ENZYMATIC ACTIVITY OF CELLOBIASE
Cellulose is the structural polysaccharide in the cell walls of plants, and is the most
abundant biological molecule on earth. Cellulose is a polymer of glucose monomers connected
together by beta-linkages (Figure 1) In general, many animals, including humans, are incapable
of digesting cellulose because they do not produce the enzyme to break these beta-linkages.
Figure 1
The enzyme, cellobiase, catalyzes the hydrolysis of β1-4 glycosidic bonds found in
cellulose, which is necessary for digestion. This enzyme can be found in organisms, such as
fungus and some bacteria, which can breakdown cellulose.
Enzymes are usually made up of linked chained amino acids, i.e. proteins, which speed
up the reaction it catalyzes. For this reason, enzymes are also known as catalyst. Enzymes are
specific to particular substrates, which are used to speed up most biological chemical reactions
that would take too long for them to occur naturally.
Below is another structural polysaccharide found in most foods, Starch, which is also a
polymer of glucose monomers but connected together by alpha-linkages instead of beta-linkages
(Figure 2) Animals, including humans, have the specific enzyme, amylase, to break down starch.
Therefore, these organisms have the ability to break down the alpha-linkages between glucose
molecules but cannot break the beta-linkages. This subtle structural difference is the reason why
amylase cannot digest cellulose.
Figure 2
In this experiment, we will observe the reaction catalyzed by cellobiase. In order to study
this enzyme’s activity, it is best to be able to either detect the substrate being used in the reaction,
or the products being produced. For this reaction, the substrate cellulose, and the product,
glucose, is clear and colorless making it difficult to directly quantify. We will utilize an artificial
substrate that has the same structural competency of beta-linkages, but will allow us to directly
observe and quantify the reaction of β1-4 glycosidic bond hydrolysis by using absorption
spectroscopy.
Measuring Enzymatic Activity of Cellobiase
To make this reaction easier to observe, an artificial substrate: p-nitrophenyl
glucopyranoside will be used. This artificial substrate will bind to the enzyme and is broken
down in a similar fashion to cellulose. When the artificial substrate, p-nitrophenyl
glucopyranoside, is broken down by cellobiase, it produces glucose and p-nitrophenol (Figure 3)
During the course of the experiment, you will be adding the reaction between enzyme
(cellobiase) and artificial substrate into Stop Buffer Solution at various time points. The stop
solution is a basic solution with a pH >7, which effectively disables the cellobiase enzyme
ceasing the production of glucose and p-nitrophenol. You will also notice that the reaction
solution will turn a yellow color, which is due to the p-nitrophenol that is produced during the
reaction.
The amount of yellow color observed in the reaction solution is proportional to the
amount of p-nitrophenol present. For every one molecule of p-nitrophenol present in the
solution, one molecule of p-nitrophenyl glucopyranoside has reacted with the cellobiase enzyme
(Figure 3). The yellow color of p-Nitrophenol can be measured using absorption spectroscopy.
Figure 3
Substrate (colorless)
p-nitrophenyl--D glucopyranoside
Products (yellow)
Glucose
p-Nitrophenol
Extraction of Cellobiase Enzyme
1.
Gather all the necessary materials for the experiment: mortar & pestle,
microcentrifuge tubes, funnel, and coffee filter. You MUST lightly wet the entire
coffee filter using DI water prior to placing onto the funnel. You MUST also
remove any excessive liquid from the coffee filter so that your coffee filter is not
dripping wet!
2.
Cut and weigh out 2 grams from the cap of a mushroom, taking care to remove dark
parts including the gills, and place that sample into your mortar & pestle.
3.
Add 4 ml of extraction buffer onto your mushroom sample and grind until you have a
slurry mixture.
4.
Once your mushroom sample has been ground into a slurry mixture, filter it through
your pre-wet coffee filter into (2) 2ml microcentrifuge tubes. After you have filtered
your sample, you may gently squeeze the remaining slurry mixture out of the coffee
filter into the microcentrifuge tubes. Take care to not break the filter.
