Enzyme Inovilitation.
Reference Document name
Fathi Mobasher, E. (2009).
Production and
Immobilization of alpha
amylase using biotechnology
techniques for use in
biological and medical
applications. B.Sc.
(Chemistry/Biochemistry).
Zagazig University.
Methodology images and graphics
Production and - Dialysis: The dissolved precipitated pellets
Immobilization of alpha obtained was introduced inside a dialysis
amylase using membrane to be dialyzed against the same
biotechnology techniques buffer for 24 hr at 4°C
for use in biological and
medical applications -Immobilization of alpha enzyme:
α-Amylase was purified from A. niger
Preparation of immobilized enzyme on
chitosan and sodium alginate (Natural
polymer): 0.5 g chitosan was dissolved in 50 ml
glacial acetic acid (1%), 0.5 g sodium alginate
was dissolved in 50 ml distilled H2O at room
temperature, then chitosan solution was added to
sodium alginate solution and blended with
magnetic stirrer for 6 hr. 6 ml of α-amylase
solution (contain 3 mg α-amylase) was added to
this solution and magnetically stirred for 1 hr.
Then the homogeny mixture was dropped into
0.27 M CaCl2 to form immobilized enzyme
beads.

Optimization of
immobilization of α-amylase
in alginate gel and its
comparative biochemical
studies with free α-amylase.
(2012). Recent Research in
Science and Technology,
[online] 4(2), pp.2-3.
Available at: http://recent-
science.com/ [Accessed 15
Apr. 2018].
Flores-Maltos A, Rodrı´guez-
Dura´n LV, Renovato J,
Contreras JC, Rodrı´guez R,
Aguilar CN (2011) Catalytical
properties of free and
immobilized Aspergillus niger
tannase. Enzyme Res. doi:
10.4061/2011/768183
Optimization of The protein content was determined from the
immobilization of α- standard curve for solutions containing known
amylase in alginate gel amounts of bovine serum albumin (BSA)(Figure 5).
and its comparative 1.- A 3% (w/v) sodium alginate solution in 50mM
biochemical studies with sodium phosphate buffer (pH 7.0) was prepared
free α-amylase by warming at 50°C.
2.- After cooling down to room temperature, 1ml
of enzyme stock solution was mixed with 9ml of
sodium alginate solution (the total volume of
matrix and enzyme mixture being 10ml).
3.- The mixture was taken into a syringe, and
beads were formed by dropping the solution into
1M calcium chloride solution with gentle stirring
at 4°C for 2 h.
4.- The formed beads were recovered by filtration
and thoroughly washed with distilled water. The
beads were dried using filter paper (Whatman no.
1).
5.- The filtered calcium chloride solution was
collected for enzyme activity determination.
Enzyme immobilization: Alginate
an overview on Alginate derived from cell walls of brown algae
techniques and support are calcium, magnesium and sodium salts of
materials alginic acid and have been extensively used for
immobilization. Cross-linking of alginate with
divalent ions (like Ca2+) and glutaraldehyde
improves the stability of enzymes.
Gargi Dey; Bhupinder Singh;
Rintu Banerjee. Microbial
Biotechnology and
Downstream Processing
Laboratory; Agricultural and
Food Engineering
Department; IIT-Kharagpur;
721302; India
Immobilization of a- Materials
amylase produced by Sodium alginate, calcium chloride and soluble
Bacillus circulans GRS starch were obtained from E Merck, and
313 dinitrosalicylic acid (DNS) was purchased from
Lancaster, England. All the other chemicals used
were of analytical grade.
Enzyme immobilization
Enzyme was prepared from Bacillus circulans
GRS 313 cultivated at 40oC with an initial pH of
5.5 for 48 hrs in a medium described previously
(Dey et al., 2000). An equal volume of enzyme
solution and sodium alginate solution was mixed
to give a 4% (w/v) final concentration of sodium
alginate solution in the mixture. The mixture
obtained was extruded dropwise through a
pastuer pipette (1mm diameter) into a gently
stirred 2% (w/v) CaCl2.2H2O solution for 2 h to
give bead size of 3mm. The calcium alginate
beads containing the enzyme were thoroughly
washed with distilled water and used for further
studies. Beads of different sizes 5mm, 4mm and
2mm were made by using pastuer pipettes of
diameter 3mm, 2mm and 70µm respectively.
Determination of immobilization efficiency
Immobilization efficiency was determined from
the difference in enzyme activity in the solution
before and after the immobilization.
Immobilization yield (%) = (I/ A-B) x 100
where A = added enzyme (U/g of bead); B =
unbound enzyme (U/g of bead);
I = immobilized enzyme (U/g of bead)
Saiyavit Varavinita, Narisa
Chaokasema and Sujin
Shobsngobb a Department of
Biotechnology, Faculty of
Science, Mahidol University,
Rama VI Road, Bangkok
10400, Thailand. b
Department of Chemistry,
Faculty of Science, Mahidol
University. 2002.
Immobilization of a Materials
thermostable alpha- Thermostable alpha-amylase of Bacillus
amylase licheneformis (Termamyl 60 L) was purchased
from Novo industries, Denmark. Bagasse used
as carrier and tapioca starch were purchased
from the local market. Sodium meta periodate,
sulphuric acid and soluble potato starch were
obtained from Merck Co Ltd.
Immobilization of thermostable alpha-amylase
A 1.0 g sample of dried oxidized bagasse was
immersed in 30 mL of thermostable alpha-
amylase solution and incubated in water bath
with constant shaking at 50 °C for 30 minutes.
Then the bagasse was filtered to remove excess
enzyme and followed by washing with 800 mL of
distilled water. Next, the immobilized enzyme
was washed again with 100 mL of distilled water.
The washed water was tested for the enzyme
activity which was found to be absent.
Determination of activity yield
The activity yield was defined here as the yield
for enzyme which was immobilized on the
bagasse and expressed by the following
equation.
𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑦𝑖𝑒𝑙𝑑 (%)
𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝑖𝑚𝑚𝑜𝑏𝑖𝑙𝑖𝑧𝑒𝑑 𝑒𝑛𝑧𝑦𝑚𝑒
= . 𝑥100
𝐴−𝐵
Where A is the activity of free enzyme added,
and B is the activity of unimmobilized enzyme
(remaining enzyme and unimmobilized enzyme
in washed water)