EXPERIMENT NO. 7A
ENZYME IMMOBILIZATION PROTOCOL
ENTRAPMENT IN POLYACRYLAMIDE GEL
Prepared by
Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111
ENCH485
Table of Contents
Method
This technique is based on the polymerization of acrylamide with
N,N'-methylene-bis-acrylamide (Bis) as the cross-linking agent.
The degree of cross-linking, thus, can be partly controlled by
adjusting the ratio of acrylamide to Bis used.
List of Reagents and Instruments
A. Equipment
- Beakers
- Pipets
- Balance
- Graduated cylinder
- Syringe and needle
B. Reagents
- Buffered Monomer Solution
- 0.1 mM EDTA
- 0.1 M Tris-HCl
- 11 g/l N,N'-Methylene-bis-acrylamide (CH2=CHCONH)2CH2
- 200 g/l Acrylamide CH2=CHCONH2
- Adjust the pH to 7.0 (See Note 1)
- Washing Solution
- 0.5 M NaCl
- 0.1 mM EDTA
- 0.1 M Tris-HCl
- Adjust the pH to 7.0
- Dimethylaminopropionitrile (polymerization initiator)
- Potassium persulphate solution, 10g/l (polymerization catalyst)
- Nitrogen gas cylinder
- Enzyme
Procedures
- Buffered Monomer Solution:
Add 1.1 g of Bis and 20 g of acrylamide to a 100 ml of buffered
solution (pH 7.0) of 0.1mM EDTA and 0.1M Tris-HCl in a beaker.
See Note 1.
- To 10 ml of the buffered monomer solution of the above
step, add enzyme powders (approximately 0.1ml of 75g/l fungal
amylase) or an equivalent concentrated enzyme solution; mix.
- For 20 minutes, purge the dissolved oxygen in the solution
that can interfere with the polymerization process with nitrogen.
This step is critical in achieving a high degree of
cross-linking.
- Add 0.1 ml of dimethylaminopropionitrile; mix.
- Add 1.0 ml of freshly prepared 10g/l potassium persulphate
solution to initiate polymerization.
- Now is the time to pour the solution into a mold if one
does not desire the gel to form in the original beaker. Leave
the solution undisturbed; gel will form in approximately 10-30
minutes. (Hardening can be accelarated by using more
dimethylaminopropionitrile.
- Cut the resulting gel into small cubes of approximately
3mm per side. Alternatively, if smaller pieces are desired, the
gel can be forced through a syringe fitted with a fine needle.
- Gently wash the free enzyme off the gel surface in 10 ml
of the Washing Solution. Repeat the washing process two
additional times.
Notes
- The pH of the buffer should be adjusted to match the
optimum value of the enzyme to be entrapped.
Discussions
The above methods of enzyme immobilization by gel entrapment can
be directly applied to live cells with minor modifications. For
example, dimethylaminopropionitrile used in forming the
polyacrylamide gel may not be employed because of its toxicity to
viable cells. The monomers of acrylamide are also somewhat toxic
to cells. On the other hand, cells can be immobilized with much
less degree of cross-linking due to its much larger size.
References
- M.D. Trevan and S. Grover,
Trans. Biochem. Soc., 7, 28, 1979.
Return to Prof. Nam Sun Wang's Home Page
Return to Biochemical Engineering Laboratory (ENCH485)
Enzyme Entrapment in Polyacrylamide Gel
Forward comments to:
- Nam Sun Wang
- Department of Chemical & Biomolecular Engineering
- University of Maryland
- College Park, MD 20742-2111
- 301-405-1910 (voice)
- 301-314-9126 (FAX)
e-mail: nsw@umd.edu