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CPB30103- Biochemical Engineering

E x p e r i m e n t 3 : I m m o b i l i z a t i o n o f E n z y m e s b y G e l E n t r a p m e n t P a g e | ii

TABLE OF CONTENTS
No.
1.
2.
3.
4.
5.
6.
7.

1.0

Contents
Abstract
Objectives
Results and Discussions
Conclusion and recommendations
Tutorials
References
Appendices

ABSTRACT

Page Number
1
1
2-8
9
10-11
11
12-14

CPB30103- Biochemical Engineering


E x p e r i m e n t 3 : I m m o b i l i z a t i o n o f E n z y m e s b y G e l E n t r a p m e n t P a g e | ii

Basically entrapment is one of the method in the immobilization of enzymes. In this


experiment, the objectives basically to study the immobilization which is entrapment itself and
also the characteristic of enzyme immobilization by gel entrapment. In order to start this
experiment, different solution of sodium alginate were prepared which are 3%, 6%, and also 9%.
Then 10 ml of those solutions were mixed with 1 g of enzymes separately. Next, 100 ml of 0.2M
CaCl2 were prepared for each mixture. The mixture then was dropped into the CaCl2 solution by
using the syringe in order for the beads to form. For each mixture the result obtained were
compared by its shape of alginate beads, color of the alginate beads, the average size of 10
alginate beads (cm), and lastly the average weight (g) for 10 alginate beads formed . For 3%
mixture, it is sphere, transparent, has size about 0.38 cm, and average weight around 0.041g. For
6% it has tadpole-shape, the color is bright, having size around 0.57 cm and its average weight
is 0.058 g. Lastly for the 9% mixture, it also has tadpole-shape, the color it milky (yellowish),
has the total size around 0.75 cm, and has the average weight 0.073 g. As the conclusion it said
that the average of total 10 alginate beads formed for the size and weight, it is directly
proportional to the concentration of the sodium alginate.

2.0

OBJECTIVES

Main focus of this experiment is in order to study:


The immobilization techniques
The characteristic of enzyme immobilization by gel entrapment

CPB30103- Biochemical Engineering


E x p e r i m e n t 3 : I m m o b i l i z a t i o n o f E n z y m e s b y G e l E n t r a p m e n t P a g e | ii

3.0

RESULTS AND DISCUSSIONS


Table 1: Three different concentrations of sodium alginate solution (wt. %) with their characteristics of alginate beads formed

CHARACTERISTICS OF
ALGINATE BEADS
FORMED

Concentrations of sodium alginate solution (wt. %)


3%

6%

9%

Observations

Shape of alginate beads

Sphere

Tadpole-shape

Tadpole-shape

Color of alginate beads

Clear transparent (colourless)

Bright white

Milky (little yellowish)

3.8

5.7

7.5

Total size of 10 alginate


beads (cm)
(from Table 2)
Average size of alginate
beads (cm)
(from Table 2)

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0.38

0.57
0.75

Total weight of 10 alginate


beads formed (g)
(from Table 2)

0.41

0.58

0.73

Average alginate beads (10)


weight formed (g)
(from Table 2)

0.041

0.058

0.073

Table 2: The average of size of alginate beads (cm) and the average alginate beads (10) weight formed (g) for three different
concentrations of sodium alginate solution (wt. %)
No.
3%

Concentrations of sodium alginate solution (wt. %)


6%

9%

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E x p e r i m e n t 3 : I m m o b i l i z a t i o n o f E n z y m e s b y G e l E n t r a p m e n t P a g e | ii

Size of alginate
beads (cm)

Weight of
alginate beads

1.

0.4

(g)
0.04

2.

0.3

3.

Size of alginate
beads (cm)

Weight of
alginate beads

0.6

(g)
0.06

0.04

0.6

0.4

0.03

4.

0.5

5.

Size of alginate
beads (cm)

Weight of
alginate beads

0.7

(g)
0.06

0.06

0.8

0.07

0.6

0.06

0.7

0.07

0.05

0.5

0.05

0.7

0.06

0.4

0.05

0.5

0.05

0.7

0.07

6.

0.3

0.04

0.6

0.06

0.7

0.06

7.

0.4

0.04

0.5

0.05

0.9

0.10

8.

