Biology Extended Essay Madeeha Kalsekar

 Idea

Many people are intolerant to the disaccharide lactose, which is found in milk.
The enzyme lactase is used commercially to catalyse the breakdown of lactose to
monosaccharides; glucose and galactose. These sugars taste sweeter and are easier
to digest than lactose.
Enzymes can be immobilised in a number of different ways, using different
materials.

Below shows three ways of immobilisation of enzymes.

A student carried out an investigation to compare the activity of the enzyme

lactase that had been immobilised in the three different ways shown in Fig. 1.1.

• A solution containing 20 mg cm3 of lactose was poured through a column

containing the immobilised enzyme.

• The solution containing the products was collected and the concentration of
glucose measured.

(i) State a null hypothesis that the student could make for this investigation.

There is no particular difference in relation to the activity of the lactase, the rate of
reaction, or amount of concentration of glucose regardless the type of
immobilisation.

(ii) State the independent variable in this investigation.

Method of immobilising the enzyme

(iii) Suggest how the dependent variable might be measured.

Using a biosensor, glucose meter/monitor, dip sticks

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Biology Extended Essay Madeeha Kalsekar

 Variables

o Independent Variable: Method of immobilising the enzyme

o Dependent Variable: Measuring with biosensor, comparing dipsticks, or
clinistix to colour chart, comparing colour using Benedict’s solution,
permanganate test
o Control Variables: Fixed volume of lactose solution, Fixed temperature for
each enzyme, temperature-controlled room, incubator, water bath, buffer to
control pH at same level for each enzyme, standard time for lactose to be in
contact with enzyme using a stop watch or timer

 Research Question

 Immobilization of Enzymes:

1. Affinity-tag binding: Enzymes can be immobilized to a surface, e.g. in a porous

material like protein tags. This is used because it doesn’t require protein
purification. Porous glass and derivatives can be used where the porous surface
can be adapted in terms of hydrophobicity to suit the enzyme.

2. Entrapment: Enzyme is attached to the outside of an inert material. In general, it

is the slowest. It is not a chemical reaction and so the active site of the
immobilised enzyme may be blocked by the matrix or bead. Calcium alginate,
polyacrylamide gel or agar can be used.

3. Covalent bond: The enzyme is covalently bound to an insoluble support such as

silica gel or microporous beads. This provides the strongest enzyme/support
interaction so the lowest protein leakage during catalysis.

4. Cross-linkage: Enzyme molecules are covalently bonded to each other to create

a matrix. The reaction makes sure that the binding site does not cover the
enzyme’s active
site.

 Test for the breakdown of lactose

- Test for the glucose concentration

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Biology Extended Essay Madeeha Kalsekar

- Before and after the experiment

- Use Benedict’s solution
- Clinistix

 Supports used in immobilization technology

1) Natural Polymers
a. Alginate:
i. Derived from the cell wall of algae
ii. Calcium or magnesium alginate
iii. Inert and have a good water holding capacity
b. Chitosan and chitin:
i. Structural polysaccharides
ii. Naturally occurring in the cell wall of fungi and exoskeleton of
arthropods
iii. The functional groups in enzymes can bind to the -OH group of
chitins and form covalent bonds
c. Collagen:
i. Proteinaceous support
ii. Good porosity and water holding capacity
iii. Side chains of amino acids in the collagen and that of enzyme can
form covalent bonds to permanently hold the enzyme to the
support
d. Carrageenan:
i. Sulphated polysaccharide obtained from red algae
ii. Good gelling properties together with its high protein holding
capacity makes it good support for immobilising enzymes
e. Agar
i. Partially hydrolysed collagen with good water holding capacity
f. Cellulose fibres- coconut fibres
i. Most abundant polymer of nature
ii. Cheapest support available as carrier of enzymes
iii. The hydroxyl group of the monomer units can form covalent
bonds with the amino acids of the enzyme
g. Starch
i. Natural polymer of amylose and amylopectin
ii. Good water holding capacity
h. Pectin
i. Structural polysaccharide of plants found in their primary cell
wall and acts as the inter-cellular cementing material in plant
tissues
ii. Good gelling agent with good water holding capacity

2) Synthetic Polymers

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Biology Extended Essay Madeeha Kalsekar

They are ion exchange resins or polymers and are insoluble with a porous
surface. This surface can hold the enzymes or the whole cell.
Example:
a. Diethylaminoethyl cellulose [DEAE cellulose]
b. Polyvinyl chloride [PVC]
c. UV activated Polyethylene glycol [PEG]

3) Inorganic materials
a. Zeolites
i. Microporous, aluminosilicate minerals with good adsorbing
properties and used for immobilising enzymes and whole cells
b. Ceramics
i. Non-metallic solids consisting of metal and non-metal atoms
held in ionic an covalent and non-covalent bonds
c. Silica
d. Glass/Marbles
e. Activated carbon
f. Charcoal

 Methods of Immobilization

 Structure of the EE

1. Acknowledgements
2. Research Question

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Biology Extended Essay Madeeha Kalsekar

3. Hypothesis
4. Introduction
5. Apparatus
6. Method
7. Results
- Experimental Results
- Uncertainties
8. Analysis and Evaluation
- Analysis of data and graphs
- Analysis of Uncertainties
9. Conclusion
10. Bibliography

 Materials

 Sources

1) http://www.nuffieldfoundation.org/practical-biology/working-immobilised-
enzymes-or-microscopic-organisms
2) https://link.springer.com/article/10.1007/s10450-014-9623-y
3) https://journals.sagepub.com/doi/abs/10.1177/0883911516637377
4) http://www.usa-journals.com/wp-content/uploads/2013/11/Viet_Vol112.pdf
5) https://pdfs.semanticscholar.org/0b57/f60c854ad6dbe5b522de4b3470b172768
b18.pdf
6) https://www.easybiologyclass.com/enzyme-cell-immobilization-techniques/

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Biology Extended Essay Madeeha Kalsekar

 Planning

1M 2M 3M 4M 5M

Agar

Alginate

Coconut
Fibres