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Effects of Temperature on

Enzyme Activity
by Ashley Straub

Partners: Salvador, Angie, Alex

Biology 101

October 29,2018
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ABSTRACT

At what temperature will the enzyme, beta galactosidase, catalyze the substrate, ONPG at

the fastest rate, in other words what is the optimal temperature of this specific enzyme? In this

experiment, this question was tested by pouring 1ml of 8% beta galactosidase into 5 different test

tubes filled with 2ml of ONPG. Each test tube was then placed into 5 separate temperatures, 0°C,

22°C, 37°C, 50°C, and 60°C for 15 minutes, utilizing water baths set at different temperatures

and a freezer. Twenty-two degrees Celsius was used as the control for the experiment. Once 15

minutes was up, the concentration of yellow was measured by using a spectrometer. It was found

that 37°C, had the highest absorbance of yellow and 60°C had the lowest. The test tube held in

0°C and 60°C were then placed into the water bath at 37°C for 15 minutes, then remeasured to

examine if either temperature denatured. It was concluded from these results that 37°C would be

the optimal temperature for beta galactosidase and at 60°C the enzyme would not survive and

become denatured.

INTRODUCTION

Enzymes work in very specific ways. Since they are so substantial to supporting life, they

must act in situations that are most optimal. According to the lab manual, enzymes are protein

catalysts that speed up the rate of chemical reactions within a cell by lowering the activation

energy (Ea) needed for the reaction to occur. They have enzyme specificity, meaning that

enzymes have a “unique globular shape” in which its active site (the site where the substrate

[reactant] will bind) will only bind to very specific substrates (“Lab Manual”). In this lab, ONPG

is the substrate to the enzyme, beta galactosidase, in which together, produces the product, O-

nitrophenol and galactose (“Lab Manual”). Since O-nitrophenol is yellow in color, the higher
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concentration of it will represent a darker shade or higher absorbance of yellow (“Lab Manual”).

In the Campbell Biology textbook, it states that since proteins are sensitive to their environment,

the protein catalyst enzyme will be too. When proteins reach high temperatures outside of their

standard conditions, they will denature and will no longer be able to go back to their original

shape (Campbell, Neal A). An example of this, is when an egg is boiled, the egg will no longer

go back to its liquid state; it will coagulate and denature. However, there is an instance in

between this high temperature and a low one where the enzyme’s reaction rate will be at its

greatest without becoming denatured. At higher temperatures, molecules move faster, meaning

that there will be more chances for a substrate to be in the optimal position to bind to an enzyme,

ultimately creating a higher rate of reaction for the system. At this temperature where the

reaction is the fastest, without changing the shape of the protein/ denaturing is considered the

optimal temperature. The overall purpose of this lab was to understand how the effects of

temperature affect enzyme activity as well as to discover the optimal temperature for beta

galactosidase to catalyze its substrate and find out at what temperature the enzyme would

denature. The hypothesis for this experiment was that if 1mL of the enzyme, 8% beta

galactosidase and 2mL of ONPG was placed into a 50°C water bath, then it would have the

highest rate of reaction and absorbance when compared to 0°C, 22°C, 37°C and 60°C without

denaturing. This hypothesis was concluded based off of learning from the text book and lab

manual using an educated guess that the optimal temperature wouldn’t be the highest

temperature tested, but possibly the second highest. The independent variable of the experiment

was temperature and the dependent variable was the micromoles of O-nitrophenol produced per

minute from the reaction.

