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

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?

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.

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)

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 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 + 0.05 g gel 3min #2 2.50 mL of phenol reagent + 0.05 g gel 0 min #3 2.50 mL of phenol reagent + 0.10 g gel 3 min #4 2.50 mL of phenol reagent + 0.10 g gel 0 min #5 2.50 mL of phenol reagent + 0.2 g gel 3 min #6 2.50 mL of phenol reagent + 0.2 g gel

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 #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) 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 + 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 units/mg=  A/min 6.58 x mg gel 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