Enzyme lab PDF

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Lab report for professor rami alsaber on enzymes...

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Anissa Zibo Identifying the role of Enzymes as Catalysts and Identifying the Substrate and Product of an Enzyme Introduction: Enzymes are special types of proteins that are made up of different amino acids in a long chain. The specific function of an enzymes is determined by their sequence of amino acids. Enzymes are substances produced by living organisms that act as catalysts to speed up chemical reactions. Enzymes also reduce the activation energy needed in a reaction by binding to reactants. The reactants of an enzyme reaction are called substrates. Substrates are what bind to active sites through hydrogen bonds. Though enzymes are useful in speeding up reactions, there are a number of factors that affect how an enzyme will work. Temperature is one of them. The higher the temperature the faster the enzyme will work, but if the temperature becomes too hot the enzyme will denature and breakdown. PH levels can also affect the ability for an enzyme to do its job, too high or low of a pH can ultimately stop a reaction. Hypothesis: The standard curve of maltose will correlate maltose concentration with absorbance and will determine the amount of maltose produced as starch is digested with amylase. Materials: The materials we used to prepare the starch was conical flask, beakers, graduated cylinders, pipettes, glass plate, hot plate, thermometer, water bath, potato starch powder, iodine, sodium phosphate and buffer. For the second, we used beakers, graduated cylinder, test tubes, test tube rack, hot plate, spectrometer, maltose, cuvettes, pipettes, pipettes bulbs, kimwipes, distilled water, dinitrosalicylic acid and a marking pencil. The same materials were used in experiment 3.

Procedures: To prepare the starch we first added 40 mL of water to a large beaker and brought it to a gentle boi. Next we mixed 1g of soluble potato starch in 5mL of coldwater. Next while stirring, we added the mixture to the boiling water. After mixing, we let the starch cool to 37 degrees centigrade. After we added water to bring the volume up to 100 mL. After doing this, we put a few drops of starch solution onto a glass plate and added 1 mL of lugol’s iodine to indicate the presence of starch in the solution. Finally, we added 100 mL of 0.02 M sodium phosphate buffer to bring the volume to 200mL. For the preparation of the standard curve for maltose we first set up a boiling water bath. Next we dissolve 200mg of maltose in 50 ml distilled water, then bring the volume with 100 ml with distilled water. After we labeled 6 test tubes we boiled them for 3 minutes, then cooled them for 5 minutes. Next we added 8 ml of distilled water to each tube and diluted the mixtures. We then used a spectrophotometer to read the absorbance of each sample. Then finally we plotted the standard curve. Finally, for experiment 3 we had to measure the enzymatic degradation of starch by amylase. The first step we did was turn on the spectrophotometer and set it at 540 nm. Secondly we labeled two 250ml conical flasks and added 100ml of the starch solution prepared in activity. Afterwards we collected about 14 test tubes and labeled seven of them as -enzyme and the other as +enzyme. We had to prepare the spectrophotometer blank for the +enzyme and the -enzyme. We then added the 1% starch solution to the first test tube of the plus and negative enzymes. After that we proceeded to prepare amylase by mixing 0.2g of amylase with 100ml of distilled water. After that we added 5ml of the amylase to the +enzyme flask and 5ml of distilled water to

-enzyme. Every 5 minutes we removed 1 mL of both enzyme reactions and placed them in test tubes. Afterwards we measures the absorbance of the enzyme reactions. Results: Experiment 1 Test Tube #

1 (blank)

2

3

4

5

6

Water (mL)

1.0

0.8

0.6

0.4

0.2

0

Maltose (mL)

0

0.2

0.4

0.6

0.8

1.0

Dinitrosalicylic acid (mL)

1

1

1

1

1

1

Water (after boiling) (mL)

8

8

8

8

8

8

Maltose concentration (mg/mL)

0

0.04

0.08

0.12

0.16

0.2

Absorbance (540 nm)

0.122

0.122

0.176

0.174

0.250

0.308

Experiment 2 Tube #

-1

-2

-3

-4

-5

-6

-7

Absorbance

0

0.021

0.039

0.054

0.081

0.121

0.145

Maltose (mg/mL)

0

0.017

0.025

0.037

0.06

0.08

0.1

Experiment 3 Tube #

1

2

3

4

5

6

7

Absorbance

0

0.008

0.009

0.011

0.013

0.015

0.016

Maltose (mg/mL)

0

0.003

0.007

0.008

0.009

0.011

0.013

Overall, the absorbance and maltose levels were significantly lower when starch was not treated with amylase. This is because amylase hydrolyses starch into maltose. Conclusion: we used amylase to break down starch into simple sugars, aka glucose molecules. Iodine was used to track to effect of amylase on starch. The enzyme in amylase has an active site which fits with our substrate which is starch. Amylase broke down those molecules. We started both solutions at the same temperature because the temperature can affect enzyme activity which is why it must be controlled. Overall, we saw the effects of amylase and how it can be related to the amylase that we have present in our bodies when aiding digestion especially in our mouths and digestive systems....