LABORATORY REPORT

PTT 202 Organic Chemistry for Biotechnology

SEM 1 2015/2016
Department of Chemical Engineering Technology
(Industrial Biotechnology)
EXPERIMENT 7: IMMOBILIZATION OF ENZYME
Name: LEE WAI SHENG
No. Matrix: 141282462
Group B6 Member : TAN WAI TENG
(141282498)
LOW HUEY CHEE
(141282466)
MUHAMMAD RIDZUAN BIN OSMAN
(141282480)
NUR ZAYANA SHAZLIN BT ZAIDI
(141283572)
Lecturer: ENCIK ABDUL LATIF ABDUL RANI
Date of Experiment : 24/11/2015
Date of Submission : 2/12/2015

Objective
1. To understand the principle of enzyme immobilization by using gel
entrapment method in alginate gel.
2. To study the immobilization enzyme activity by using gel entrapment
method.

Introduction
An immobilized enzyme is an enzyme that is attached to an inert, insoluble material such
as calcium alginate (produced by reacting a mixture of sodium alginate solution and
enzyme solution with calcium chloride). Enzyme immobilization can be defined as the
process of confining the enzyme molecules to a solid support over which a substrate is
passed and converted to product. This can provide increased resistance to changes in
conditions such as pH or temperature. It also allows enzymes to be held in place
throughout the reaction, following which they are easily separated from the products and
may be used again - a far more efficient process and so is widely used in industry for
enzyme catalysed reactions. An alternative to enzyme immobilization is whole cell
immobilization.
Entrapment is caging of enzymes by covalent or non-covalent bonds within gels or
fibers . Efficient encapsulation has been achieved with alginategelatincalcium hybrid
carriers that prevented enzyme leakage and provided increased mechanical stability.
Alginate derived from cell walls of brown algae are calcium, magnesium and sodium
salts of alginic acid and have been extensively used for immobilization as xanthan
alginate beads, alginatepolyacrylamide gels and calcium alginate beads with enhanced
enzyme activity and reusability. Cross-linking of alginate with divalent ions (like Ca 2+)
and glutaraldehyde improves the stability of enzymes.

Methodology
Part 1
1. 10 ml of 3% sodium alginate solution and 0.015g of -amylase is
mix well to form polymer solution.
2. 100 ml of 0.2 M calcium chloride is taken using measuring cylinder.
3. The polymer solution prepared is dripped from a height of about 20
cm into the stirred 0.2 M calcium chloride.
4. The beads is leaved for 1 hour.
5. The formed beads is filtered and washed thoroughly using distilled
water.
6. The beads is dried in open air for 1 hour.
7. The filtered calcium chloride solution is collected.

Part 2
1. 0.5 g of beads is immersed in a test tube.
2. 5 ml of sodium phosphate buffer and 0.25 ml of starch solution is put
into the test tube.
3. The test tube is incubated in water bath at 37C for 10 minutes.
4. After 10 minutes, 2.5 ml of 0.1 M HCl is added and the solution is
mix well.
5. Then, 5 ml of DNSA reagent is added and mix well.
6. The test tube is boiled for 15 minutes in 100C water bath.
7. The sample is ready to be analyzed.
8. The sample is analyzed using UV spectrophotometer and the
absorbance value is recorded.

9. The filtrate CaCl2 is carried out the same steps as above.

10.A control is prepared and using the same steps as above but without
starch.

