Prepared by Leong Chean Ring CPB 30103 Biochemical Engineering

Experiment 3: IMMOBILIZATION OF ENZYMES BY GEL ENTRAPMENT

Overview

Immobilization

Enzymes are biological catalysts that can perform various chemical conversions and have tremendous
industrial applications. Invariably, a major cost factor in the industrial application of enzymes is the
production of the enzymes. The enzyme when used in soluble form, not only will it contaminate the
product but also cannot be economically recovered for subsequent use. At present, applications of
immobilized biocatalysts include:

1) The production of useful compounds by stereospecific or regiospecific bioconversion

2) The production of energy by biological processes
3) The selective treatment of specific pollutants to solve environmental problems.
4) Continuous analysis of various compounds with a high sensitivity and a high specificity
5) Medical uses such as new types of drugs for enzyme therapy or artificial organs.

Carrier Binding
Enzyme proteins have amino acid residues containing chemically reactive groups, ionic groups, and/or
hydrophobic groups. These amino acid residues and the hydrophobic domains can participate in the
immobilization of enzymes through covalent linkage, ionic binding, or physical adsorption. Various types
of insoluble supports (carriers) are utilized after proper modification.

Covalent Binding
This method involves the formation of covalent bonds between the chemical
groups in enzyme and to the chemical groups on the support or carrier. It is
one of the widely used methods of enzyme immobilization. Hydroxyl groups
and amino groups of support or enzyme form covalent bonds more easily.
Chemical groups and amino groups of support or enzyme form covalent
bonds more easily. Chemical groups in the support are:

 Amino groups, Imino groups

 Hydroxyl groups
 Carboxyl groups
 Thiol groups and Methythiol groups
 Guanidyl groups and Imidazole groups
 Phenol rings
Ionic Binding
There is no permanent bond formation between carrier and the enzyme. Only weak
bonds stabilize the enzymes to the support or carrier. The weak bonds (low energy
bonds) involved are mainly

 Ionic interaction
 Hydrogen bonds
 Van der Waal forces

Cross-linking
This method is also called as copolymerization. In this method
of immobilization enzymes are directly linked by covalent
bonds between various groups of enzymes via polyfunctional
reagents. Unlike other methods, there is no matrix or support
involved in this method. Commonly used polyfunctional
reagents are glutaraldehyde and diazonium salt.

Entrapment
In this method enzymes are physically entrapped inside a porous
matrix. Bonds involved in stabilizing the enzyme to the matrix may
be covalent or non-covalent. The matrix used will be a water
soluble polymer. The form and nature of matrix varies with
different enzymes. Pore size of matrix is adjusted to prevent the
loss of enzyme. Pore size of the matrix can be adjusted with the
concentration of the polymer used. Agar-agar and carrageenan
have comparatively large pore size. Examples of commonly used
matrix for entrapment are:
 Polyacrylamide gels
 Cellulose triacetate
 Agar
 Gelatin
 Carregeenan
 Alginate
Encapsulation
This type of immobilization is done by enclosing the enzymes in a
membrane capsule. The capsule will be made up of semi permeable
membrane like nitro cellulose or nylon. In this method, the
effectiveness depends upon the stability of enzymes inside the
capsule.
INTRODUCTION

Alginate, commercially available as alginic acid, sodium salt, commonly called sodium alginate, is a linear
polysaccharide normally isolated from many strains of marine brown seaweed and algae, thus the name
alginate. The copolymer consists of two uronic acids: D-mannuronic acid (M) and L-guluronic acid (G).
Because it is the skeletal component of the algae it has the nice property of being strong and yet flexible.
Alginic acid can be either water soluble or insoluble depending on the type of the associated salt. The salts
of sodium, other alkali metals, and ammonia are soluble, whereas the salts of polyvalent cations, e.g.,
calcium, are water insoluble, with the exception of magnesium. The alginate polymer itself is anionic (i.e.,
negatively charged) overall. Polyvalent cations bind to the polymer whenever there are two neighboring
guluronic acid residues. Thus, polyvalent cations are responsible for the cross-linking of both different
polymer molecules and different parts of the same polymer chain. The process of gelation, simply the
exchange of calcium ions for sodium ions, is carried out under relatively mild conditions. Because the
method is based on the availability of guluronic acid residues, which will not vary once given a batch of
the alginate, the molecular permeability does not depend on the immobilization conditions. Rather, the
pore size is controlled by the choice of the starting material.