5.
Centrifuge your filtered crude mushroom extract for 2 minutes at the highest RPM to
pellet the debris.
6.
After centrifugation, transfer the supernatant (the liquid layer) into the 15 ml tube that
your extraction solution came in. This is your enzyme extract! Make sure that you do
not transfer any pelleted debris.
7.
The entire class will combine their individual enzyme extract into a single tube or
beaker so that each group in the class will begin with the same enzyme concentration.
Save your 15 ml tube for the next step. Speculate on the variation you might see if
you were to not pool the enzyme. Where might variation be introduced?
8.
Once your class has successfully combined all of their enzyme extract, the teacher
will distribute 2 ml of the Enzyme Extract to each of the groups into their 15 ml tube.
Determining Cellobiase Enzymatic Activity
1. Gather all the necessary materials for this portion of the experiment: Timer, Cuvettes,
Cuvette racks, 15 ml tube, and spec200.
2. Next, label 8 cuvettes #1-8 and pipet 500 μl of Stop Buffer into each cuvette. These will
be your different time points. What is the purpose of the Stop Buffer?
3. Your teacher will make a “zero” cuvette by adding 500 μl of stop buffer, then add 165 μl
of enzyme extract, and then 335 μl of reaction substrate. This will be the zero for the
spec readings of your 8 cuvettes of reactions. More than one group can share a zero.
4. You reaction will be conducted in the 15ml tube labeled ‘reaction’ which contains 4 ml
1.5 mM p-nitrophenyl--D glucopyranoside substrate.
IMPORTANT: You will be adding your enzyme extract to the 15 ml tube labeled Reaction and
immediately start a timer.
5. Quickly add and mix the 2 ml of enzyme extract to your substrate solution. Immediately
start the timer as the Enzyme Extract is going into the Substrate. This is the time from
when everything needs to be measured!
6. After 1 min, remove 0.5 ml of your reaction and add it to cuvette labeled #1. Mix briefly
after each step!
7. 2 min from the start, remove 0.5 ml of your reaction and add it to cuvette labeled #2.
8. 4 min from the start, remove 0.5 ml of your reaction and add it to cuvette labeled #3.
9. 6 min from the start, remove 0.5 ml of your reaction and add it to cuvette labeled #4.
10. 8 min from the start, remove 0.5 ml of your reaction and add it to cuvette labeled #5.
11. 10 min from the start, remove 0.5 ml of your reaction and add it to cuvette labeled #6.
12. 15 min from the start, remove 0.5 ml of your reaction and add it to cuvette labeled #7.
13. 30 min from the start, remove 0.5 ml of your reaction and add it to cuvette labeled #8.
Cuvette
1
2
3
4
5
6
7
8
Time
1 min
2 min
4 min
6 min
8 min
10 min
15 min
30 min
Absorption
p-Nitrophenol Concentration
Determining Standard Curve of p-Nitrophenol
The concentration of p-nitrophenol produced in your reaction can be determined by comparing to
a standard curve of known concentrations of p-nitrophenol. You will make this standard curve
using p-nitrophenol ranging from 12.5 M to 100 M p-nitrophenol. When a graph of
absorbance vs. concentration is plotted for the standard solutions, a linear relationship should
result. The linear relationship between absorbance and concentration for a solution is known as
Beer’s law. You can determine the p-nitrophenol concentration produced in your reaction by
measuring its absorbance and then finding the corresponding concentration on the standard
curve. Because the relationship is linear, you could also calculate the p-nitrophenol concentration
using the formula for the standard curve best fit line (y=mx+b).
1.
2.
3.
4.
5.
6.
7.
8.
9.
For the p-Nitrophenol standards, label 5 cuvettes 1-5.
Add 1.0 ml of 0 (blank) to cuvette 1.
