Enzyme Lab Report

Effects of Temperature and pH on Enzyme Function
Chelsie Mesa
Section 0479
Robin Cotter Introduction The purpose of this experiment is to identify three unknown enzymes. This is done by using different temperature and pH to affect the function of the enzyme, which ultimately, will affect how much maltose is produced.
Enzymes are proteins that help catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, the products. Enzymes are extremely selective for their substrates and speed up only a few reactions from among many possibilities (Encarta, 2007). Activity is affected by temperature, chemical environment
(pH), and the concentration of substrate.
Temperature affects enzyme function by changing its shape/structure, which ultimately changes the function. It affects the rate of the chemical reaction; if temperature is too high, the enzyme is denatured/inactive. pH also alters enzyme shape and reaction rates. Optimal pH for most enzymes is between 6-8. The enzyme in this experiment is amylase. It is mainly the ingredient of pancreatic juice and saliva, needed for the breakdown of long-chain carbohydrates (such as starch) into smaller units. The primary function of the enzyme amylase is to break down starches into food so that they can be used by the body (Advanced Enzyme Technologies Ltd, 2006).
The enzyme alpha-amylase from Bacillus licheniformis (bacteria) is active at a relatively low pH and high temperatures. The alpha-amylase derived from the fungus Aspergillus oryzae is most active in warm temperatures and an acidic pH. The enzyme alpha-amylase from porcine
(pig) is most active at body temperature (37ºC) and a neutral pH of 7. Materials and Methods
For this experiment each group was given a tube of concentrated enzyme labeled either
A, B, or C. To test against temperature, six test tubes were marked: blank, 4ºC, 23ºC, 37ºC,
65ºC, and 100ºC. Next, one ml of 1% starch solution (pH7) was added to each tube. These tubes were then placed in a water bath that had a temperature equal to how they were marked.
The “blank” and “23ºC” tubes were left at room temperature and the 4ºC tube was set on ice.
They remained at these temperatures for 10 minutes. After the 10 minutes, 1ml of dH2O was added to the blank tube only. The other five tubes received 1 ml of a diluted enzyme (100µl of concentrated enzyme + 9.9 ml of dH2O). All six tubes were placed back in their appropriate temperature for 12 minutes. After the 12 minutes 1 ml of Maltose Color Reagent was added to all six tubes then the tubes were placed in a boiling water bath for 15 minutes. After retrieving the tubes, they were placed on ice until cooled, then 9 ml of dH2O was added to each tube. The last step was to measure the absorbance (at 540nm) off all the samples and the record the data in tables. To test against pH, another six test tubes were marked: blank, pH 5, pH 6, pH 7, pH 8, and pH 9. Next, 1 ml of the corresponding pH was added to each tube. For the blank tube, 1 ml of 1% starch solution (pH7) was added. The next step was to start a timer at 12 minutes then begin adding 1 ml of diluted enzyme (100µl of concentrated enzyme + 9.9 ml of dH2O) to each pH tube. The blank tube received 1 ml dH2O. After the 12 minutes 1 ml of Maltose Color
Reagent was added to all six tubes. They were then put into a boiling water bath for 15 minutes.
The tubes were then cooled on ice and then 9 ml of dH2O was added to each tube. Using a spectrophotometer, the absorbance was measured then recorded in tables. Maltose Standards y = 5.6339x + 0.0235
R
2 = 0.994
0
0.5
1
1.5
2
2.5
0 0.1 0.2 0.3 0.4
Maltose (mg)
Absorbance (540nm)
Maltose (mg)
Linear (Maltose (mg))
Effects of Temperature on Maltose Production
0
0.5
1
1.5
2
2.5
0 20 40 60 80 100 120
Temperature (C)
Maltose Production (mg) enzyme a enzyme b enzyme c
Effect of pH on Maltose Production
0
0.5
1
1.5
2
2.5
56789
pH
Maltose Production (mg) enzyme a enzyme b enzyme cResults
Enzyme A produced the most maltose at 65 ºC and a pH of 5.
Enzyme B produced the most maltose at a high temperature (100 ºC) and an acidic pH.
Enzyme C produced the most maltose at 37 ºC (body temperature) and a pH of 7 (neutral).
Conclusion
In a prior lab, it was given that the enzyme Aspergillus oryzae (fungus) works best at warm temperatures and an acidic pH, porcine (pig) works best at body temperature (37 ºC) and
a neutral pH (7), and Bacillus licheniformis (bacteria) works best at high temperatures and an acidic pH.
With this information and the results of the lab tests, it is apparent that enzyme A is
Aspergillus oryzae (fungus), enzyme B is Bacillus licheniformis (bacteria), and enzyme C is porcine (pig). This experiment shows how enzyme function can be affected by concentration, temperature, and pH. The function of this particular enzyme is to breakdown starch and produce maltose. Each alpha-amylase functions best at an optimal temperature and pH, producing a larger amount of maltose in their preferred environment. This was a good way of identifying the unknown enzymes because temperature and pH are major factors in changing an enzymes shape/structure, which ultimately affects the enzyme’s function.
References
Bacterial Alpha Amylase [Internet]. c2006. India: Advanced Enzyme Technologies Ltd; [cited 2008 Mar 5]. Available from: http://www.enzymeindia.com/enzymes/bacterial-alpha.asp
Structure and Function of an Enzyme [Internet]. c2007. Encarta. Microsoft Corporation; [cited 2008 Mar 5]. Available from: http://encarta.msn.com/media_461517494/structure_
and_function_of_an_enzyme.html