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MATERIALS AND METHODS

Materials:

• 1 container of parafilm

• 1 spectrophotometer

• 1 box of Kim wipes

• 1 flask of pH 7 buffer

• 1 flask of 8% beta galactosidase

• 1 flask of pH 7 ONPG

• 3 water baths at temperatures 37°C, 50°C, and 60°C

• 1 freezer at 0°C

• 1 test tube rack

• 5 test tubes

• 6 cuvettes

• 1 wax pencil

• 1 box 1 mL pipets

• 1 box 1mL pipet pumps

• 1 tray for used pipets

• Test tube brushes

Methods:

The experiment started out by labeling 5 test tubes with a wax pencil, A, B, C, D,

and E. Two mL of the substrate, ONPG was then added to each test tube using a 1mL

pipette and pipette pump. One mL of 8% beta galactosidase was then added into each test
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tube using the same method as before and then parafilm was carefully placed over each

tube. Test tube A was placed into the freezer at 0°C, test tube B was left alone in the test

tube rack on the table at a room temperature of 27°C, test tube C was placed into a 37°C

water bath, test tube D was placed into a water bath at 50°C and test tube E was placed

into a water bath at 60°C. These five test tubes were left in their various placements for

15 minutes. During this time, the spectrometer was turned on to allow it to heat up before

measuring the test tubes for their absorbance. It was then set to the SPEC 20 D+

Emulation option and placed at 420nm using the nob. Using the navigator button, the

spectrometer was then set to measure Absorbance. A set of SPEC 20 tubes were then

taken out of the spectrometer and their rack was labeled A-E with one left blank. The

blank cuvette was filled with 3mL of pH 7 buffer using a 1 mL pipette and pipette pump.

After 15 minutes were up, test tubes A-E were carefully poured into the cuvettes on the

rack with the respective letter. The blank cuvette was then placed into the spectrometer

and the lid was shut. To “tare” out the system, the Auto Zero button was pushed. Once

the display read Absorbance 0, the cuvette was taken out and test tube A was placed into

the spectrometer with the lid shut. Once the absorbance meter stopped moving, the

absorbance was recorded on paper. These previous two steps were repeated for the rest of

the SPEC 20 tubes, switching between the blank cuvette, zeroing it out, then measuring

the corresponding tube in sequence. The highest absorbance measured was then

considered to be the optimal temperature in which the lowest absorbances were carefully

placed back into their corresponding test tubes covered in parafilm. The two test tubes

were then placed back into the water bath at the found optimal temperature for another 15

minutes to determine any other conclusions that could be made about enzyme activity.
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After the time was up, the test tubes were placed back into their original cuvettes and

remeasured the same way. A conclusion was then determined about the effects of

temperature on enzyme activity. Further calculations were made using the original

absorbances found to find micromoles produced per minute and micromoles produced

per 15 minutes. After the experiment was concluded, the spectrometer was turned off, test

tubes were cleaned adequately using test tube brushes, trash was thrown away, and

everything was placed back into their respective places.

RESULTS

Effect of Enzyme Temperature on Production of 0-nitrophenol

Tube Enzyme Intensity of Absorbance µmoles 0- µmoles 0

Temperature Yellow nitrophenol nitrophenol

(°C) produced/15 produced/min

min

A 0°C Light yellow 0.48 120 8

B 22°C Medium 0.59 147.5 9.83

yellow

C 37°C Very Bright 1.39 347.5 23.17

yellow

D 50°C Bright yellow 1.17 292.5 19.5

E 60°C Very light 0.36 90 6

yellow
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Temperature of Beta-Galactosidase Vs. O-

Nitrophenal Produced per Minute
25
µmoles 0-nitrophenol produced/min

20

15

10

0
0 10 20 30 40 50 60 70
Temperature °C

The data shows that as temperature increases, the absorbance of O-nitrophenol and the

micromoles of O-nitrophenol produced per minute and O-nitrophenol produced per 15 minutes

from the reaction increases at a rapid rate then decreases instantly. The control for the

experiment was 22°C which produced an absorbance of 0.59, 147.5 micromoles of O-

nitrophenol per 15 minutes, and 9.83 micromoles of O-nitrophenol per minute. At a lower

temperature, the rate of reaction is slower resulting with an absorbance of 0.48, 120 micromoles

of O-nitrophenol per 15 minutes, and 8 micromoles of O-nitrophenol at 0°C. At a temperature

only 15°C more than 22°C, the rate of reaction increased rapidly resulting with an absorbance of

