Investigating the activity of enzymes through

immobilization

INTRODUCTION
Research question: How will the immobilization of lactase in sodium alginate affect it’s
reactivity with lactose present in milk and change the amount of glucose produced?

In my biology HL class we were introduced to the immobilization of enzymes, specifically

of the catalyst enzyme lactase that is responsible for the digestion of lactose (a disaccharide
sugar). Immobilization is the “imprisonment” of an enzyme in a matrix, which still allows the
exchange of molecules, such as effector or inhibitor molecules (Moo-Young).

Immobilization of enzymes is used in many industries such as food, chemical,

pharmaceutical, biomedical and environmental sectors. It allows a way to develop biocatalysts
with superior catalytic properties, which are more stable, stronger and recoverable (Bilal and
Iqbal). Due to their characteristics, biocatalysts are considered environmentally green, which
makes them highly popular amongst scientists that strive to make society become more
environmentally sustainable. There is a great number of natural immobilizing agents that are
biopolymers (alginate, xanthan, cellulose etc.) which have many resourceful properties, in
particular: non-toxicity, biocompatibility, biodegradability, flexibility, renewability, and the
availability of numerous reactive sites (Bilal and Iqbal). Other advantages of immobilization are
the possibility to immobilize multiple enzymes and most importantly the reduction of the cost of
downstream processing. Biopolymers have the capacity to be recyclable and salvageable and
due to their non-toxic nature, the disposal of their waste is much easier. However,
immobilization also has its downsides. The disadvantages include the incineration of the enzyme,
fouling, carrier and immobilization costs and, above all, lower reaction rates and lower enzyme
activity, in particular of native enzymes (Basso and Serban).

This experiment is important to the dairy industry. Milk has a great number of
advantages, the main one being that it is an almost irreplaceable source of dietary calcium
(Silanikove et al.). Due to milk’s popularity in the food industry, many substitutes and alternative
ways of digesting lactose have been introduced for people suffering from lactose intolerance.
There are numerous ways in which people lacking the lactase producing gene could be able to
digest lactose. For example, they could take lactase tablets or substitute milk products (oatmeal
milk, rice milk); however, this experiment will be focusing on a way of removing lactose from
milk products through immobilization.

Variables
Independent variable: Immobilization of lactase in sodium alginate

Dependent variable: Reactivity of the enzyme

Controlled variables: Reactivity of sodium alginate 3% (controlled by checking glucose levels for
water immobilized by sodium alginate), temperature (can be controlled by putting substances in
a water bath in order to keep their room temperature), time (can be controlled by measuring the
time accurately with a stopwatch).

Hypothesis
The null hypothesis for this experiment would be that there will be no observed change between
the immobilized lactase and the independent lactase.

The alternative hypothesis that is intended to be proven through this experiment is that
immobilizing lactase will lower its reactivity.

MATERIALS AND METHODS

Materials and Apparatus
 Calcium chloride 2% m/V solution 150ml
 Sodium alginate 3% m/V solution 10ml
 Demineralized water
 Lactase enzyme solution 10ml
 200ml of milk
 7 syringes (at least 5ml) [+/- 0.1ml]
 Sieve
 Glucose stripes (for urine testing)
 Glucose solutions (0; 100; 300; 500; 1000mmol/L)
 Stirring rod
 15 test tubes (25ml) [+/- 0.1ml]
 4 beakers (200ml) [+/- 10ml]
 2 watch glasses
 Stopwatch [+/- 0.01s]
 5 Pipettes (2.5ml) [+/- 0.1ml]

Procedure
In order to immobilize lactase, we prepared 150ml of calcium chloride 2% in a beaker.
Then we mixed (measuring the solutions with syringes) 5ml of enzyme solution with 5ml of
sodium alginate 3% in a different beaker. For negative control, we did the same, but instead of
the enzyme solution we used water. Using a syringe we transferred 5ml of alginate-enzyme
solution in droplets into the CaCl2 to form beads (we did the same for negative control with the
alginate-water solution). We sieved the enzyme beads and water beads and put them onto 2
watch glasses. Next, in order to prepare the samples we separated all the alginate beads with
the enzyme into equal amounts and put an equal number of them in 5 test tubes (we did the
same for the alginate beads with water). Then, using a pipette, we added 0.5ml of just the
enzyme, into each of the 5 additional tubes.

For the second step we first checked the concentration of glucose in the milk by
transferring one droplet of milk using a pipette onto the glucose strip. Then into each of the 5
test tubes with enzyme beads, in 10 second intervals, measured by a stopwatch, we added 5ml
of milk using 5 syringes*. Lastly, at the appropriate times (1min; 2.5min; 5min; 10min) we stirred
the solution and added, using a pipette, one droplet of it to the strip and read the results. We
repeated this for the control beads and only the enzyme.

*We used 5 in order to make the process easier since we didn’t have enough time in between
intervals to collect the milk

Risk Assessment, Ethical and Environmental Issues

Safety: Calcium chloride on its own can cause conditions like eye irritation or skin irritation but in
such a low concentration it’s considered harmless, so no safety measures were taken.

Ethical: There were no ethical implications to be considered because no living animals were used
in the experiment.

Environmental: Since the solutions had a very low percentage concentration and other
substances used had a pH close to neutral, they were safe to pour down the drain.

