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Experiment No: Immobilization of Enzyme Using Sodium Alginate

This experiment aims to immobilize an enzyme using sodium alginate. Sodium alginate forms a gel when calcium ions are added, trapping the enzyme within. This is done by dripping a mixture of enzyme and sodium alginate into a calcium chloride solution, forming beads 0.5-2 mm in size. The activity of the immobilized enzyme is then tested using a starch solution and measuring absorbance with DNSA reagent to quantify glucose production.

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Vijayasarathy Sampath Kumar
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473 views

Experiment No: Immobilization of Enzyme Using Sodium Alginate

This experiment aims to immobilize an enzyme using sodium alginate. Sodium alginate forms a gel when calcium ions are added, trapping the enzyme within. This is done by dripping a mixture of enzyme and sodium alginate into a calcium chloride solution, forming beads 0.5-2 mm in size. The activity of the immobilized enzyme is then tested using a starch solution and measuring absorbance with DNSA reagent to quantify glucose production.

Uploaded by

Vijayasarathy Sampath Kumar
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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Experiment No:

IMMOBILIZATION OF ENZYME USING SODIUM ALGINATE

Aim

To immobilize the given enzyme using Sodium Alginate

Principle

Alginate is a linear polysaccharide normally isolated from many strains of marine


brown seaweed and algae. It is commercially available as sodium 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. The permeability depends on the concentration of the
alginate used.

2 Na(Alginate) + Ca++ -------> 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.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 film-forming. 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-M-G-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.

Procedure:

1. Prepare 3% solution of Sodium Alginate.


2. Mix approximately 0.015 g of enzyme with 10 ml of 3% (wt.) sodium alginate
solution.
3. 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 bead size can be controlled by pump
pressure and the needle gauge. A typical hypodermic needle produces beads of 0.5-2
mm in diameter. Other shapes can be obtained by using a mold whose wall is
permeable to calcium ions. Leave the beads in the calcium solution to cure for 0.5-3
hours.
4. Estimate the activity of amylase by using starch as substrate.
(i) Add 1 gm of alginate beads in 10 ml of 1% starch solution.
(ii) Incubate for 15 minutes
(iii) Take 1 ml of this solution and 1 ml of distilled water.
(iv) Add 1ml of DNSA
(v) Incubate in water bath for 10 mins
(vi) Then, add 0.5 ml of Rochelle salt.
(vii) Read the OD at 570 nm
5. Calculation of enzyme activity
The amylase enzyme activity =-------- Units /ml (consider Dilution factor as 1)

Glucose estimation by DNSA method (standard graph)

Test S0 S1 S2 S3 S4 S5
tubes/reagent
s

Glucose 0 200 400 600 800 1000


(µg/ml)

Glucose 0 0.4 0.8 1.2 1.6 2


solution of
1000 µg/ml in
ml

Phosphate 2 1.6 1.2 1.6 1.4 0


buffer (ml)

DNSA(ml) 1 1 1 1 1 1

Rochelle 0.5 0.5 0.5 0.5 0.5 0.5


salt(ml)
0.179 0.281 0.432 0.567 0.681
OD at 570nm 0

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