0.4

0.04

0.5

0.05

0.7

0.07

9.

0.4

0.04

0.6

0.07

0.7

0.07

10.

0.3

0.04

0.7

0.07

0.9

0.10

TOTAL

3.8

0.41

5.7

0.58

7.5

0.73

AVERAGE
0.38
0.041
(see full samples of calculations in Appendices)

0.57

0.058

0.75

0.073

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Average size of alginate beads (cm) against concentrations of sodium alginate (wt. %)
1
0.9
0.8
0.7

f(x) = 0.06x + 0.2

0.6
Average size of alginate beads (cm)

Average size of alginate


beads (cm)

0.5

Linear (Average size of


alginate beads (cm))

0.4
0.3
0.2
0.1
0

Concentration of sodium alginate (wt. %)

Figure 1: Average size of alginate beads (cm) against concentrations of sodium alginate (wt. %)
Total size of 10 alginate beads (cm) against concentration of sodium alginate (wt. %)
10
9
8
7
6
Total size of 10 alginate beads (cm)

5
4
3

f(x) = 0.62x + 1.97


Total size of 10 alginate
beads (cm)
Linear (Total size of 10
alginate beads (cm))

2
1
0
Concentration of sodium alginate (wt. %)

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Figure 2: Total size of 10 alginate beads (cm) against concentrations of sodium alginate (wt. %)
Figure 1 shows the average size of alginate beads (cm) against concentration of sodium
alginate (wt. %) and Figure 2 shows the total size of 10 alginate beads (cm) against
concentrations of sodium alginate (wt. %) where the increase the concentrations of sodium
alginate (wt. %) the increase the size of alginate beads (cm) where 9 % concentration of sodium
alginate solution has the biggest size of alginate beads (cm) compared to 3 % and 6 %
concentration of sodium alginate solution.
Immobilized enzymes also experienced a mass transfer limitations. The size of beads is
the parameters of immobilization of enzyme. The smaller the beads the higher catalytic activity
due to reduced substrate transfer resistance. Thus, the smallest beads has the highest catalytic
activity and the biggest beads has the lowest catalytic activity. As expected, specific activities of
enzyme entrapped in the beads decreased as the bead size increased. The activity of immobilized
enzyme also decreases with increasing bead size due to mass transfer resistance. As the bead size
decreased, immobilization increased, but loading efficiency remained constant.

Total weight of 10 alginate beads forme d (g) against conce ntration of sodium alginate (wt. %)

Total weight of 10 alginate beads formed (g)

Concentration of sodium alginate (wt. %)

Total weight of 10 alginate


beads formed (g)
Linear (Total weight of 10
alginate beads formed (g))

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Figure 3: Total weight of 10 alginate beads formed (g) against concentrations of sodium alginate
(wt. %)
Figure 3 shows the total weight of 10 alginate beads formed (g) against concentration of
sodium alginate (wt. %) where the increase the concentrations of sodium alginate (wt. %), the
increase the total weight of alginate beads (g) where 9 % concentration of sodium alginate
solution has the highest amount of total weight of alginate beads (cm) compared to 3 % and 6 %
concentration of sodium alginate solution.
The higher the concentration of sodium alginate, the heavier the total weight of 10 beads
formed. 10 ml of 3% of sodium alginate reacts with 1ml enzyme and produce 0.4 g of total
weight of the beads. Meanwhile 10 ml of 9% of sodium alginate, produced 0. 73 g which was
the heaviest total weight of the beads. This is due to the unreacted or excess substrate because all
substrate had occupied all the active site.

Ave rage alginate be ads (10) we ight formed (g) against conce ntration of sodium alginate solution (wt. %)

Average alginate beads


(10) weight formed (g)
Average alginate beads of 10 weight formed (g)

Concentration of sodium alginate (wt. %)

Linear (Average alginate


beads (10) weight formed
(g))