Results and analysis

Results
Table of raw obtained results

Sample 0 min 1min 2.5min 5min

(mmol/L) (mmol/L) (mmol/L) (mmol/L)
Control beads1 0 0 0 0
Control beads2 0 0 0 0
Control beads3 0 0 0 0
Control beads4 0 0 0 0
Control beads5 0 0 0 0
Bead w/ 0 5.5 14 14
enzyme1
Bead w/ 0 14 14 14
enzyme2
Bead w/ 0 5.5 5.5 28
enzyme3
Bead w/ 0 0 5.5 28
enzyme4
Bead w/ 0 0 5.5 14
enzyme5
Enzyme1 0 5.5 56 111
Enzyme2 0 28 56 111
Enzyme3 0 28 28 111
Enzyme4 0 28 56 111
Enzyme5 0 28 28 111

Mean and Standard Deviation

Beads with enzyme:

0min [+/- 1min [+/- 0.01s] 2.5min [+/- 5min [+/- 0.01s]
 0.01s] 0.01s]
Mean [mmol/L] 0 5 8.9 19.6
Standard 0 5.73 4.66 7.67
deviation
mean=8.38

Enzyme:

0min [+/- 1min [+/- 0.01s] 2.5min [+/- 5min [+/- 0.01s]
0.01s] 0.01s]
Mean [mmol/L] 0 23.5 44.8 111
Standard 0 10.1 15.3 0
deviation
mean=44.82

Graph of the glucose level of Beads with Enzyme compared to time

y=0.2551x

r2=0.991

r=0.996
Graph of the glucose level of Enzyme compared to time

y=0.045x

r2=0.993

r=0.99

Uncertainties
Syringe uncertainty: +/- 0.1ml

Beaker uncertainty: +/- 10ml

Pipette uncertainty: +/- 0.1ml

Stopwatch uncertainty: +/- 0.01s

Analysis
As observed in the raw data table no effect was measured when the control beads were
tested therefore meaning that sodium alginate on it’s own doesn’t react with lactase and
doesn’t produce glucose.

In the raw data table, there are some values that are slightly different from the trend. For
example, in the 1 minute column for beads with enzyme there are values ranging from 0-14
mmol/L which may have an effect on the results.

In the graph representing the beads with enzyme, a positive trend line is drawn showing
that when time increases so does the level of glucose. The coefficient of determination is 0.996
which means that the trend line almost perfectly fits the data points and Pearson’s correlation
coefficient of 0.991 shows that the correlation is very strong. The second graph represents
almost the same values.

As seen in the raw data table the values for the beads with enzyme turned out to be
smaller than the enzyme itself, therefore showing that the reactivity of the immobilized enzyme
is decreased. The graphs show that the reactivity of the independent enzyme was much faster
than the reactivity of the immobilized enzyme. The immobilized enzyme at the 1 minute mark
had a spike of glucose where the average equaled 5 mmol/L, whereas the independent enzyme
at the same time reached an average glucose level of over 20 mmol/L. The final results observed
after 5 minutes were also very different: the immobilized enzyme reached an average of 19.6
mmol/L and the maximum glucose level reached was 28 mmol/L, while the independent enzyme
reached an average and maximum of 111 mmol/L.

Discussion
As hypothesized, the immobilized lactase enzyme turned out to be much less reactive
than the enzyme by itself. As mentioned in previously cited sources, some enzymes will become
more reactive when immobilized, although this does not seem to be the case for lactase. This
may be because of the fast kinetic of indigenous enzymes, which are naturally obtained from
bovine milk, experiences a dramatic reduction when immobilized due to diffusion restrictions
(Basso and Serban). These results show how effective this method of removing lactose from milk
products is.

Strengths

1. The high r2 value of 0.99 indicates high linearity of the data, which means that the
enzyme reactivity was linearly increasing with the time passed.
2. The Pearson’s correlation coefficient was very close to 1.00, meaning there was precision
and strong correlation between the two variables
3. The control group showed that sodium alginate did not have an effect on the glucose
levels, proving that the results are not skewed by it.

Limitations

Since we took no measures to insure the
temperature of the milk was at room
temperature this could have had an effect on
the enzyme reactivity.
There could have been random errors coming
from the way the solutions were measured
which was by using pipettes and syringes.
There could have been potential random
errors when measuring the results from the
glucose strips due to the small windows of
time that we had in between putting the
droplets of milk onto the strips.
This can be controlled by putting substances
in a water bath in order to ensure their room
temperature and measuring the experiments
shortly after taking the milk out of the water
bath.
This can be prevented using a scale instead.
This could be avoided by for example having
more people to help perform the experiment
or performing the measurements separately
for every repetition.
 Additionally, the slight inconsistencies that
occurred may have had an effect on the
result, and they occurred due to keeping the
milk droplets on the glucose strips for a
prolonged amount of time.

References

Basso, Alessandra, and Simona Serban. “Industrial Applications of Immobilized Enzymes—a

Review.” Molecular Catalysis, vol. 479, 18 Sept. 2019, p. 110607.,
doi:10.1016/j.mcat.2019.110607.

Bilal, Muhammad, and Hafiz M.N. Iqbal. “Naturally-Derived Biopolymers: Potential

Platforms for Enzyme Immobilization.” International Journal of Biological
Macromolecules, vol. 130, 28 Feb. 2019, pp. 462–482.,
doi:10.1016/j.ijbiomac.2019.02.152.

Moo-Young. “Immobilization of Enzymes.” Immobilization of Enzymes - Enzyme

Technology, 1985,
biocyclopedia.com/index/biotechnology/microbial_biotechnology/enzyme_technology/
biotech_enzyme_immobilization.php.

Silanikove, Nissim, et al. “The Interrelationships between Lactose Intolerance and the
Modern Dairy Industry: Global Perspectives in Evolutional and Historical
Backgrounds.” Nutrients, vol. 7, no. 9, 31 Aug. 2015, pp. 7312–7331.,
doi:10.3390/nu7095340.