Experiment: 5 Immobilization of α-amylase enzyme by gel entrapment

Experiment: 5
Immobilization of α-amylase enzyme by gel entrapment

Aim:
To learn the technique of immobilizing enzyme in alginate beads and determine its
enzymatic activity by assay.
Introduction:
Immobilization is a technique for the combination of enzyme in an insoluble support
matrix. The matrix is usually a high molecular weight polymer such as polyacrylamide,
starch, cellulose, etc. The advantage of immobilizing enzymes over free enzyme is to
increase their stability and efficiency. The immobilized enzymes can also be recovered at
the end of the reaction and can be used repeatedly.
Principle:
An enzyme is usually immobilized onto an inert, insoluble material e.g. calcium alginate.
This is produced by the reaction of a mixture of sodium alginate solution with calcium
chloride. These beads provide increased resistance to changes in conditions such as pH
or temperature. They also allow enzymes to be held in place throughout a reaction,
following which they are easily separated from the products and may be used again. The
enzyme can be easily immobilized with a number of methods: entrapment, ion exchange
adsorption, porous ceramics, and even covalent bonding. There are several methods for
immobilization of enzymes.
Adsorption: It is a method which involves electrostatic interaction such as Van der waals
forces, ionic and hydrogen bonding between the enzymes and the support matrix.
Figure 1: Different immobilization techniques.

Covalent Binding: This method involves formation of covalent bonds between the
enzymes sand the support matrix. The bond is normally formed between the functional
groups present on the surface of the support and functional groups belonging to amino
acid residues on the surface of the enzyme.
Entrapment: In this method the enzyme molecules are mixed with a polyionic polymer
material and then crosslinking of the polymer with multivalent cations in an ion exchange
reaction to form a lattice structure that traps the enzymes.
Encapsulation: This can be achieved by enveloping the enzymes within various forms
of semi permeable membranes.
Crosslinking: This involves joining of enzymes with each other to form large three
dimensional complex structures and can be achieved by physical or chemical methods
without any support system.
External and internal mass transfer limitation:

Mass transfer is one of the major factors affecting activity of immobilized enzyme since
reactant and products have to be transported through external boundary layers (external
mass transfer or film mass transfer) and diffusion through the porous matrix (internal
mass transfer). Thus, the effectiveness of immobilized enzyme systems can be reduced
by mass transfer limitations. With proper design of an immobilized reactor, external mass
transfer resistance can be made negligible, for example, by increasing the velocity of the
bulk solution relative to the gel particle. However, internal mass transfer resistance, which
is affected by system design parameters such as particle size, enzyme loading, bulk
movement, reactant concentration and reactor type, generally is not negligible. Diffusion
through the immobilization matrix (Deff) cannot be characterized by the bulk diffusivity
(Daq) because the pore space is only a fraction of the total volume (exclusion effect) and
matrix increase the path length for a diffusing substrate (obstruction effect). Thus,
information about effective diffusivity is essential for describing the kinetics of
immobilized enzyme reactions.
internal mass transfer resistance
enzyme Bulk phase
Substrate: cellulose
Porous
matrix Product: glucose
Film:
external mass transfer resistance
Figure 2: Mass transfer through immobilized enzyme

Some parameters could be calculated in order to quantify the contribution of mass
transfer by diffusion into support particles on the overall transformation process rate.
Diffusional limitations can be quantitatively expressed by the effectiveness factor, η,
defined as the ratio between the average reaction rate and the rate that would be
obtained if all enzyme molecules inside the particles were exposed to the same substrate
concentration as the bulk liquid, that is, in the absence of diffusional effects.
reaction rate with intraparticle diffusion limitation

𝜂=
reaction rate with out diffusion limitation
observed reaction rate with immobilized enzyme

=
reaction rate with free enzyme
The effectiveness factor is a dimensionless parameter that measures how effectively the
catalyst is being used. For near unity, the entire volume of the particle is reacting at the
same high rate because the reactant is able to diffuse quickly through the support
material. For near zero, the reaction is conducted at the lowest rate. The reactant is
unable to penetrate significantly and the reaction rate is limited in a small portion of the
particle volume. The diffusional resistance is predominant which lowers the overall
reaction rate.
List of Reagents and Instruments:

