Immobilized Enzyme Analysis
Activity and Thermal Stability of Gel-Immobilized Peroxidase.
Taylor Thompson taylor.thompson72895@gmail.com Department of Chemistry, University of North Florida, 1UNF Drive, Jacksonville, FL 32224 _____________________________________________________________________________________________
Immobilizing an enzyme provides various analytical benefits, and can be done in a myriad of ways, with the most common being entrapment. For this study peroxidase (from horseradish), an enzyme that catalyzes the cleavage of hydrogen peroxide into water, was entrapped within a polyacrylamide gel matrix. The gel matrix was formed by the addition of methylene bis-acrylamide (a cross linking agent) to acrylamide. The immobilized enzyme was then tested via spectrophotometric assay at 510nm for kinematic activity and stability relative to its free enzyme counterpart. The enzymatic reaction was induced and analyzed via the addition of hydrogen peroxide (H2O2), phenol, and 4-aminoantipyrine. To test stability a spectrophotometric assay (again at 510 nm) was conducted after both immobilized and free enzymes were heated at 70˚C for four minutes. The results of the immobilized enzyme yielded a significant stability increase with a difference in activity remaining of 44% when compared with free enzyme. However, the comparison also gave the immobilized peroxidase a significantly lower kinematic rate. Thus proving that immobilizing enzymes aids in stabilization as well as the ability to take control of reactions during analysis. Key Words: enzymatic activity, peroxidase immobilization, polyacrylamide gel, spectrophotometric assay, thermal stability. _____________________________________________________________________________________ INTRODUCTION As biotechnology becomes even greater of a dynamic impact on modern science, the more its parameters are pushed and defined. Immobilization of enzymes has become a proficient aid in analytical experiments to scientist in
Taylor Thompson taylor.thompson72895@gmail.com Department of Chemistry, University of North Florida, 1UNF Drive, Jacksonville, FL 32224 _____________________________________________________________________________________________
Immobilizing an enzyme provides various analytical benefits, and can be done in a myriad of ways, with the most common being entrapment. For this study peroxidase (from horseradish), an enzyme that catalyzes the cleavage of hydrogen peroxide into water, was entrapped within a polyacrylamide gel matrix. The gel matrix was formed by the addition of methylene bis-acrylamide (a cross linking agent) to acrylamide. The immobilized enzyme was then tested via spectrophotometric assay at 510nm for kinematic activity and stability relative to its free enzyme counterpart. The enzymatic reaction was induced and analyzed via the addition of hydrogen peroxide (H2O2), phenol, and 4-aminoantipyrine. To test stability a spectrophotometric assay (again at 510 nm) was conducted after both immobilized and free enzymes were heated at 70˚C for four minutes. The results of the immobilized enzyme yielded a significant stability increase with a difference in activity remaining of 44% when compared with free enzyme. However, the comparison also gave the immobilized peroxidase a significantly lower kinematic rate. Thus proving that immobilizing enzymes aids in stabilization as well as the ability to take control of reactions during analysis. Key Words: enzymatic activity, peroxidase immobilization, polyacrylamide gel, spectrophotometric assay, thermal stability. _____________________________________________________________________________________ INTRODUCTION As biotechnology becomes even greater of a dynamic impact on modern science, the more its parameters are pushed and defined. Immobilization of enzymes has become a proficient aid in analytical experiments to scientist in
References: [1] Boyer, Rodney. Modern Experimental Biochemistry, 3rd ed.; Cummings: San Francisco, 2000 [2] Diaz, Felipe J.; Enzyme Immobilization in MCM-41 Molecular Seive; Journal of Molecular Catalysis; Online; 1996 [http://ac.els-cdn.com/S1381117796000173/1-s2.0-S1381117796000173-main.pdf?_tid=05e520f0-bb90-11e3-95a4-00000aab0f02&acdnat=1396571480_b96da4aefdd33c95738c11f3ae20c8f7] Accessed April 3rd, 2014.