Presentation on theme: "Activity and Thermal Stability of Gel-Immobilized Peroxidase"— Presentation transcript:
1 Activity and Thermal Stability of Gel-Immobilized Peroxidase Experiment #12
2 HOW COULD THIS BE IMPROVED? ENZYMES………..have high catalytic activitiescatalyze a great variety of reactionsBUT………..enzymes are very expensive for commercial useenzymes are very fragile and often unstableHOW 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 matrixBenefits of immobilizationpossibly 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 insightability 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 solutionimmobilization may force the enzyme to take on a different conformationthe 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 carriersCross-Linking: intermolecular cross-linking of enzymes by multifunctional reagentsEntrapping: 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 enzymegel 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 escapeMatrix must be inert to limit disintegration
8 GEL MATRIX:A cross linked polymer formed by acrylamide and methylene bisacrylamidePOLYMER = a combination of many smaller molecules to form a larger molecule2 major classes of polymerization reactionsADDITION: monomers added on top of one another. All of the starting atoms of the monomer remain as part of the polymerCONDENSATION: a portion of the monomer is split out when forming the polymerCatalysts 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 waterH2O2 + AH2 2H2O + AperoxidaseENZYME ACTIVITY (Free and Immobilized)The concentration of peroxidase is assayed in the following mannerH2O2 + phenol+4-aminoantipyrine quinonemimine + 2H2Othe 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 enzymeIII. Thermal Stabilitythe 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 tube3.25 mL of potassium phosphate buffer2.7 mL of acrylamide/bis-acrylamide solution1.0 mL of 0.1 mg/mL peroxidase80 uL of 10% ammonium persulfateMix well on vortex mixerand add 10uL of TEMEDGently mix by inversion and vortexingBubble 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 liquidTransfer gel to a beaker containing 5 mL of waterAspirate the gel using a Pasteur pipet (8-10 times)Filter the gel on Buchner funnel. Rinse 2x with deionized waterDry the gel by vacuum filtering for 5 minutesTransfer 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 activitySet 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 referenceIMMOBILIZED ENZYME0 min #1 2.50mL of phenol reagent g gel3min # mL of phenol reagent g gel0 min # mL of phenol reagent g gel3 min # mL of phenol reagent g gel0 min # mL of phenol reagent g gel3 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 nmFREE 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 nmThermal StabilityFree 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 tubesPlace one test tube in a 70 degree C bath for 4 minutes. Allow the other tube to sit at room temperatureAfter 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. InvertAllow 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 bufferPlace one test tube in a 70 degree bath for 4 minutes. Allow other tube to sit at room temperatureAfter 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 mixAfter 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 minPlot change of A/min vs. mg of gelPlot change of A/min vs. mL of free enzymeCalculate the activity for free enzyme and immobilized enzyme for each assayImmobilized Freeunits/mg = A/min units/mg= A/min6.58 x mg gel x .010 x ml enzyme
19 Data AnalysisCompare the % Activity remaining in free and immobilized enzymeAssume that A0min = 0.000Calculate 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 CCalculate the % activity remaining for free and immobilized%Activity remaining = A at 70o X 100% Aat room temp
Your consent to our cookies if you continue to use this website.