UNIVERSITI TEKNOLOGI MARA

FAKULTI KEJURUTERAAN KIMIA

BIOPROCESS ENGINEERING LABORATORY
(CBE 661)
NAME:
1. INTAN NUR JANNAH BT ROSLAN
2. MASLINDA IDAYU BT MOHD NOR
3. MUHAMMAD HAFIZ B HUSSIN
4. NOOR HANINI BT HASHIM
5. SITI NABILAH BT MOHD NADZRI
GROUP
:
EXPERIMENT
:
DATE PERFORMED :
SEMESTER
:
PROGRAMME/CODE

BIOPROCESS / EH222

No.
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EH222 5B
IMMOBILIZED ENZYME
13 OCTOBER 2015
5
:
CHEMICAL
ENGINEERING

Title
Abstract/Summary
Introduction
Aims
Theory
Apparatus
Methodology/Procedure
Results
Calculations
Discussion
Conclusion
Recommendations
Reference /Appendix
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STUDENT I/D:
2013409384
2013244384
2013822938
2013851264
2013820288

Allocated Marks
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5
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10
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10
10
10
20
10
5
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100

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1.0 ABSTRACT.........................................................................................................................3
2.0 INTRODUCTION............................................................................................................3-4
3.0 OBJECTIVES......................................................................................................................4
4.0 THEORY..........................................................................................................................4-5
5.0 APPARATUS & MATERIALS............................................................................................6
6.0 PROCEDURES................................................................................................................... 7
7.0 RESULTS............................................................................................................................ 7
8.0 DISCUSSIONS..............................................................................................................8-10
9.0 CONCLUSION................................................................................................................. 10
10.0 RECOMMENDATIONS...........................................................................................10-11
11.0

REFERENCES & APPENDICES.........................................................................11-12

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1.0 ABSTRACT
This experiment is to analyse the mass transfer effects of kinetics of immobilized enzyme
reactions. Other than that, the experiment also studies about the effect of sodium alginate
concentration on the gel capsule permeability and also the effect of CaCl 2 concentration on
the rigidity of the beads. In this experiment, an amount of sodium alginate was dissolved in
distilled water in order to make a 3% solution. Then, the solution is mixed with an amount of
enzyme. The beads are formed by dripping the polymer solution from a height of
approximately 20 cm into an excess of stirred 0.2M CaCl 2 solution by using the syringe and
needle gauge. The absorbance was obtained by using three different blank sample. For beads
solution blank sample, the average result for absorbance is 0.333. Whereas, for the mixture of
beads with starch and the mixture of amylase with starch as the blank sample, the average
absorbance obtained are 0.267 and 0.133 respectively.

2.0 INTRODUCTION
Generally,

immobilized enzyme is an enzyme that attached to the inert and insoluble

material such as calcium alginate which is being produced by a reaction of sodium alginate
solution and enzyme solution with calcium chloride. This immobilized enzyme able to resist
the changes in pH and temperature. Besides, this type of enzyme able to be held in place
when the reaction occurs. What makes this enzyme is beneficial, it can easily be separated
from products after a reaction and can be used again. ]1
[

Basically, the immobilized enzyme is responsible in enhancing the limited

characteristics of normal enzyme. Most of the enzymes have low stability, easily being
inhibited by high concentration of substrates and products and low activity in reaction.
Therefore, immobilization of enzyme is a technique by which it allows the modified enzyme
to be re-used in more than one reaction.]

For this experiment, there is a method to immobilize the enzyme. To conduct this
experiment, first of all, the 30 g of sodium alginate were dissolved in 1 litre distilled water to
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make a 3% solution which later 0.015 g of enzyme were mixed with 10 ml of 3 wt. % sodium
alginate solution. After that, the beads were formed by dripping the polymer solution from a
height of approximately 20 cm into an excess of stirred 0.2M CaCl2 solution with a syringe
and the needle gauge at room temperature. However, the beads size can be controlled by
pump pressure and needle gauge. Generally, a typical hypodermic needle produces beads of
0.5 2.0 mm in diameter. Later, the beads were left in the calcium solution to cure for 0.5 3
hours. Then, a few of beads were put into a cuvette in order to take the reading of absorbance
by using beads solution, starch, and amylase with starch as blank samples.

3.0 OBJECTIVES
1) To familiarize with the techniques of enzyme immobilization matrix entrapment,
encapsulation, ionic and cross linking, column packing.
2) To analyse the mass transfer effects of kinetics of immobilized enzyme reactions.
3) To study the effect of sodium alginate concentration on the gel capsule permeability.
4) To study the effect of CaCl2 concentration on the rigidity of the beads.