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Figure 4: Average alginate beads (10) weight formed (g) against concentration of sodium alginate
solution (wt. %)
Figure 4 shows the average of alginate beads (10) weight formed (g) against
concentration of sodium alginate (wt. %) where the increase the concentrations of sodium
alginate (wt. %), the increase the average of alginate beads (10) weight formed (g) where 9 %
concentration of sodium alginate solution has the highest amount of average alginate beads of 10
weight formed (g) compared to 3 % and 6 % concentration of sodium alginate solution.
Immobilization of enzymes are enzymes that were attached to an insoluble support
medium or enclosed by the support medium which is also known as a carrier. Immobilization
was the preferred process as it was economic friendly as it use a continuous use of bio-catalyst
and on the bright side the separation of bio-catalyst and product was so much easier than
conventional batch process. Immobilized enzymes typically have greater thermal and operational
stability than the soluble form of the enzyme. In this experiment, the method of immobilization
that was used was called the gel entrapment. In entrapping method, the enzymes was trapped into
the lattice of a semi permeable polymer membrane.
The effects of enzyme entrapment efficiency and immobilization yield were determined.
An increase in alginate concentration can increased the efficiency, but show a decreased in
immobilization yield. As the bead size decreased, immobilization increased. Among the tested
parameters, alginate concentration and bead size were important for enzyme entrapment in
sodium alginate beads. In order to prevent enzyme leakage, the gel beads were coated with
chitosan and silicate. The surface coating provided the alginate beads with higher operational
stability compared to non-coated beads.
Besides the leakage of enzymes, another problem associated with the entrapment method
of immobilization is the mass transfer resistance to substrates, products, and inhibitors. Because
the size with high concentration of sodium alginate cannot diffuse deep into the gel matrix, as in
any other conventional non-biological immobilized catalysts. At the same time, the diffusional
resistance encountered by the product molecules can sometimes cause the product to accumulate

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near the center of the gel to an undesirable high level, leading to product inhibition for some
enzymes.

4.0

CONCLUSION AND RECOMMENDATIONS


Based on the objectives of this experiment, immobilization of enzyme has been carried

out by using gel entrapment method. As the concentration of sodium alginate solution is higher,
the weight of beads obtained is also higher. Means, the size of beads form becomes larger
according to high concentration of sodium alginate solution. Sodium alginate solution at low
concentration has regular spherical in shape compare to the high concentration. The colour
appearance is also differ to each samples where at high concentration of sodium alginate
solution, the colour turn to be yellowish compare to lower concentration of sodium alginate
solution that has the clearest colour. So this indicates that large size beads will save the additional
cost required for small size beads to accommodate the same amount of enzyme in terms of
bioreactor size. It is also difficult to work with at high concentration as the gels can withstand
breakage or else the gels are fragile and the enzyme was loosely bound to the gel at low
concentration.
While conducting the experiment, there are several possible errors that may occur and
here some precaution may be recommended. Firstly, make sure all apparatus are well cleaning
before using it. The contamination may occur in the solution that resulting inaccurate of reaction.
Next, when sucking up the sodium alginate solution into the syringe the plunger must be pulled
up slowly to prevent the air bubbles form in the syringe. Lastly, the beaker containing 0.2M
CaCl2 solution must be swirled slowly when dripping out the sodium alginate solution into it, so
that the beads formed did not stick together.

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5.0

TUTORIAL
1. List of the advantages and disadvantages of the immobilized enzymatic conversion
process versus a free one in the experiment.
Advantages

Disadvantages

Immobilized enzymatic conversion process the

Immobilization required additional time, more

enzyme are not present in the products, so no

equipment and material

need of purification
Immobilized enzymatic conversion process the

These enzymes are less active has they dont

enzyme is immediately available for reuse. So

mix freely with substrate

a continuous process can be carried out


Immobilized enzymes are more stable than the Any contamination will affect all the whole
free one. It is also biodegradable.

system.

2. Suggestion on other materials which can be used to replace sodium alginate for enzyme
immobilization by gel entrapment.
Sodium alginate is the sodium salt of the compound alginic acid. It is a natural gum and
is used to thickening various compounds. It is also used in various chemical processes to bind
together other compounds without reacting to them. Natural and synthetic polymers such as
agar and gelatin can also be used for enzyme entrapment.
Gelatin consists of proteins and peptides produced by the denaturation of collagen, which
breaks down into smaller fragments. Gelatin immobilization methods have been developed
for entrapment of microbial cells and enzymes, especially when enzyme is placed in a whole
cell. Apart from that, agar was also introduced for immobilization of bacterial cells and

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enzymes in the form of crosslinked gels. It is used to solidify the culture medium to provide a
solid surface for bacterial growth. The structural function of agar in bacteriological media is
similar to the function that gelatine has in Jell-O. Agar is commonly used rather than gelatine
because agar is not broken down by most bacteria.