Add 1.0 ml of 12.5 M to cuvette 2.
Add 1.0 ml of 25 M to cuvette 3.
Add 1.0 ml of 50 M to cuvette 4.
Add 1.0 ml of 100 M to cuvette 5.
Set the wavelength to 410 nm.
Blank the spectrophotometer with the cuvette labeled #1.
Read the absorbance of your standards and record data below:
Cuvette
1
2
3
4
5
p-Nitrophenol
concentration
0 (blank)
12.5 M
25.0 M
50.0 M
100 M
Absorption
@ 410 nm
0
Use Excel to find out the equation for the linear best fit line and write it below (these instructions
are for Excel in Windows 7, yours may differ):
1. Open an Excel spread sheet containing a column for p-Nitrophenol standard
concentrations, and an additional column with their corresponding absorbances (as
shown above).
2. Highlight the values, click on insert, and choose the scatter plot with no line function.
3. Go to chart layouts, choose trendline. In order to see the equation, go into trendline
options and click “display equation on chart”.
4. Using this equation, determine the concentration of p-Nitrophenol in your Enzyme
reaction tubes.
Did your enzyme extraction allow the breaking of the β1-4 glycosidic bond between glucose and
p-Nitrophenol (p-Nitrophenyl--D glucopyranoside)? Explain.
Using excel plot your product concentration (p-Nitrophenol) versus time. Is the plot linear or
exponential, does the concentration of product reach a plateau, and not change with any more
time?
Come up with the chemical reaction for this reaction, using E for the enzyme (cellobiase) and S
for the substrate (of p-Nitrophenyl--D glucopyranoside) and products (p-nitrophenol and
glucose:
Thinking in terms of the enzyme acting as a catalyst, what do you think would happen to your
above reaction if you added more enzyme? If you added more substrate? If you boiled the
reaction? If you changed the pH? Design an experiment asking one or these questions or one of
your own questions, and predict your outcome, if time permits, conduct the experiment.
Cellulose is an untapped source of glucose. Glucose is not only extremely important for animal
metabolism, but it can be used to make ethanol, which can be used as fuel. Currently, glucose
from corn is utilized for ethanol production for fuel. Instead of using corn to make biofuel, if we
could access the glucose in cellulose, ethanol could be synthesized without impacting animal
food sources. Name several sources of cellulose:
Do you think it is feasible to use cellobiase to generate biofuel? Why?
Solutions
Extraction buffer and reaction buffer
50 mM MOPS Buffer pH 6 (MW 209.26 g/ mole)-Store at room temp indefinitely
Weigh out 2.6 g
Dissolve in 150 mL of distilled water
Adjust pH to 6
Bring to final volume of 250 mL with distilled water
Substrate
1.5 mM p-nitrophenyl-B-D glucopyranoside (pNPG) (MW 301.25 g/mole) in
50 mM MOPs Buffer pH 6.0-Store at 4°C, shelf life 1 week.
Weigh out 0.023 g
Dissolve in 40 mL of 50 mM MOPS Buffer
Bring to final volume of 50 mLs with 50 mM MOPS Buffer
Stop Solution
500 mM Na2CO3.H2O (MW 124.00 g/mole)-Store at room temp indefinitely
Weigh 6.2 g
Dissolve in 200 ml of dH2O
Bring to final volume of 250 ml with dH2O
p-Nitrophenol Standard Stock Solution
2.0 mM p-nitrophenol (MW 139.11 g/mole)-Store in at room temp indefinitely
Weigh 0.0278 g of p-nitrophenol
Dissolve in 75 mL of water
Bring to a final volume of 100 mL
0.2 mM p-nitrophenol Standard solution-Store at room temp indefinitely
Dilute 2.0 mM p-nitrophenol 1:10 using distilled water by adding 1 mL of 2 mM p-nitrophenol
and 9 mL of distilled water in 15 mL conical tube.