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
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
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
• 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 aminoantipyrine-phenol 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
#2 2.50 mL of phenol reagent + 20uL diluted free enzyme
#3 2.50 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 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 + 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 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 + 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