Activity and Thermal Stability of

Gel-Immobilized Peroxidase
Experiment #12

 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?

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

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

 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

Practical Applications of Enzyme

Immobilization
We developed a new technique to use natural clays which have layerlike 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

 Process

Enzyme Entrapment

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

 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

 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.

 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
peroxidase

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)

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

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

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

Procedure

ASSAY OF ENZYME ACTIVITY

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 aminoantipyrinephenol solution and 2.5mL of DI water as the reference

IMMOBILIZED ENZYME
0 min #1
2.50mL of phenol reagent + 0.05 g gel
3min #2
2.50 mL of phenol reagent + 0.05 g gel
0 min #3
3 min #4

2.50 mL of phenol reagent + 0.10 g gel

2.50 mL of phenol reagent + 0.10 g gel

0 min #5
3 min #6

2.50 mL of phenol reagent + 0.2 g gel

2.50 mL of phenol reagent + 0.2 g gel

Procedure
Assay of Enzyme Activity (continued)
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

#1 2.50 mL of phenol reagent + 10uL diluted free enzyme

#22.50 mL of phenol reagent + 20uL diluted free enzyme
#32.50 mL of phenol reagent + 40uL diluted free enzyme

Procedure
Free Enzyme Assay (continued)
Transfer solution to a cuvette, insert in spectrophotometer, add 2.50 mL of H 2O2, 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 + 2.0 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

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 H 2O2 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 + 2.25 mL DI water

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
6.58 x mg gel

units/mg= A/min
6.58 x .010 x ml enzyme

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