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Kinetic Properties of β-glucosidase/

Cellobiase from Aspergillus Niger

Ethan G. Parker

Department of Biochemistry, University of New Mexico, Albuquerque, New Mexico 87106

Received May 1, 2018

glucohydrolase, EC 3.2.1.21) have been determined. The Michaelis constants (Km) for

cellobiose is 1.1 mM, respectively, at pH of approximately 5 and 50°C. Glucose is shown to be a

competitive inhibitor with inhibitor constants (Ki) of 1.7 mM when 4NPG is the substrate and 1

mM when cellobiose is the substrate. D-Glucono-1,5-lactone is shown to be a potent inhibitor (Ki

= 8 μM; 4NPG as substrate) while D-fructose exhibits little inhibition. Cellulose hydrolysis is

best fit in these conditions, which can be attributed to the activity of the β-D-glucosidase in this

preparation in maintaining the cellobiose at low concentrations during cellulose hydrolysis.

β-glucosidase or Cellobiase is an enzyme associated with bacteria and protozoa

(particularly in ruminants) catalyzing the hydrolysis of β1→4 glycosidic bonds of the

disaccharide. It catalyzes the hydrolysis of the glycosidic bonds to terminal non-reducing

residues in beta-D-glycosides and oligosaccharides, with release of glucose. These enzymes are

powerful tools for degradation of plant cell walls by pathogens and other organisms consuming

plant biomass. Cellulases break down the cellulose molecule into monosaccharides ("simple

sugars") such as beta-glucose, or shorter polysaccharides and oligosaccharides (carbohydrate

polymers comprise three to ten monosaccharides. They are linked together mostly by O-

glycosidic bond through condensation reaction between an anomeric carbon of a monosaccharide

and the other). Cellulase is used for commercial food processing in coffee and performs

hydrolysis of cellulose during drying of beans. Furthermore, cellulases are widely used in textile
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industry and in laundry detergents, have been used in the pulp and paper industry for various

purposes, and are even used for pharmaceutical applications. Cellulase is used in the

fermentation of biomass into biofuels, although this process is relatively experimental at present,

Cellulase is also used as a treatment for phytobezoars (a type of bezoar, or trapped mass in the

gastrointestinal system, that consists of components of indigestible plant material, such as fibres,

skins and seeds), a form of cellulose bezoar (a small stony concretion that may form in the

stomachs of certain animals, especially ruminants, and which was once used as an antidote for

various ailments) found in the human stomach. The purpose for this lab was to determine how

factors such as pH, temperature, substrate concentration, and inhibitors affect the reaction rate of

the enzyme, and what conditions are most suitable for it.

MATERIALS AND METHODS

General Assay and Preliminary Activity

For the preliminary activity, first calibrate the spectrophotometer using a blank cuvette with 500

µL water and 500 µL of stop solution. Then, perform the general assay twice. To perform the

general assay, start by adding 500 µL of stop solution to a small cuvette. Add 0.45 mL of

resuspension buffer, 0.15 mL substrate solution, and 0.40 mL enzyme solution to a

microcentrifuge tube. Start a timer when the enzyme is added. Cap and gently mix the tubes.

After ten minutes, transfer o.5 mL from the microcentrifuge tube to the cuvette. Read the

absorbance of the contents of the cuvette at 410 nm.

Repeat the general assay using 0.45 mL of pH buffers 3 through 7 instead of the resuspension

buffer.

Temperature Studies

Use a similar procedure to test the effect of increasing temperature. Use the same amounts of

chemicals as in the general assay. Place the resuspension buffer and substrate solution in one

microfuge tube, and the enzyme in another microfuge tube. Incubate the tubes in a water bath at

the desired temperature for 5 minutes. Add the enzyme to the other microfuge tube, and start a

10 minute timer. Continue to incubate.

Effect of Substrate on Reaction Rate

Use the same procedure as the general assay. Except add resuspension buffer and then add

substrate and water. Run trials from 0mL of substrate to 0.15mL of substrate, and add water to

bring the total volume to 0.15mL.

Effect of an Inhibitor on Reaction Rate:

Redo the increasing substrate activity with and without the inhibitor (30mM glucose). Choose a

few points from the increasing substrate studies to use. For the ones with inhibitors, add 50µL of

inhibitor and 50 µL less of water.

Using enzyme from Another Source

Place approximately 1g of portobello mushroom in a mortar. Add 2 mL of extraction buffer for

every gram of mushroom. Grind into a slurry. Scoop the slurry into a microcentrifuge tube.

Centrifuge it for about 2 minutes. Remove 1 mL of the supernatant and put it into a clean small
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test tube. Add 3 mL of resuspension buffer to the supernatant, Mix gently. Use this suspension

instead of the enzyme solution to repeat the Increasing substrate study.

RESULTS

It is previously established that Cellobiase is involved in the breaking down of Cellulose,

a molecule that is made up of long chains of glucose in plant walls and is used as a way to

produce food by many fungi. Therefore it provokes the interest to many the conditions that best

suit the enzyme’s reaction rate of establishing a free reducing end. Such factors are pH,

temperature, substrate concentration, and the presence of an inhibitor.

The graph Figure 1 you will see on the next page is the General Assay that was used to

complete the other test. The purpose of the general assay that was completed first is to have a

general sense of how each of the studies was completed. When adding the 500 µL of stop

solution to a cuvette is to create a blank sample that is used to calibrate the spectrophotometer.

By doing this we are able to measure the amount of absorbance of light that the enzyme has

when performing later experiments. Another reason that we complete a general assay is to assure

that our R2 value is at a minimum of .95 to assure that our measurements are accurate and will

have little error when performing later test.

