
3) After heating the water bath, we set up the 2 tubes and labelled them. One was for
amylase and the rest was for starch.
4) After labelling the test tubes, we added the 5 ml of starch in one tube and 5 ml amylase
in another tube with the help of syringe.
5) We put these two test tubes in water bath so that they can reached their designated
temperature (Approximately 3 minutes).
6) While waiting for test tubes to reach their temperatures, we added 3 drops of Iodine
solution into the wells of spotting tiles.
7) Set the time zero on stop watch and mix the starch solution with the amylase solution
using glass rod and simultaneously start the watch.
 8) After the one minute of mixing, collect 3 drops of of mixture and added them in spotting
tiles where we added iodide and mix it with the glass rod.
9) After two minutes of mixing, added another three drops of mixture in another spotting
tiles and repeat at 3,4 and 5 etc minutes.
10) Record the iodine color and continue until the sample produced the yellowy-orange than
the blue black colour.
11) Record the time to reach the end point and repeat the same procedure for 0 oC, 20 oC,
40 oC, 60 oC, 80 oC.

(Fig. 1)
Precautions:

 Do not turn off the timer while mixing and heating the tubes.
 Mix thoroughly with the glass rod.
 Clean the glass rod with distilled water before each sampling.
Discussion:
The purpose of doing this experiment was to check the effect of temperature on the activity of
enzyme amylases. We successfully did this experiment and while seeing the results, it is
cleared from the results that the time to reach at the end point of the solution decreases as the
temperature increases. The trend is inverse till the amylases reach it’s optimum temperature
value.
It can be observed from the result’s that the trend gets reversed after the optimum temperature
that is about 40 in this experiment. We can also compare the temperature effect on the rate of
reaction. The rate of reaction increased with the increase of the temperature and reached at
constant value and then it again decreases with the further increase in the temperature.
 Temperature Vs Rate of reaction
7

6 5.56 5.56

Rate of Reaction (10^-3) 5

3
2.08
1.67 1.67
2

0
0 10 20 30 40 50 60 70 80 90
Temperature (oC)

Temp vs rate of reaction

(Fig. 2)

Temperature vs Average Time

700

600

500
Time (Sec)

400

300

200

100

0
0 20 40 60 80
Temperature oC

Temperature vs Average Time

(Fig. 3)
References:

 https://sebiology.weebly.com/blog/the-effects-of-the-temperature-of-amylase-on-the-
breakdown-of-starch
 https://journals.sagepub.com/doi/abs/10.1177/00220345510300010701?journalCode=jdr
b
 https://www.researchgate.net/figure/Effect-of-different-temperature-on-amylase-activity-
from-different-isolates-of_fig1_281633885