4.0 THEORY
Enzyme immobilization refers to confinement of enzyme to a phase (matrix/support) different
from the one for substrates and products.(1) This technique provides an excellent base for
increasing availability of enzyme to the substrate with greater turnover over a considerable
period of time which will increase the production of product. (1) Enzymes are immobilized to
increase its functional efficiency and one advantage is that immobilized enzymes can be
reused again for the next cycle.(1) The immobilized enzymes can either be enzymes or whole
cells and it use inert polymers and inorganic materials as carrier matrices. (1) There are some
characteristics for good matrices such as affordable, inert, good stability and have the ability
to increase enzyme specificity or activity and reduce product inhibition.(1)
There are many methods available for enzyme immobilization that includes
adsorption method, covalent binding method, cross-binding method, and entrapping method.
In this experiment, only the demonstration for the entrapping method was carried out. In
entrapment, the enzyme molecules are held or entrapped within suitable gels or fibres. (2)
There may or may not be covalent bond formation between the enzyme molecules and the
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matrix.(2) When the enzyme molecules were trapped in the form of gels or beads, the reaction
occur by diffusion of substrate to the enzyme and diffusion of product away from the
enzyme.(2) The most widely used is the entrapment in calcium alginate. (2) In this experiment,
the material used was sodium alginate that was added with the enzyme amylase. At the end of
the experiment, formations of beads were to be expected after the solution mixture were to be
dropped into excess of stirred calcium chloride (CaCl2) solution.

Figure 4.1: Methods for enzyme immobilization(2)

5.0 APPARATUS AND MATERIALS

Beakers
Measuring cylinder
Weighing balance
Pipets

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Syringe
Conical flask
Hot plate with stirrer
Cuvettes
Sodium alginate
Amylase enzyme
Calcium chloride (CaCl2) solution

Figure 5.1: Some materials used

6.0 PROCEDURES
1) 30 g of sodium alginate were dissolved in 1 litre distilled water to make a 3% solution.
2) 0.015 g of enzyme were mixed with 10 ml of 3 wt. % sodium alginate solution.
3) The beads were formed by dripping the polymer solution from a height of approximately
20 cm into an excess of stirred 0.2M CaCl 2 solution with a syringe and the needle gauge
at room temperature.

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4) The beads size was controlled by pump pressure and needle gauge. A typical hypodermic
needle produces beads of 0.5 2.0 mm in diameter.
5) The beads were left in the calcium solution to cure for 0.5 3 hours.
6) Then, the number of beads were put into a cuvette in order to take the reading of
absorbance.
7) The first reading of absorbance were read by using beads solution as the blank sample.
8) The absorbance was read three times to take the average.
9) Steps (6) to (8) were repeated by using the mixture of beads with starch, and also the
mixture of amylase with starch as the blank sample.
10) The result was recorded for all blank sample

7.0 RESULT

Beads
Beads + starch
Amylase + starch

-0.322 209.8%
-0.003 100.8%
-0.124 133.0%

Absorbance
-0.341 219.3%
-0.130 134.9%
-0.129 134.5%

-0.335 216.3%
-0.134 136.0%
-0.147 140.4%

8.0 DISCUSSION
Immobilized enzyme is an enzyme that is physically attached to a solid support over which a
substrate is passed and converted to product. There are some methods or techniques of
immobilization which are adsorption method, covalent binding method, cross-binding
method, entrapping method and entrapping by micro-encapsulation. This experiment is
carried out to analyse the mass transfer effects of kinetics of immobilized enzyme reactions.
Other than that, it is also carried out to study about the effect of sodium alginate
concentration on the gel capsule permeability and the effect of CaCl 2 concentration on the
rigidity of the beads. (SachinTalekar and Sandeep Chavare, 2012)
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From this experiment, the absorbance readings were obtained by using three different
blank samples. For beads solution blank sample, the average result for the absorbance is
0.333. Whereas, for the mixture of beads with starch and the mixture of amylase with starch
as the blank sample, the average absorbance obtained are 0.267 and 0.133 respectively. It
shows that, the absorbances reading of beads was the highest from others. It is due to the
effect of sodium alginate concentration and CaCl 2 concentration. Starch is made of linear
amylase and branched amylopectin units. The starch has been used as enzyme immobilizer.
Calcium alginatestarch hybrid supports were applied for surface immobilization and
entrapment of bitter gourd peroxidase. Entrapped enzyme was more stable in the presence of
denaturants like urea due to internal carbohydrate moieties, while surface-immobilized
enzyme had superior activity (Matto and Husain 2009). It happens in the mixture of beads
with starch.
There are many lack of techniques or methods while conducting the Immobilized
Enzymes Experiment. It is due to the lack of knowledge about the immobilized enzymes and
late preparation of laboratory manual. Therefore, it is important to do some research from any
source (such as senior lecturers, website and books) before the experiment session. Other
than that, apparatus and materials are the most important things for any experiments. The lack
of CaCl2 concentrations and sodium alginate concentrations were caused the objectives of
this experiment cannot be achieved. Therefore, we need to ensure that the apparatus and
materials are prepared well before conducting the experiment. Then, the experiment will be
run smoothly and better results can determine.

As for the Immobilized Enzymes Experiment, we can determine the effect of sodium
alginate concentration, effect of concentration calcium chloride, effect of curing time of
calcium alginate beads, effect of pH and Temperature on -amylase activity, effect of bead
size on rate of starch hydrolysis, and kinetic analysis. Because of problems that cannot be
avoided, only the average absorbance readings were took from three different samples. To get
better results from the experiment, reducing errors as much as possible is required.

