Lab Enzyme Essay

Submitted By mmich513
Words: 837
Pages: 4
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Observation: Enzymes are proteins that help speed up chemical reactions and as such, are subject to change when heat, salt or pH are involved. Enzymes have evolved to withstand such factors in which they reside so, if there are different factors like pH and heat involved, the effect of a specific enzyme may not work as well or even better in different conditions compared to other types of enzymes. Another type of factor as well is what kind of organism uses the enzyme and if they have any conditions to adapt to.
Question:
1. Does amylase from different organisms have the same optimal temperature?
2. Does amylase from different organisms have the same optimal pH?
Prediction:
1. Amylase of different organism have different optimal temperature
2. Amylase of different organism have different optimal pH
Hypothesis:
1. If I place bacterial or fungal amylase in different temperatures, then it will have a lower optimal temperature.
2. If placed in a different pH, bacterial and fungal amylase will probably respond different than human amylase.
Equipment and material
1. Spectrophotometer and pair of cuvettes
2. Starch solution (stock solution)
3. Frozen amylase of human-bacteria and fungi
4. Water bath fixed at 3°C, 25°C, 37°C, 45°C, 65°C, 85°C
5. pH specific buffer 450
6. Hot plate and glass beaker for boiling water bath
7. Six testing tubes
8. DNS reagent
9. Micropipettors with appropriate tips
10. 10mL glass pipette and plastic pipettor
11. Deionized water

Optimal temperature
I will use human amylase to determine its optimal temperature and others will use fungi so I can compare them to the bacteria amylase optimal temperature 45®C data that I have obtained in the lab.
1. Dispose of glass beaker about ½ full water and turn it on the hot plate
2. Label seven tubes
3. Add a same volume of starch stock solution in all six tubes (900µL of 2% stock solution for example)
4. Place all tubes into appropriate temperature
5. Add an equal amount of amylase in five tubes to hydrolyze the starch (100µL for example)
6. Add (100µL) of water in the remain tubes that will be the control tubes
7. Add a volume of DNS into six tubes (five test tubes and one control tube) to stop the hydrolysis and detect the presence of maltose
8. Place the tubes into the beaker and boil for 5min to observe the color change of the oxidation-reduction reaction
9. Add (8mL) of water in each tube to dilute the solution using the 10mL glass pipette
10. Mix the contexts
11. Measure the absorbance at 540nm

Data
Activity of Amylase directly related to Maltose in Humans
Tube/Temperature
Initial Absorbance reading
Final Absorbance
3°C
.043
.43
25°C
.286
2.86
37°C
.589
5.89
45°C
.690
6.90
65°C
.284
2.84
85°C
.002
.02 Tubes
Amount of Maltose in (mg)

Volume of Maltose Solution (ml)
Final Absorbance (initial absorbance x dilution)
1
0 mg
0 ml
0
2
0.4 mg
0.2 ml
0.7
3
0.8 mg
0.4 ml
1.7
4
1.2 mg
0.6 ml
2.8
5
1.6 mg
0.8 ml
3.7
6
2 mg
1 ml
4.8
Temperature
Amount of Maltose
(mg)
3°C
0.875 mg
25°C
1.075 mg
37°C
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