Immobilized Enzyme Analysis

Topics: Enzyme, Immobilized enzyme, Catalysis Pages: 6 (1841 words) Published: April 15, 2014
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.
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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 all biochemical fields, for various different reasons. Enzymes alone are valuable and versatile reagents. However, they have advantages and disadvantages. On one hand they have high catalytic activity, generally are able to function under mild reaction conditions and there are generally no side reactions or products1. On the other, they are highly expensive and are only available in small quantities not to mention quite fragile and unstable1. In order to use enzymes efficiently and effectively commercial users have developed a method that enables companies to recover and reuse enzymes. This method is immobilization. In addition to recycling enzymes immobilization provides other advantages as well. (1) It allows for facile separation from product which helps to

eliminate protein contamination2; (2) the reaction can be controlled by simply removing the enzyme from solution1; (3) the enzyme is usually more stable in regards to thermal activity. These advantages allow enzymes to be used in advanced biochemical research. Because most enzymes in human cells are immobilized in cells, tissues and membranes, the study of immobilized enzymes helps gives insight to how free enzymes work in the human body. This study focuses on the activity of immobilized enzymes and the stability of an immobilized enzyme after thermal studies. The results will exemplify the advantages of immobilized enzymes concentrating in stability. Thus showing how enzymatic catalyzed reactions can be heated whilst immobilized. This study also utilizes the entrapment method of immobilization, particularly that of cross linking matrices. This method of entrapment often leads to a 90% decrease in activity1, while this can be seen as a disadvantage it also provides control over reactions as seen during analysis.


METHODS AND MATERIALS

Measurements
The chemicals used in this experiment were commercial products of high purity. The spectrophotometric UV-VIS assays were completed on a Thermo...

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.
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