1.39, 347.5 micromoles of O-nitrophenol per 15 minutes, and 23.17 micromoles of O-

nitrophenol. In which at this temperature yielded the brightest color and the highest absorbance

recorded for all temperatures. At 50°C the rate of reaction was still faster than the two lowest
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temperatures but slower than 37°C meaning that 37°C was the optimal temperature according to

the results. At 50°C the absorbance was at 1.17, 292.5 micromoles of O-nitrophenol per 15

minutes, and 19.5 micromoles of O-nitrophenol. At 60°C, the rate of the reaction decreased

immensely in which produced the lightest color, had an absorbance of 0.36, 90 micromoles of O-

nitrophenol per 15 minutes, and 6 micromoles of O-nitrophenol, meaning that at this temperature

the enzyme must have denatured.

DISCUSSION

According to the data, the optimal temperature for beta galactosidase is 37°C. It’s

absorbance, and O-nitrophenol per minute was recorded to be the highest out of all temperatures

at an absorbance of 1.39, 347.5 micromoles of O-nitrophenol per 15 minutes, and 23.17

micromoles of O-nitrophenol. This data concludes that the hypothesis, “if 1mL of the enzyme,

8% beta galactosidase and 2mL of ONPG was placed into a 50°C water bath, then it would have

the highest rate of reaction and absorbance when compared to 0°C, 22°C, 37°C and 60°C

without denaturing” is rejected. At 50°C the absorbance was at 1.17, 292.5 micromoles of O-

nitrophenol per 15 minutes, and 19.5 micromoles of O-nitrophenol concluding that its reaction

happened at a slower rate and, therefore not the optimal temperature for the enzyme, beta

galactosidase. There were many possible errors that may have happened during the duration of

this experiment. One possible source of error would be the amount of time the test tubes were

left with both the enzyme and substrate at room temperature before being placed into their

corresponding positions. This error may have caused the amount absorbed to be higher than if

the experiment was corrected by placing the test tubes into the water baths and freezer directly

after beta galactosidase was added to each tube, for at room temperature each tube will have
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already reacted some. Another similar source of error may have happened directly after taking

the test tubes out of their directed places after 15 minutes. Before the test tubes were recorded for

their absorbance, they sat on the table once again reacting more, creating higher absorbances.

This could have led to the cuvettes measurement of absorbance to be higher than what it was

right at 15 minutes. This error could be avoided by doing the experiment in time intervals so that

each test tube can be measured directly after being removed from its area.

CONCLUSION

The effects of temperature on enzymatic activity was thoroughly learned throughout this

experiment. It was concluded that enzymes are sensitive to temperature and will work best in

specific conditions. Specifically, for the enzyme, beta galactosidase it was discovered that 37°C

was this enzyme’s optimal temperature when catalyzed with its substrate, ONPG. It was also

learned that within a small temperature threshold from 50°C to 60°C the enzyme became

denatured and would not become usable again. When the enzyme was originally placed into the

60°C water bath, then placed back into the 37°C water bath, it no longer could react and no

longer could create the product, O-nitrophenol. However, when the enzyme that was originally

put into the 0°C was then placed into the 37°C water bath, the product O-nitrophenol increased,

meaning at the lower temperature, the enzyme did not denature. Overall, it can be concluded that

it is best for enzymes to operate at their optimal temperatures so that they may work at the fastest

rate feasible, but are very sensitive from their protein structures, and therefore cannot reach too

high of a temperature or else the enzyme will become denatured.

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LITERATURE CITED

Campbell, Neal A., et al. Campbell Biology. Pearson, 2017.

“Laboratory Manual” BIO 181: General Biology, Glendale Community College, The Biology

Department, manual.