Practical no.
: Date:
Aim: To immobilize yeast cells and demonstrate its biological activity by invertase assay.
Introduction:
Enzymes in water-insoluble form show catalytic activity. This activity is retained by
coating these enzymes or cells producing these enzymes on a solid surface and their activity are
also restricted at that site. This phenomenon of coating enzyme on a solid surface is called
immobilization of enzymes.
Methods for enzyme immobilization can be classified into three basic categories as
follows:
1. Carrier- binding method: the binding of enzymes to water insoluble carriers such as
polysaccharide derivatives, synthetic polymers and porous glass, etc.
2. Cross-linking method: Intermolecular cross linking of enzymes by means of bifunctional
or multifunctional reagents such as glutaraldehyde, bisdiazobenzidine and hexamethylene
diisocyanate.
3. Entrapping method: Incorporating enzymes into the lattice of a semipermeable gel on
enclosing the enzymes in a semipermeable polymer membrane, such as collagen, gelatin
cellulose triacetate polyacrylamide and k-carrogeenan, etc.
These three methods are shown schematically in figure 1.
Because of the special characteristics of immobilized enzymes, a considerable
amount of work has been carried out on the application of immobilized enzymes on solid
catalysts having high substance specificity. Immobilized enzymes are used in various
fields, such as chemical processes, analysis, medical treatment, food processing, wine
production, affinity chromatography and so forth.
Requirements:
Immobilization of yeast cells
1. 1gm dry baker’s yeast
2. 2gm% sterile chilled calcium chloride (CaCl2 )(200 ml)
3. 2gm% sterile sodium alginate (200 ml)
4. Sterile Pasteur pipettes
A. For standard graph preparation
1. DNSA reagent
2. Glucose stock solution (10mM)
3. Glass tubes
4. Pipettes
5. Distilled water
6. Graph paper
B. Invertase assay
1. Sterile 0.3 M sucrose solution
2. Sterile saline
3. Pipettes
Procedure :
A) Immobilization of whole cell
1. 1gm baker’s yeast was weighed and suspended in 100 ml of sterile 2gm% sodium
alginate solution. One more 250-ml flask containing 100 ml of sterile 2gm% sodium
alginate solution without addition of baker’s yeast is kept as a control.
2. With the help of sterile Pasteur pipette, above mixture is added to sterile chilled 2
gm % calcium chloride, so as to form uniform size beads.
3. The beads were kept as it is in chilled calcium chloride solution for 1hr and then
refrigerated overnight.
B) Invertase assay:
1. Yeasts are well known to produce invertase enzyme. The production of invertase
enzyme was observed by incubating 100 beads of immobilized yeast cells with 0.3
M sucrose solution. 50 ml of 0.3 M sucrose solution was added to 50 ml saline and
100 beads were mixed.
2.
a) Positive and negative controls were simultaneously prepared as per protocol.
b) The samples at different time interval 0, 30 ,60, 90 and 120 min. were removed
and assayed to estimate the reducing sugar formed, by DNSA method.
C) Standard graph preparation
Standard graph was prepared using DNSA method with 10 mM glucose solution
as stock as per the protocol.
References:
1. Wilson K. and Walker J. (2005)
2. Hill F. (1991) Immobilization of yeast in alginate beads for production of alcoholic
beverages, 5,070,019, United States Patent.
3. http://www.scribd.com/doc/12707839/Enzyme-Immobilisation