2 Na(Alginate) + Ca2+ -------> Ca(Alginate)2 + 2 Na+

The ionically linked gel structure is thermostable over the range of 0-100oC; therefore heating will not
liquefy the gel. However, the gel can be easily redissolved by immersing the alginate gel in a solution
containing a high concentration of sodium, potassium, or magnesium. Maintaining sodium:calcium <=
25:1 will help avoid gel destabilization. In fact, it is recommended by alginate vendors to include 3mM
calcium ions in the substrate medium. On the other hand, citrate or phosphate pH buffers cannot be
effectively used without destabilizing the alginate gel. Alginate is currently widely used in food,
pharmaceutical, textile, and paper products. The properties of alginate utilized in these products are
thickening, stabilizing, gel-forming, and filmforming. Alginate polymers isolated from different alginate
sources vary in properties. Different algae, or for that matter different part of the same algae, yield
alginate of different monomer composition and arrangement. There may be sections of homopolymeric
blocks of only one type of monomer (-M-M-M-) (-G-G-G-), or there may be sections of alternating
monomers (-M-G-MG-M-). Different types of alginate are selected for each application on the basis of the
molecular weight and the relative composition of mannuronic and guluronic acids. For example, the
thickening function (viscosity property) depends mainly on the molecular weight of the polymer;
whereas, gelation (affinity for cation) is closely related to the guluronic acid content. Thus, high
guluronic acid content results in a stronger gel.

OBJECTIVES
● To study the immobilization techniques.
● To study the characteristic of enzyme immobilization by gel entrapment.
LABORATORY PROCEDURE
A. Equipment
1. Erlenmeyer flasks
2. Pipettes and pipette bulbs
3. Volumetric flask
4. Beakers
5. Graduated cylinder
6. Test tubes
7. Funnel
8. Spatula
9. Stirring hot plate and stir bars
10. Weighting paper
11. Temperature bath
12. Syringe
B. Reagents
1. CaCl2
2. Sodium alginate
3. Amylase enzyme (commercial)
4. Starch solution
5. Distilled water

Procedure

Preparation of Immobilized Enzyme Beads

a) Prepare 3%, 6%, and 9% solution of sodium alginate. Dissolve 30g of sodium alginate in 1 liter to make
a 3% solution, and etc. See Appendix 1.
b) Mix approximately 0.015 g of enzyme with 10 ml of 3% (wt.) sodium alginate solution. Repeat step (b)
for 6% and 9% solution of sodium alginate.
c) Prepare three 250 mL beakers and into each beaker put in 100 mL of 0.2M CaCl2.
d) The beads are formed by dripping the polymer solution from a height of approximately 20 cm into an
excess (100 ml) of stirred 0.2M CaCl2 solution with a syringe and a needle at room temperature. The
beaker must be swirled slowly to prevent the beads of alginate-enzyme mixture coalescing upon
solidifying in the CaCl2 solution. Leave the beads in the calcium solution to cure for 0.5-3 hours.
e) The beakers will now contain amylase enzyme trapped in 3%, 6%, and 9% alginate beads respectively.
f) Filter the contents of all beakers and check the weight of every 10 beads formed.

Appendix 1
a) Sodium alginate solution is best prepared by adding the powder to agitated water, rather than vice
versa, to avoid the formation of clumps. Prolonged stirring may be necessary to achieve the complete
dissolution of sodium alginate. After sodium alginate is completely dissolved, leave the solution
undisturbed for 30 minutes to eliminate the air bubbles that can later be entrapped and cause the beads
to float.
b) Although not necessary, the beads may be hardened by mixing some amines in the sodium alginate
solution and cross-linking with glutaraldehyde.
Results/Discussion
☼ Discuss the predicted/expected results for the investigation of the effect of alginate concentrations on
physical characterization (total bead weights formed, average beads (10) weight formed, size, shape, color,
and etc). Did you obtain predicted/expected results? If not, what are the predicted/expected results and
can you make any reasonable suggestions why you did not get them?
☼ Discuss your observations in the preparation of immobilized enzymes.
☼ Plot appropriate curves to relate your observations.

Tutorial
☼ List the advantages and disadvantages of the immobilized enzymatic conversion process versus a free
one in the experiment.
☼ Give suggestions on other materials which can be used to replace sodium alginate for enzyme
immobilization by gel entrapment.