Effect of Temperature on Catecholase (Solanum Tubersom)
Activity
Shirley Moon
BI 112
Abstract
Catecholase (solanum tuberosm) activity was tested in four separate experiments and
four different substrate temperatures ranging from 5 degrees Celsius to 70 degrees
Celsius. A spectrometer was used to measure the production of benzoquinone in a tow
minute period. The results of each test were charted and the rate of reaction was
figured. It was discovered that the rate of catecholase-catalyzed reaction was best in
temperatures of 37 degrees Celsius and 22 degrees Celsius and slow in temperatures
of 70 degrees Celsius and 5 degrees Celsius.
Introduction
Enzymes are protein catalysts that are present in all living things. Their main function is
to speed up reaction time by lowering reaction energy. They are used to break down
food consumed by humans from start to finish, for example, amylase breaks down
starch in the mouth, and pepsin breaks apart protein in the stomach. Enzymes do all
this with extreme precision. They are three-dimensional with an active site that fits its
target substrate perfectly to allow reactions to begin, but if the active site is altered or
the cofactor is removed the enzyme will no longer work. Some of the factors that may
contribute to the dysfunction of an enzyme reaction are salt concentration, pH, and
temperature. Temperature can affect the way an enzyme works by changing the shape
of the enzyme and changing the energy level of the substrates.
The subject of this experiment was catecholase, an enzyme found in most
vegetables and fruit, and it facilitates in the browning of produce when it is damaged or
exposed to oxygen. The catecholase came from a potato (solanum tuberosm).
Catechol and molecular oxygen was used as substrates to facilitate the browning
reaction. The browning product is called benzoquinone. As stated in Foundations of
Life-Chemistry, Cells, and Genetics (Losos, Singer and Mason), most human enzymes
work best at 35-40 degrees Celsius, this is the normal body temperature.
The purpose of this experiment was to test the effects of temperature on
catecholase (solanum tuberosm). According to the International Journal of Food
Science and Technology, work done by Dogan, Dogan, and Arslan, at the University of
Balikesir, in Turkey, showed that ployohenol oxidase with catechol as a substrate had a
decrease in activity at temperatures above 40 degrees Celsius. Further testing showed
the optimum temperature for this enzyme activity was 30 degrees Celsius (Dogan,
Arslan and and Dogan). It is hypothesized that because humans do not change their
body temperature organic enzymes, or enzymes in foods consumed by humans, should
react best at temperatures closest to the human body (37 degrees Celsius). The
hypothesis was tested by observing the activity of catecholase (solanum tuberosm) at
four different substrate temperatures ranging from 5-70 degrees Celsius.
Materials and Methods
The rate of the benzoquinone formation in a catecholase (solanum tuberosm) catalyzed
reaction was measured by making reagents that was put into a spectrophotometer. The
spectrometer was then set to read the absorbance of the solution (wavelength set to
540 nm). A higher concentration of benzoquinone will make the color of the reagent
darker, which results in higher absorbance, the rate of reaction was then measured by
the increase of absorbance over two minutes (Briggs, Cawley and Brown-Istvan).
Catecholase was prepared with 117.2 grams mashed potato (solanum tuberosm) in 500
ml. H2O (instructions of catecholase preparation was given by BI 112 lab instructor).
Three large tubes were labeled 1, 2, and 3 with a grease pencil. Three pipettes are
labeled H20, potato (solanum tuberosm) extract, and catechol so no cross
contamination occurred during measuring. First, a reagent blank was made of 3.0 ml.
potato (solanum tuberosm) extract and 9.0 ml. distilled H2O and was measured into
tube 1. Next, 4.0 ml. of catechol was measured into tube 2. Third, 3.0 ml. potato
(solanum tuberosm) extract and 5.0 ml. distilled (22 degrees Celsius) H2O was
measured into tube 3. Some of the solution in tube 1 was put in a cuvette and was used
to zero out the spectrometer, then the contents of the cuvette was dumped out, the
cuvette was rinsed thoroughly with distilled H2O and placed upside to dry on a paper
towel. In 20 seconds the contents of tube 1 and tube 2 were mixed, placed into the
curvette, and placed into the spectrometer. The first reading was read at 20 seconds
exactly (Briggs, Cawley and Brown-Istvan). Readings were then taken every 20
seconds for a full two minutes.
To perform the second experiment, first, a reagent blank was made of 3.0 ml.
potato (solanum tuberosm) extract and 9.0 ml. distilled H2O and measured into tube 1.
Next, 4.0 ml. of catechol was measured into tube 2. Third, 3.0 ml. potato (solanum
tuberosm) extract and 5.0 ml. distilled (5 degrees Celsius) H2O was measured into tube
3. Some of the solution in tube 1 was put in a cuvette and was used to zero out the
spectrometer, then the contents of the cuvette was dumped out, the cuvette was rinsed
thoroughly with distilled H2O and placed upside to dry on a paper towel. In 20 seconds
the contents of tube 1 and tube 2 were mixed, placed into the curvette, and placed into
the spectrometer. The first reading was read at 20 seconds exactly (Briggs, Cawley
and Brown-Istvan). Readings were taken for a full two minutes.
To perform the third experiment, first, a reagent blank was made of 3.0 ml. potato
(solanum tuberosm) extract and 9.0 ml. distilled H2O and measured into tube 1. Next,
4.0 ml. of catechol was measured into tube 2. Third, 3.0 ml. potato (solanum tuberosm)
extract and 5.0 ml. distilled (37 degrees Celsius) H2O was measured into tube 3. Some
of the solution in tube 1 was put in a cuvette and was used to zero out the spectrometer,
then the contents of the cuvette was dumped out, the cuvette was rinsed thoroughly
with distilled H2O and placed upside to dry on a paper towel. In 20 seconds the
contents of tube 1 and tube 2 were mixed, placed into the curvette, and placed into the
spectrometer. The first reading was read at 20 seconds exactly (Briggs, Cawley and
Brown-Istvan). Readings were taken for a full two minutes.