6.0

REFERENCES
1. Optimization Of Lipase Entrapment In Ca-alginate Gel Beads. Keehoon Wona , Sangbum
Kima , Kwang-Je Kima , Hong Woo Parkb , Sang-Jin Moon. Department of Chemical
Engineering, Hanyang University, 17 Haengdang-Dong, Seongdong-Gu, Seoul 133-791,
South Korea. 16 August 2004.
2. Immobilization of Invertase Within Calcium Alginate Gel Capsules by Aziz Tanriseven ,
Senay Dogan from Department of Biochemistry. Gebze Institute of Technology, Gebze,
Kocaeli, Turkey 8 February 2001.
3. Experiment No. 7 : Enzyme Immobilization Protocol Entrapment In Alginate Gel.
Prepared by Nam Sun Wang from Department of Chemical & Biomolecular Engineering.
University of Maryland College Park.
https://terpconnect.umd.edu/~nsw/ench485/lab7b.htm

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6.0

APPENDICES
Table 2: The average of size of alginate beads (cm) and the average alginate beads (10) weight formed (g) for three different
concentrations of sodium alginate solution (wt. %)

No.

1.

Concentrations of sodium alginate solution (wt. %)


3%
6%
9%
Weight of
Weight of
Weight of
Size of alginate
Size of alginate
Size of alginate
alginate beads
alginate beads
alginate beads
beads (cm)
beads (cm)
beads (cm)
(g)
(g)
(g)
0.4
0.04
0.6
0.06
0.7
0.06

2.

0.3

0.04

0.6

0.06

0.8

0.07

3.

0.4

0.03

0.6

0.06

0.7

0.07

4.

0.5

0.05

0.5

0.05

0.7

0.06

5.

0.4

0.05

0.5

0.05

0.7

0.07

6.

0.3

0.04

0.6

0.06

0.7

0.06

7.

0.4

0.04

0.5

0.05

0.9

0.10

8.

0.4

0.04

0.5

0.05

0.7

0.07

9.

0.4

0.04

0.6

0.07

0.7

0.07

10.

0.3

0.04

0.7

0.07

0.9

0.10

TOTAL

3.8

0.41

5.7

0.58

7.5

0.73

AVERAGE

0.38

0.041

0.57

0.058

0.75

0.073

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The concentration of sodium alginate solution (wt. %):


1. 3 %
i.

Average size of alginate beads (cm) =

Total alginate beads (cm)


No . ofalginatebeads

=
0.4+0.3+ 0.4+0.5+0.4 +0.3+0.4 +0.4+ 0.4+0.3
10
=

ii.

3.8
10

= 0.38 cm
Total weight of alginate beads ( g )
Average weight of alginate beads (g) =
No . of alginate beads
=
0.04+0.04+ 0.03+0.05+0.05+ 0.04+0.04 +0.04+ 0.04+0.04
10

0.41
10

= 0.041 g
2. 6 %
i.

Average size of alginate beads (cm) =

Total alginate beads (cm)


No . ofalginatebeads

=
0.6+0.6+0.6 +0.5+0.5+0.6 +0.5+0.5+ 0.6+0.7
10
=

35.78
10

= 0.57 cm

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ii.

Average weight of alginate beads (g) =

Total weight of alginate beads ( g )


No . of alginate beads

=
0.06+0.06+0.06+ 0.05+0.05+0.06+ 0.05+0.05+0.07+ 0.07
10

0.58
10

= 0.058 g

3. 9 %
i.

Average size of alginate beads (cm) =

Total alginate beads (cm)


No . ofalginatebeads

=
0.7+0.8+0.7+ 0.7+0.7+ 0.7+0.9+ 0.7+0.7+ 0.9
10
=

7.5
10

= 0.75 cm
ii.

Average weight of alginate beads (g) =

Total weight of alginate beads ( g )


No . of alginate beads

=
0.06+0.07+0.07 +0.06+0.07+ 0.06+0.10+0.07 +0.07+0.10
10

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0.73
10

= 0.073 g