Results
With beads
Times
Absorbance
(540nm)

1
0.5504

2
0.5773

Average absorbance value, y =

0.5504+0.5773+0.5743
3

Average
0.5673

= 0.5673

y + 0.0135
1.0594

maltose concentration, x =
=

3
0.5743

0.5673+ 0.0135
1.0594

= 0.5482mol/ml
Enzyme activity = maltose amount x reaction time
1
10 min

= 0.5482mol/ml x 342.3g/mol x (0.25+5+2.5)ml x

= 145.43g/min
Without beads (filtered CaCl2)
Times

Average

Absorbance
(540nm)

0.3367

0.3590

Average absorbance value, y =

maltose concentration, x =
=

0.4317

0.3367+0.3590+0.4317
3

0.3758

= 0.3758

y + 0.0135
1.0594
0.3758+ 0.0135
1.0594

=0.3675mol/ml
Enzyme activity = maltose amount x reaction time
1
10 min

= 0.3675mol/ml x 342.3g/mol x (0.25+5+2.5)ml x

= 97.49g/min
The maltose concentration is calculated using the standard curve line
equation which is y = 1.0594x-0.0135 where y is the absorbance value while
x is the maltose concentration.
EXPERIMENT 7

PREPARED BY: YEAR 2 STUDENT RY21 ACADEMIC SESSION 2015/16

Discussion
In this experiment, we are required to study the immobilized enzyme activity by using gel
entrapment method in alginate gel. The alginate that used is sodium alginate salt which is
the alginate acid.

The structure of alginate acid

Alginic acid, also called algin or alginate, is an anionic polysaccharide distributed widely
in the cell walls of brown algae, where through binding with water it forms a viscous
gum. Its colour ranges from white to yellowish-brown. It is sold in filamentous, granular
or powdered forms. The chemical compound sodium alginate is the sodium salt of alginic
acid. Its empirical formula is NaC6H7O6. Sodium alginate is a gum, extracted from the
cell walls of brown algae. It is mix with sodium phosphate buffer to make 3% solution
and stirred until it completely dissolved. A buffer solution is an aqueous solution
consisting of a mixture of a weak acid and its conjugate base, or vice versa. Buffer
solutions are used as a means of keeping pH at a nearly constant value so that the enzyme
can survive.
After mix, the solution are leave for 30 minutes. This is to eliminate the air bubbles that
can later be entrapped and cause the beads to float. After 30 minutes, 10ml of the sodium
alginate solution is mix with 0.015g of -amylase to form a polymer solution. Then,
100ml of 0.2M calcium chloride is measured and the syringe containing the alginate
solution and amylase is prepared. The alginate solution which contain the amylase is
dropped from about 20cm height into the calcium chloride. Calcium chloride (CaCl 2) is
the ionic compound of calcium and chlorine. It is a salt that behaves as a typical ionic
halide, being solid at room temperature and highly soluble in water. It is used to harden
the beads form which is the entrapment of enzyme. After that, the beads is left for 1 hour
so that it can sink to the bottom of the measuring cylinder. The beads is filtered with
distilled water, dried and put inside the fridge, the filtered CaCl 2 also collected and put
inside the fridge. This is to determine the amount of enzyme that may leaching into the
calcium chloride solution. The first part of the experiment is done.
For the next part, 0.5g of beads is put into a test tube. Then, 0.25ml of starch solution and
5ml of phosphate buffer solution are added together into the test tube containing the
beads. This is to ensure that the enzyme can survive even though there is small change if
pH. The mixture is put inside the water bath using the sonicator for about 10 minutes.
During this time, the -amylase will start to work by hydrolyze the starch into maltose.
After 10 minutes, 2.5ml of HCl is added as stopping solution to stop the enzyme from
continue hydrolyze the starch. The -amylases are calcium metalloenzymes, completely
unable to function in the absence of calcium. By acting at random locations along the
starch chain, -amylase breaks down long-chain carbohydrates, ultimately yielding
maltotriose and maltose from amylose, or maltose, glucose and "limit dextrin" from
amylopectin. It has optimum pH which is range from 6.70-7.0. Therefore, adding HCl
will change the pH and altered the bonds between the enzyme and finally the enzyme
denatured.
Next, 5ml of DNSA reagent is added and mix well. 3,5-Dinitrosalicylic acid (DNS or
DNSA, IUPAC name 2-hydroxy-3,5-dinitrobenzoic acid) is an aromatic compound that
reacts with reducing sugars and other reducing molecules to form 3-amino-5nitrosalicylic acid, which absorbs light strongly at 540 nm. It is used to detect the
formation of reducing sugar such as glucose, maltose etc. except sucrose. This is because
sucrose is not a reducing sugar. The test tube is then boiled for 15 minutes in water bath.
This is to kill all the enzyme in the test tube since the amylase cannot survive after 40C.