QUESTIONS
1) Find the suitable method for entrapment of amylase enzyme in ca-alginate.
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The suitable method for entrapment of amylase enzyme in ca-alginate is matrix

entrapment. Enzyme immobilization by entrapment produces the particle structure
which allows contact between the substrate and enzyme to be achieved and
additionally, it is possible to immobilize several enzymes at the same time. The
technique is one of the most frequently used. Other than that, it is reasonably safe,
simple, cheap and offering good mechanical strength, high porosity for substrate and
product diffusion and above all the simple procedural requirements for
immobilization. (SachinTalekar and Sandeep Chavare, 2012)
2) Discuss the effect of sodium alginate concentration on the gel capsule permeability.
The immobilization of enzyme depends on concentration of sodium alginate, the
porosity of the calcium alginate beads depends upon the alginate type and the gelling
agent concentration. By using various concentrations of sodium alginate for calcium
alginate beads, it will vary the relative degree of cross linking which would create
different pore size. The decreasing immobilization yield with increase in sodium
alginate concentration has been due to the decrease in the porosity of the gel beads,
which caused diffusion limitation of the substrate. By reducing the porosity can
reduce leakage but some initial leakage of the enzyme molecule is certain to occur.
The lower immobilization yield in case of lower concentration of sodium alginate
solutions might be due to larger pore size and consequently greater leakage of the
enzyme from matrix. (SachinTalekar and Sandeep Chavare, 2012)
3) Discuss the effect of CaCl2 concentration on the rigidity of the beads.
The concentration of CaCl2 was also varied in order to acquire stable beads capable to
secure maximum enzyme. It was found that, increasing the CaCl 2 concentration will
affect increasing of amylase enzyme until it reach the stability. When the CaCl 2
concentration is increased to beyond of stability of amylase enzyme, therefore the
immobilization is decrease. It might be due to the changes in pH of calcium chloride
solution with changes in calcium chloride concentration. It is an important in
immobilization as it produces stable calcium alginate beads which could reduce the
enzyme leakage and increase the immobilization yield of enzyme. (SachinTalekar and
Sandeep Chavare, 2012)

9.0 CONCLUSIONS
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The experiment of immobilized enzymes was conducted with several aims. The techniques of
enzyme immobilization matrix entrapment, encapsulation, ionic and cross linking, column
packing is able to practice throughout this experiment. Other than that, the mass transfer
effects of kinetics of immobilized enzyme reactions can be analysed by the reaction involved
in the experiment. The effect of sodium alginate concentration on the gel capsule
permeability can be studied in the method to produce immobilized enzymes. The effect of
CaCl2 concentration on the rigidity of the beads can be studied. Therefore, the aims were
studied and the experiment was not successful.

10.0 RECOMMENDATIONS
1) Make sure to continuously stir the water on a hot plate during preparation of sodium
alginate solution to avoid any clump formation.
2) Make sure to continuously shake the conical flask during dropping of the mixture of
sodium alginate and amylase solution into the CaCl 2 solution so that the beads did not
clump together.
3) Make sure during measurement, the readings are read perpendicular to the eye level to
avoid any parallax error.
4) To avoid enzyme deactivation, make sure the amylase enzyme solution is kept at low
temperature.
5) When using the syringe, make sure the solution inside the syringe is being expelled in
the form of droplet not stream to produce the beads.
6) During absorbance measurement, make sure the cuvette is clean to avoid any
contamination.

11.0 REFERENCES / APPENDICES

[1] Raghu, H. S. and Rajeshwara, N. A., Immobilization of - Amylase (1, 4--DGlucanglucanohydralase) by calcium alginate encapsulation,International Food
Research Journal 22(2): 869-871 (2015)
[2] Jose M. Guisan, Immobilization of Enzymes and Cells, Springer science &Bussiness
Media, 1 Jan 2006, pg. 1-4

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[3] Matto M, Husain Q. Calcium alginatestarch hybrid support for both surface
immobilization and entrapment of bitter gourd (Momordicacharantia) peroxidase. J
MolCatal B-Enzym. 2009;57:164170.
[4] SachinTalekar and Sandeep Chavare, 2012, Recent Research in science and Technology.
[5] S. Datta., L. R. Christena., & Y. R. S. Rajaram. (2012). Enzyme immobilization: an
overview on techniques and support materials. 3 Biotech. 3(1): 1-9
[6] S. Pujari. (n.d). Immobilization of Enzymes: Methods, Effects on Enzymes and
Ribozymes.

Retrieved

November

2015

from:

www.yourarticlelibrary.com/biology/enzyme/immobilization-of-enzymes-methodseffects-on-enzymes-and-ribozymes/33689/

APPENDICES

Figure 13.0-1: It is how dripping the polymer solution from a height of approximately 20 cm
into an excess of stirred 0.2M CaCl 2 solution with a syringe and the needle
gauge.

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Figure 13.0-2: The reading of absorbance for three different samples were read from the
spectrophotometer.

Figure 13.0-3: The beads were formed while dripped polymer solution into CaCl 2 solution
and swirled at the same time.

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