FIG. 1. General Assay Studies using 450 µL of the Resuspension Buffer, 105 µL of Substrate Solution, and 400 µL of the β-glucosidase Enzyme
Solution, and run through a spectrophotometer set a 410nm. Tested three times.
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Based of the General Assay, our technique is something that needs to be improved as

referred to in out R squared value.

pH Studies:

The optimal pH of an enzyme is the pH where the conditions best suit it, however can be

different for different areas of the organism. By changing the preferred pH that a certain enzyme

likes you could do one of three things. Optimal pH is determined by the activity that the enzyme

performs while still accounting for stability where one is a part of the enzyme, and one is the

enzyme as a whole. By changing the pH you could denature the protein in a way that it can not

work properly. Another possible outcome is that by with the wrong pH you will change or affect

the charge of the molecule’s hydrophilic groups. The third possible outcome is that by with the

wrong pH you change the conformation of the enzyme to a point where the Cellobiase is no

longer able to recognize or bind to the substrate. In Figure 2 you will note that we used four

different pHs (3,4,5,6,7) in comparison to their absorbance as a way to measure their activity and

stability.
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According to Figure 2 our enzyme has an optimal pH of approximately 4.47 based on the

line that was given. With the pHs that were more acidic to this it is possible that they did one of

the three things that was mentioned earlier with the effects of pH on an enzyme. The same goes

for the pHs that we tested that were more basic. According to what we found online, the average

optimal pH for our specific enzyme is approximately at a 4.7pH. To calculate the where our

optimal pH was we used the equation y= -0.1349x^2+1.2052x-1.2667. A question that we are yet

to solve is what setting is this a optimal pH and if we tested it in other organisms like fungi, what

would it be then?

Temperature Studies:

Figure 3 is a graph that relates to temperature. Temperature can affect enzymes in a

similar way to the pH as it can change the activity of the enzyme along with the stability. It may

also change the conformation of the enzyme with the addition or subtraction if energy; though it

may be in a different conformation state, it might still be active before it is completely denatured.

In order to do this we made several different types of water baths, and followed through with the

general assay.*
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With online resources we were able to determine that the optimal temperature for our

enzyme was 50°C, or approximately 122°F.

*Note that the graph is a straight line and at the end is still increasing. The reason for this is that

our group had difficulty at keeping the water bath that the enzyme was in at a specific

temperature, and did not yield results for temperatures higher than 49°C. Theoretically, figure

three should increase to the 50 and then after that begin to level off as we have denatured or

changed the conformation too much. Other questions that occur are what we might have done

wrong in the procedure.

Increasing Substrate Studies:

The goal of the increasing substrate study is to determine the maximum velocity that the

enzyme can work at without becoming overwhelmed. The way that our group completed this

experiment was by increasing the concentration of the substrate. The substrate that was used in

all of these cases was p-Nitorphenyl glucopyranosidase. We were able to determine the general

concentration levels of these because as you increased the concentration, when adding it to the

stop solution, it would become a yellow color. The deeper the color, the higher amount of the

product that you made. Figures 4, and 5 below shows the results that we got for this part of the

procedure.
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Inhibitor Studies:

The reason for completing this experiment is to determine if a source that we choose to

use is either competitive, noncompetitive, or uncompetitive. It is commonly used a a way for

determining the rate at which the enzyme binds to the substrate, and through the translation

process becomes the enzyme and the product. This can commonly be shown in a the formula

E+S→ 𝐸𝑆 → 𝐸𝑃 → 𝐸 + 𝑃.

Both the results and the Lineweaver Burk graph are below in Figures 6 and 7.
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Using the Enzyme from Another Source:

For this study we isolated cellobiase from portobello mushrooms and repeated our increasing

substrate study using the enzyme retrieved from the mushroom. This was to determine the

differences between the pure enzyme and the one from an alternative source . As can be seen in

Figures 8 and 9.
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The action of the enzyme peaked at a lower substrate concentration than the pure enzyme. The

enzyme from an alternative source peaked in its activity at approximately 0.13 mM concentration

of substrate.

DISCUSSION

Through our studies we were able to determine many of the factors that impact the reaction rate

of the enzyme cellobiase. We found the optimum pH of cellobiase as well as the optimum

temperature and substrate concentration. Our study showed 4.7 to be the optimal pH for

cellobiase. Unfortunately, due to many errors in our methods, we were not able to determine

accurate values for the optimal temperature and substrate concentration. By finding these we

were able to determine the effects on the enzyme if requirements were not met, and how it was

affected. With the inhibitors studies we were able to conclude that glucose was a competitive

inhibitor only changing Km and leaving Vmax constant.

Due to many issues with our methods, we got very inaccurate and incomplete data for some of

the studies. Our temperature study did not include a aide enough range of temperatures, and our

data for the inhibitor study was bizarre. Our increasing substrate study did not test quite enough

values of substrate concentration. Ideally, in the future, we would be able to repeat many of these

studies with more time. If we had more time to complete the studies, we could be more careful

and more accurate in our studies. We would like to test a more complete range of values for all

of these studies.
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RESOURCES

"Cellulase." Wikipedia. Wikimedia Foundation, n.d. Web. 20 Apr. 2016.

"Beta-glucosidase." Wikipedia. Wikimedia Foundation, n.d. Web. 20 Apr. 2016.

Peneva, Petrana. A Study of the Enzyme Beta-glucosidase. Ithaca, NY: n.p., 1937. Print.

Hu, A.s.l., R. Epstein, H.o. Halvorson, and R.m. Bock. "Yeast β-glucosidase:

Comparison of the Physical-chemical Properties of Purified Constitutive and Inducible Enzyme."

Archives of Biochemistry and Biophysics 91.2 (1960): 210-18. Web. 29 Apr. 2016.