At high temperature the enzyme denature as the 3D structure of the enzyme altered. After
boiling, the sample is collected and analyzed using the spectrophotometer at 540nm
wavelength. 3 readings of absorbance value are taken so that the average reading can be
determined.
A control is prepared as the steps above without adding the starch. The filtered CaCl 2 act
as the beads and the steps follow the steps of the original beads. This is to determine how
much the enzyme leaching into the CaCl2 solution. Based on the result obtained, when
using the beads, the average absorbance value is 0.5673. By using the absorbance value,
the maltose concentration can be determined using the maltose standard curve equation
which is y = 1.0594x 0.0135 where y is the absorbance value, x is the maltose
concentration. The maltose concentration that found is 0.5482mol/ml. The enzyme
activity is determined using maltose amount x reaction time. The molecular weight of the
maltose is 342.3g/mol and the total volume that added is 7.75ml. The maltose
concentration is times with the molecular weight then times with volume and finally
times 1/10 minutes. The enzyme activity that calculated is 145.43 g/min.
For the filtered CaCl2, the average absorbance value is 0.3758. The maltose concentration
is 0.3675mol/ml . The enzyme activity is calculated which is 97.49mol/ml. Based on
the calculation, we found that the enzyme activity in the beads is higher than the filtered
CaCl2 which means that more enzyme is entrap in the gel. Lower enzyme activity in
CaCl2 means that less amount of enzyme leaching into it which is a good experiment
result. For the DNSA, we cannot see the colour change because it can only see at UV- vis
spectrophotometer with 540nm wavelength. With the presence of reducing sugar, it will
change colour from yellow to orange or red.
In this experiment, there is some precaution steps that should follow. During dropping the
sodium alginate gel with amylase into the calcium chloride, make sure that it is drop by
drop so that we can get beads without any error occurs. The syringe cannot push too hard
or else it will cause large amount of enzyme flow into the calcium chloride and form a
string not beads. We need to cover the test tube with aluminium foil after adding the
DNSA coloring reagent to prevent it from exposing to light which will contaminate the
DNSA reagent. When doing the boiling process, make sure wear gloves to prevent the hot
water spilled out burn the hand.

Conclusion
Based on the experiment carried out, the objectives of this experiment is
achieved which are to understand the principles of enzyme immobilization
and study the immobilized enzyme activity by using gel entrapment method.
We have used sodium alginate salt as alginic acid and -amylase as enzyme.

The -amylase is mix with sodium alginate salt and form a polymer solution.
The solution is then dropped into calcium chloride to form beads. After 1
days. the beads is mix with starch solution and sodium phosphate buffer. The
mixture is put inside water bath and then follow the procedures. The sample
is analyzed and the average absorbance value is taken. The filtered CaCl 2 is
carried out using the same steps. For the beads, the enzyme activity that
gained is 145.43g/min while for the CaCl 2 the enzyme activity is
97.49mol/min. This means that more enzyme is entrapped in the beads than
leaching to the CaCl2 which is good. This means that we successfully
achieved the objectives which is the enzyme entrapment. There are some
precautions steps that need to follow which are wear gloves when putting the
sample in the water bath, wrap the test tube with aluminium foil when
DNSA is put into the sample. In conclusion, the experiment is successfully
carried out.

References
1. https://en.wikipedia.org/wiki/Alginic_acid, retrieved on 6/12/2015.
2. http://www.eng.umd.edu/~nsw/ench485/lab7b.htm,
retrieved
on
6/12/2015.
3. https://en.wikipedia.org/wiki/3,5-Dinitrosalicylic_acid, retrieved on
6/12/2015.
4. https://en.wikipedia.org/wiki/Amylase, retrieved on 6/12/2015.