Equipment: Flasks, Spectrophotometer, Sample tubes, Micropipette, beaker,
shaker/incubator (50 οC)
Reagents/chemical/enzyme:
CaCl2 (3%), Na-alginate, alpha amylase (Sigma), starch (stock: 20 g/L), glucose
oxidase kit (GOD-POD kit), Acetate Buffer (pH 6) using 0.1 (M) acetic Acid and 0.1 (M)
sodium acetate.
Procedure:
Day-1
Task1: Immobilization of enzyme
1. Prepare 10 ml of 1.5 mg/ml alpha amylase solution
2. Prepare 20 ml of 2% sodium alginate solution
3. In a 50 ml centrifuge tube take 5 ml of the enzyme solution and to it add 20 ml
of the 2% sodium alginate solution. Put the cap tightly. Keep remaining 5 ml
enzyme solution for free enzyme experiments.
4. Mix the enzyme solution with sodium alginate solution properly.
5. Take 50 ml of Calcium chloride solution (3%) in a conical flask/beaker.
6. With a 1 ml pipette, take the alginate and enzyme solution mix and add drop
wise to the calcium chloride solution. While adding make sure that the flask is
swirled gently.
7. Leave the immobilized enzyme beads to harden in the calcium chloride solution
for overnight at 4 οC.
8. The alginate will be ionically cross-linked by the calcium ions.
Day-2
Task 1: Ca-alginate bead washing/preparation for enzyme activity
experiments
1. Isolate the beads after discarding the solution.
2. Wash with acetate buffer (pH 6)
3. Dry the beads at room temperature. Use tissue paper for that.
4. Measure the diameter of the beads.
5. Take the picture of the beads (If possible).
6. Take the weight of the alginate beads and divided into five equal portions for five
different substrate concentration experiments.
Task 2: Determination of enzymatic activity of immobilized enzyme and free

enzyme by GOD-POD Assay:
1. Prepare 20 ml of starch solution in 50 ml flask of different concentrations (0.1,
0.5, 1.0, 2.0, 5.0 g/L) from stock starch solution (20 g/L in water) in acetate
buffer (pH: 6)
2. Add alginate beads in each flask and incubate at 50 οC with shaking at 200 rpm.
3. Take 10 uL sample each time point (0, 10, 30, 60 min) and put 1 ml GOD-POD
buffer. Keep it either 15 min in 37 οC or 20 min in 30 οC for glucose measurement
using spectrophotometer. [Check GOD-POD kit manual].
Simultaneously do the same experiments (five experiments, 0-5 g/l starch, 20 ml each)
with free enzyme. Make sure equal amount of enzyme added in each flask as does for
immobilized enzyme. For that add 1 ml enzyme solution in each flask. (check total 5 ml
of enzyme has been added during immobilization).
Tasks:
1. Plot glucose released Vs t for different concentration of starch for both free
and immobilized enzyme.
2. Plot reaction rate (glucose released/ time/mg of enzyme) Vs t for different
concentration of starch for both free and immobilized enzyme
3. Plot initial reaction rate (r) Vs initial starch concentration (S0). Take 0-10 min
rate as initial rate.
4. Plot 1/r Vs 1/S0.
5. Determine Km and rmax for free and immobilized enzyme based on initial rate
S
data using Michaelis-Menten model: r = r max K +S , (use plot 4)
m
6. Determine Effectiveness factor for immobilized enzyme. Plot effectiveness
factor with time for different concentrations of starch. Specify the size of
beads.
Table-1 (OD data)
Time Glucose concentration (OD) for Glucose concentration (OD) for

(min) immobilized enzyme system free enzyme system
0.1g/l 0.5 g/l 1 g/l 2 g/l 5 g/l 0.1 g/l 0.5 g/l 1 g/l 2 g/l 5 g/l
starch starch starch starch starch starch starch starch starch starch
0
10
30
60
Table-2 (glucose concentration data)

Time Glucose concentration for Glucose concentration for
(min) immobilized enzyme system free enzyme system
0.1g/l 0.5 g/l 1 g/l 2 g/l 5 g/l 0.1 g/l 0.5 g/l 1 g/l 2 g/l 5 g/l
starch starch starch starch starch starch starch starch starch starch
0
10
30
60

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Experiment: 5

Immobilization of α-amylase enzyme by gel entrapment

Figure 1: Different immobilization techniques.

External and internal mass transfer limitation:

Figure 2: Mass transfer through immobilized enzyme

reaction rate with intraparticle diffusion limitation

observed reaction rate with immobilized enzyme

List of Reagents and Instruments:

Task 2: Determination of enzymatic activity of immobilized enzyme and free

Table-1 (OD data)

Time Glucose concentration (OD) for Glucose concentration (OD) for

Table-2 (glucose concentration data)

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