Immobilized Enzyme Analysis Essay Example

on the activity and stability of immobilized enzymes under thermal studies using the entrapment method of manipulation, particularly cross-linking matrices. The results illustrate the stability advantages of immobilized enzymes and how enzymatic reactions can occur at elevated temperatures while being immobilized.The entrapment method used in this study results in a significant decrease in activity, with a reduction of up to 90%. While this may be perceived as a disadvantage, it allows for more control over reactions during analysis. The chemicals utilized were of high purity and spectrophotometry assays were conducted using a Thermo Fisher Scientific Geneses 20 (Model 400114). The immobilizers gel was filtered through a Boucher funnel. Horseradish peroxides was the specific enzyme used for entrapment within a polysaccharide gel. To properly synthesize the gel, the following order of steps was required: (1) Adding 3 mL of potassium phosphate buffer; (2) Adding 2.7 mL of creamily and methyl ibis- creamily solution; (3) Adding 1.0 mL of 0.1 MGM/ml peroxides; (4) Adding a full amount of 10% ammonium resurface; (5) Adding a full amount of TEEMED as a catalyst for the polymerization process. Carefully mixing the solution between each step while minimizing exposure to oxygen facilitates entrapping the enzyme peroxides within the polysaccharide cross-linked gel, effectively immobilizing it. Once polymerization is complete, the gel is transferred to a beaker, where diluted water is added before filtering it using a Boucher funnel to remove any non-entrapped enzyme. Figure l provides an overview of the process.After filtering the gel, the amount of immobilizer enzyme recovered can be determined by weighing the product. To analyze the enzyme's activity, it undergoes three different tests using an IV-VISA spectrophotometer. The

gel is divided into 0.05, 0.10, and .20 grams and mixed with 2.Ml of 4-mountaineering and phenol reagent, along with 2.Ml of H2O to activate the immobilizer enzyme. Each mass of immobilizer enzyme is analyzed at zero and three minutes at Mann. The same procedure is followed for analyzing the free enzyme, substituting the immobilizer mass with free enzyme volumes: 10, 20, and lull. The stock solution used for free enzyme is .MGM/ml with an activity of 300 manipulation. To complete the thermal stabilization assay, the stock enzyme solution is diluted to a ratio of 1:300 with downsized water. One ml of the diluted enzyme is left in a heat bath at approximately 70;C for four minutes and then cooled to room temperature. After that, 2.Ml of the 4-mountaineering and phenol reagent is added to the thermally tested enzyme and absorbency is measured at zero and three minutes.To ensure accuracy, both free and immobilized enzymes should undergo thermal analysis. To begin, the immobilized enzyme must be weighed and mixed with phosphate buffer. After undergoing heating for four minutes and cooling to room temperature, the solution can be analyzed using the same procedure as the free enzyme. It is important to also test a control sample of the immobilized enzyme at room temperature. Through this analysis, it can be determined if the immobilized enzyme is stabilized during heating while free enzyme activity declines. To quantify the amount of enzyme activity present, a graph should be plotted comparing absorbency versus the amount analyzed. The activity of immobilized enzymes can be calculated using the equation: slope/6.58=units/MGM. Additionally, to determine thermal stability, the equation (Deadheaded/Control)*100% can be used to

find the percent activity remaining for both free and immobilized enzymes.After conducting the necessary analyses, the results of this study were calculated and graphed to yield actual results. Figure III/IV displays the change in absorbency, while taking note of an error in the weighing of the 0.1 Gram mass during the analysis of the immobilizers enzyme, causing an omission from the results. The activity of each immobilizer and free enzyme was calculated using the slopes from these graphs and the equations provided. The tables shown in Figure V, Mathematical Analysis display these results, showing that the immobilizers enzyme has a much smaller activity than the free enzyme due to the polysaccharide cross-linked gel matrix being an intermolecular cross-linking of enzymes by multifunctional polymer cage which limits substrate diffusion. However, this manipulation leads to more control over reactions and maintains enzyme purity despite reduced activity.The thermal analysis revealed that monopolizing an enzyme helps stabilize it during heating. Both immobilized and free enzymes underwent the same treatment, with immobilization resulting in a 135% increase in activity compared to a 91% increase for the free enzyme. The data analysis showed that the immobilized enzyme had lower cinematic activity due to the cross-linked polymer cage, which restricted substrate intake and product release. Roger A. Sheldon cautioned against the use of cross-linking, which dilutes activity and reduces production yields. The low activity of the immobilized enzyme when compared to the free enzyme supports Sheldon's point. Caution should be exercised when handling phenol, as it is a chronic poison that can be fatal if inhaled, swallowed, or absorbed through the skin. The results of this study are consistent with previous research

on immobilized enzymes and their thermal properties.According to Feline Ditz and Kenneth J. Baulks Junior, the activity of the immobilizers enzyme is maintained when there is allowance for it.