To perform the fourth experiment, first, a reagent blank was made of 3.0 ml.
potato (solanum tuberosm) extract and 9.0 ml. distilled H2O and measured into tube 1.
Next, 4.0 ml. of catechol was measured into tube 2. Third, 3.0 ml. potato (solanum
tuberosm) extract and 5.0 ml. distilled (70 degrees Celsius) H2O was measured into
tube 3. Some of the solution in tube 1 was put in a cuvette and was used to zero out the
spectrometer, then the contents of the cuvette was dumped out, the cuvette was rinsed
thoroughly with distilled H2O and placed upside to dry on a paper towel. In 20 seconds
the contents of tube 1 and tube 2 were mixed, placed into the curvette, and placed into
the spectrometer. The first reading was read at 20 seconds exactly (Briggs, Cawley
and Brown-Istvan). Readings were taken for a full two minutes.
Results
After conducting four different experiments on catecholase (solanum tubersom)
catalyzed reaction by changing only the temperature of the distilled H20 substrate in
each experiment it was concluded that rate of reaction was best at 37 degrees Celsius
with a rate of reaction of 0.004/s. This data is displayed in Figure 3. It was also
concluded that the substrate temperature of 22 degrees Celsius had a good rate of
reaction rate of 0.003/s. This data is displayed in Figure 1. The worst reaction rate was
observed in the last experiment, when the substrate temperature was 70 degrees
Celsius with a reaction rate of 0.00375/s. This data is displayed in Figure 4. The
reaction rate was also slow when the substrate temperature was 5 degrees Celsius with
a reaction rate of 0.0035/s. This data is displayed in Figure 2.
Slope=rise/run = 0.12/40s=0.003/s
Rate of Reaction=0.003/s
0.35
0.32
0.3
0.28
Absorbance
0.25
0.23
0.2
0.17
0.15
0.11
0.1
0.05
0.02
0
20
40
60
80
100
120
Time (in seconds)
Figure 1. Production of benzoquinone in a catecholase catalyzed reaction.
Substrate temperature 22 degrees Celsius.
Slope=rise/run = 0.14/40s=0.0035/s
Rate of Reaction=0.0035/s
0.5
0.45
0.43
0.4
0.38
Absorbance
0.35
0.32
0.3
0.25
0.25
0.2
0.18
0.15
0.12
0.1
0.05
0
20
40
60
80
100
120
Time (in seconds)
Figure 2. Production of benzoquinone in a catecholase catalyzed reaction.
Substrate temperature 5 degrees Celsius.
0.5
Slope=rise/run = 0.16/40s=0.004/s
Rate of Reaction=0.004/s
0.46
0.45
0.42
0.4
0.38
Absorbance
0.35
0.3
0.3
0.25
0.22
0.2
0.15
0.13
0.1
0.05
0
20
40
60
80
Time (in seconds)
100
120
Figure 3. Production of benzoquinone in a catecholase catalyzed reaction.
Substrate temperature 37 degrees Celsius.
0.4
Slope=rise/run = 0.15/40s=0.00375/s
Rate of Reaction=0.00375/s
0.35
0.32
0.3
Absorbance
0.37
0.35
0.26
0.25
0.2
0.17
0.15
0.1
0.09
0.05
0
20
40
60
80
100
120
Time (in seconds)
Figure 4. Production of benzoquinone in a catecholase catalyzed reaction.
Substrate temperature 70 degrees Celsius.
Discussion
Enzymes are only able to do their job effectively when everything is in the exact order it
should be, when anything occurs to take the enzyme out of its optimum state it no
longer can do its job, even if its something as small as temperature change. In the four
experiments conducted the production of benzoquinone in a catecholase catalyzed
reaction was very slow with substrate temperatures at 5 degrees Celsius and
insubstrate temperatures at 70 degrees Celsius. This data is represented in Figures 2
and 4. And the production of benzoquinone in a catalyzed reaction was good with
substrate temperatures at 22 degrees Celsius and 37 degrees Celsius. This data is
represented in Figures 1 and 3.
With the four experiments conducted the hypothesis was supported that
catecholase (solanum tuberosm) rate of reaction was best in temperatures closest to
the human body (37 degrees Celsius). The data is consistent with the works of Dogan,
Arslan, and Dogan, who stated that the effects of substrate temperature on polyphenol
oxidase slowed reaction rate when temperatures where above 40 degrees Celsius and
below 20 degrees Celsius (Dogan, Arslan and and Dogan).
Although this data is clear and concise it would be interesting to further testing on
other organic enzymes found in produce consumed by humans to verify and solidify
these findings with experiments using more extreme temperatures. It would be
interesting to conduct studies on the enzyme bromelain, found in pinapple (ananas
cosmosus) which can digest protein.
Conclusion
There was a significant difference in the catecholase catalyzed reaction when the
substrate temperatures where changed. Production of benzoquinone was best when
substrate temperatures where 37 degrees Celsius and 22 degrees Celsius, and low
when substrate temperatures where 5 degrees Celsius and 70 degrees Celsius. This
information supports the hypothesis that catecholase (solanum tuberosm), an organic
enzyme consumed by humans, has the best rate of reaction in temperatures closest to
the human body, 37 degrees Celsius.