University of Guyana

Faculty of Natural Sciences

Department of Biology

BIO 3110: Biochemistry 11

Lecturer: Ms. Dianna Seecharran

Laboratory Exercise # 5

Enzyme Immobilization

Candy Anderson
1028633
01/11/2019
Title: Enzyme Immobilization

Aim:

 To immobilize enzyme by gel entrapment

 To compare the effectiveness of different methods of enzyme immobilization

Introduction:

‘An enzyme is defined as a biomolecule that speeds up biochemical reactions. It therefore acts as
a biocatalyst that causes the conversion of a substrate into a product while never changes or used
up,’ (Supriya, 2019). Whereas, ‘enzyme immobilization is defined as a process by which an
enzyme molecule is enclosed/ adsorbed to the surface of an insoluble support or is incorporated in
to a gel matrix. This implies that an immobilized enzyme movement is restricted,’ (Supriya, 2019).

Enzymes are highly effective and efficient catalysts of a wide variety of processes. Also, enzymes
may reduce the number of reaction steps and quantities of hazardous solvents needed and thus
make a process more inexpensive and environmentally friendly. Therefore, enzymes have become
an important catalyst which exhibit great potential in many practical applications in industries, that
ranges from food to pharmaceuticals. One of the most important and widely used techniques is
enzyme immobilization. The greatest advantage of immobilization is that it significantly improves
the stability of the biomolecules under various reaction conditions and enhances the reusability of
biomolecules over successive catalytic cycles, (Archer, Palmer, 1989).

Moreover, after binding the enzyme molecules, the catalysts change from a homogeneous to a
heterogeneous form, which facilitates simple separation of the biocatalytic system from the
reaction mixture and results in products of higher purity. There are various immobilization
techniques such as adsorption, covalent binding, entrapment, encapsulation and cross-linking.
Selection of the most appropriate immobilization method and support material depends strongly
on the type and conditions of the catalytic process as well as the type of the enzyme. However, in
this laboratory exercise gel entrapment method was utilized and was also compared with other
methods, (Archer, Palmer, 1989).
Materials:

 Beakers
 Graduated cylinders
 Balance
 Pipets
 Syringe
 Alginic acid, sodium salt
 CaCl2
 Enzyme (Promalt)

Methodology:

6g of sodium alginate was dissolved in a 100ml to male a 6% solution. It was then leave for 30
minutes. 4g of promalt was then mixed with 10 ml of the 6% sodium alginate solution (polymer
solution).

The beads were then made by dripping the polymer solution from a height of approximately 20cm
into an excess (100ml) of stirred 5% CaCl2 solution using a syringe and a needle at room
temperature. The bead size was controlled using a pump pressure and a needle gauge. The beads
were then leave for half an hour and observed.

Results:

It was observed that upon adding the beads to get a better scope to drop the beads, drops were done
at 1-2cm at first and the shapes were observed to be spherical in shape. After raising the height to
20cm the beads were observed to be larger and different shapes could be seen. The beats were
small orange brown in colour.

Discussion:

5.

6. Methods of Immobilization used:

 Carrier binding method- this involves the binding of enzymes with water – insoluble
carriers. Also, this is the oldest immobilization technology.
  Physical Adsorption- Physical adsorption of enzyme protein on the surface of water –
insoluble carriers. This results in little to no conformational change of the enzyme protein
or its active center does not change.
 Ionic binding – involves the ionic binding of enzyme protein to water insoluble carriers
containing ion exchange residues.
 Entrapment- confining enzymes in lattice of a polymer matrix opr enclosing enzymes min
semi-permeable membrane.
 Cross linking – formation of intermolecular cross linkages between the enzymatic
molecules by bi/multi-functional reagents.

7. Advantages:

 Can be easily removed from the reaction, therefore, causing it to be recycled in the reaction.
This means that the reaction mixture does not get contaminated with the enzyme protein,
it only contains the solvent and reaction products.
 Immobilized enzymes have a greater thermal and operational stability than the free
enzymes.
 It is greatly efficient in constructive multi-step reactions.
 It reduces cost of biologically active enzymes; therefore, its use is more economical.
 Also, labour input is decreased.

Disadvantages:

 Industrial use is limited

 Enzymes can lose their catalytic properties
 Some proteins may lose their catalytic property
 Some may become inactive by heat generation.

8. Different concentration of CaCl2 solution should have been used, so as to compare the different
types and size of beads produce and also to determine which concentration would be more effective
in conversion of substrate into products.

Also, atleast two other methods, under the same condition should have been used to compare the
different types of beads produced and also how it would convert substrates into products.
Moreover, more time should have been given to get better results and understanding of the lab
exercise.

Sources of Error/ Limitation:

- It was difficult to drop the beads from the height of 20cm to the CaCl2 solution, this
results in lost of beads.
- Time was a limiting factor, since observation could not have been made at the given
procedural time range.
- Also, the immobilized enzyme was prepared, but there was no substrate given for it to
act on so as to observe its effectiveness.

Conclusion:

References:

Supriya, N. (2019). Immobilization of Enzyme. Retrieved from

https://biologyreader.com/immobilization-of-enzyme.html

Archer, C. Palmer, J. (1989). Practical Biochemistry for Colleges. Wood, E J, Pergamon Press;
Oxford.

Guisan, M. (2006). Immobilization of Enzymes. Retrieved from

www.ijpsonline.com/Article