1. Activity and Thermal Stability of Gel-Immobilized Peroxidase
Experiment #12

2. HOW COULD THIS BE IMPROVED?
ENZYMES………..
have high catalytic activities
catalyze a great variety of reactions
BUT………..
enzymes are very expensive for commercial use
enzymes are very fragile and often unstable
HOW COULD THIS BE IMPROVED?

3. Enzyme Immobilization
“Immobilization”--a process that limits the movement or free diffusion of the enzyme molecule by attaching the enzyme to an inert matrix
Benefits of immobilization
possibly increase the stability of the enzyme allowing the enzyme to be recycled (repetitive use of a single batch of enzymes)
to mimic the environment of enzymes in the body to gain better insight
ability to stop the reaction rapidly by removing the enzyme from the reaction solution

4. Immobilized enzyme Immobilized enzyme vs. Free Enzyme
Immobilized enzyme is expected to behave differently in solution
immobilization may force the enzyme to take on a different conformation
the surrounding chemical environment differs (depending on the enzyme, polymer environment will either make the enzyme more stable or will slightly denature it)
kinetic rates of a reaction will be affected by how well the substrate diffuses through the gel

5. Techniques for enzyme immobilization can be classified into three categories
Carrier-Binding: binding of enzymes to water insoluble carriers
Cross-Linking: intermolecular cross-linking of enzymes by multifunctional reagents
Entrapping: incorporating enzymes into the lattices of a semipermeable gel or enclosing the enzymes in a semipermeable polymer membrane*
* Our method of choice

6. Practical Applications of Enzyme Immobilization
“We developed a new technique to use natural clays which have layer-like structures, as matrices for enzyme immobilization. We developed a process to cross-link clay layers for trapping hydrogen peroxidase, an enzyme that catalyzes the decomposition of organic materials by hydrogen peroxide. The cross-linked layers of the clay formed a sieve-like structure, with hydrogen peroxidase entrapped in its pore network. The entrapped enzyme exhibited its normal activity but with significantly improved shelf-life and reusability. The immobilized peroxidase can be used in the detection and removal of pesticides and other organic pollutants in water. This new technique may be further developed to trap cell-associated enzymes, antibodies or bacteria for other industrial or environmental applications. “ --research for EPA

7. Enzyme Entrapment Process enzyme is added to the polymer
chemical reagent or temperature is applied that initiates polymerization and the gel/matrix forms around the enzyme
gel is then disrupted to form smaller units to increase the rate of reaction
**Pore size must not limit diffusion into and out of the matrix, but must not be large enough to allow the enzyme to escape
Matrix must be inert to limit disintegration

8. GEL MATRIX:
A cross linked polymer formed by acrylamide and methylene bisacrylamide
POLYMER = a combination of many smaller molecules to form a larger molecule
2 major classes of polymerization reactions
ADDITION: monomers added on top of one another. All of the starting atoms of the monomer remain as part of the polymer
CONDENSATION: a portion of the monomer is split out when forming the polymer
Catalysts such as riboflavin, ammonium persulfate, and fluorescent light catalyze the reaction by forming free radicals from the monomers

9. Peroxidase enzyme will be entrapped in a polyacrylamide matrix as shown in the reaction below. Polyacrylamide is formed by an addition reaction of acrylamide molecules which are then cross-linked by methylene bisacrylamide. Ammonium persulfate and TEMED will serve as catalysts.

10. H2O2 + phenol+4-aminoantipyrine  quinonemimine + 2H2O
ENZYME:
Peroxidase:
known to catalyze the cleavage of hydrogen peroxide into water
H2O2 + AH2  2H2O + A
peroxidase
ENZYME ACTIVITY (Free and Immobilized)
The concentration of peroxidase is assayed in the following manner
H2O2 + phenol+4-aminoantipyrine  quinonemimine + 2H2O
the reaction mixture is assayed within 3 minutes to assess the quantity of chromogen (the concentration of peroxidase in solution is directly proportional to the quinoneimine produced ( = 510nm)

11. Procedure I. Preparation of Immobilized Enzyme
II. Assay of Immobilized enzyme (compare to free enzyme
III. Thermal Stability
the stability in terms of decrease in activity of free enzyme and gel immobilized enzyme will be compared at room temperature and an elevated temperature

12. Procedure IMMOBILIZATION OF PEROXIDASE
Mix together the following in a 50mL screw-capped tube
3.25 mL of potassium phosphate buffer
2.7 mL of acrylamide/bis-acrylamide solution
1.0 mL of 0.1 mg/mL peroxidase
80 uL of 10% ammonium persulfate
Mix well on vortex mixer
and add 10uL of TEMED
Gently mix by inversion and vortexing
Bubble N2 gas through the mixture (if necessary) for 2 minutes and then blow on the surface of the mixture for 2 minutes

13. Procedure Immobilization of Peroxidase (cont.)
Transfer the gel to a vacuum filtration system and filter any remaining liquid
Transfer gel to a beaker containing 5 mL of water
Aspirate the gel using a Pasteur pipet (8-10 times)
Filter the gel on Buchner funnel. Rinse 2x with deionized water
Dry the gel by vacuum filtering for 5 minutes
Transfer the semi-wet gel to a tared test tube and analytically weigh the gel

14. Procedure ASSAY OF ENZYME ACTIVITY IMMOBILIZED ENZYME
Set up 6 test tubes for immobilized enzyme activity
Set wavelength of spectrophotometer to 510 nm. Set 0 and 100%T using 2.5 mL of aminoantipyrine-phenol solution and 2.5mL of DI water as the reference
IMMOBILIZED ENZYME
0 min #1 2.50mL of phenol reagent g gel
3min # mL of phenol reagent g gel
0 min # mL of phenol reagent g gel
3 min # mL of phenol reagent g gel
0 min # mL of phenol reagent g gel
3 min # mL of phenol reagent g gel

15. Procedure Assay of Enzyme Activity (continued) FREE ENZYME ASSAY
For 0 minute point, add 2.50 mL of H2O2 to tube. Within 10 seconds, rapidly mix and filter through a syringe. Record absorbance at 510 nm.
For 3 minute point, add 2.50 mL of H2O2 to tube and start timing. Invert mixture continuously for 3 minutes for the gel. After 3 minutes rapidly filter through syringe and record absorbance at 510 nm
FREE ENZYME ASSAY
** Dilute free enzyme 1:10. Set up 3 test tubes
# mL of phenol reagent uL diluted free enzyme
# mL of phenol reagent uL diluted free enzyme
# mL of phenol reagent uL diluted free enzyme

16. Procedure Free Enzyme Assay (continued) Thermal Stability
Transfer solution to a cuvette, insert in spectrophotometer, add 2.50 mL of H2O2, start timer, and immediately set 0 and 100%T.
Let reaction continue. At 3 minute point, record absorbance at 510 nm
Thermal Stability
Free Enzyme (reference = 2.0 mL phenol reagent mL water)
Dilute peroxidase stock solution 1:300 with deionized water
(Use 10uL of peroxidase stock solution diluted to a total of 3000uL)
Add 1 mL of this diluted enzyme to 2 test tubes
Place one test tube in a 70 degree C bath for 4 minutes. Allow the other tube to sit at room temperature
After 4 minutes, cool the hotter tube to room temperature. Add 2.0 mL of phenol reagent to both and 2.0 ml of H2O2 to both. Invert
Allow to sit at room temperature for 3 minutes and immediately record absorbance at 510 nm

17. Procedure Thermal Stability (continued) Immobilized Enzyme
Weigh out 0.1 g of enzyme gel to 2 test tubes containing 0.5 mL phosphate buffer
Place one test tube in a 70 degree bath for 4 minutes. Allow other tube to sit at room temperature
After 4 minutes, cool the hotter tube to room temperature, add 2.25 mL phenol reagent to both and 2.25 mL of H2O2 to both. Invert to mix
After 3 minutes, immediately filter the solution through a syringe and record absorbance at 510 nm
* Reference = 2.25 mL of phenol reagent mL DI water

18. Data Analysis Compare activity of free enzyme vs. immobilized enzyme
A/min = Abs3min - Abs0 min
Plot change of  A/min vs. mg of gel
Plot change of  A/min vs. mL of free enzyme
Calculate the activity for free enzyme and immobilized enzyme for each assay
Immobilized Free
units/mg =  A/min units/mg=  A/min
6.58 x mg gel x .010 x ml enzyme

19. Data Analysis
Compare the % Activity remaining in free and immobilized enzyme
Assume that A0min = 0.000
Calculate
 A1 = change for free enzyme at room temperature
 A2 = change for free enzyme at 70 degrees C
 A3 = change for immobilized enzyme at room temperature
 A4 = change for immobilized enzyme at 70 degrees C
Calculate the % activity remaining for free and immobilized
%Activity remaining =  A at 70o X 100%
 Aat room temp