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The gel matrix was formed y the addition of methyl bliss-scrambled (a cross linking agent) to creamily. The immobilizers enzyme was then tested via spectrophotometers assay at 51 Non for cinematic activity and stability relative to its free enzyme counterpart. The enzymatic reaction was induced and analyzed via the addition of hydrogen peroxide (H2O), phenol, and 4-mountaineering. To test stability a spectrophotometers assay (again at 510 NM) was conducted after both immobilizers and free enzymes were heated at 70′ C for four minutes.
The results of the Immobilizers enzyme yielded a significant debility increase with a difference in activity remaining of 44% when compared with free enzyme. However, the comparison also gave the immobilizers peroxides a significantly lower cinematic rate. Thus proving that monopolizing enzymes aids in stabilization as well as the ability to take control of reactions during analysis. Key Words: enzymatic activity, peroxides manipulation, polysaccharide gel, spectrophotometers assay, thermal stability.
INTRODUCTION As biotechnology becomes even greater of a dynamic impact on modern science, the more its parameters are pushed and defined. Manipulation of enzymes has become a proficient lad 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 products .
On the other, they are highly expensive and are only available in small quantities not to mention quite fragile and unstable . In order to use enzymes efficiently and effectively commercial users have plopped a method that enables companies to recover and reuse enzymes. This method Is Manipulation. In addition to recycling enzymes Manipulation provides other advantages as well. (1) It allows for facile separation from product which helps to eliminate protein contamination; (2) the reaction can be controlled by simply removing the enzyme from solutions; (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 Immobilizers In to how free enzymes work in the human body. This study focuses on the activity of immobilizers enzymes and the stability of an immobilizers enzyme after thermal studies. The results will exemplify the advantages of immobilizers enzymes concentrating in stability. Thus showing how enzymatic catcalled reactions can be heated whilst immobilizers. This study also utilizes the entrapment method of manipulation, particularly that of cross linking matrices.
This method of entrapment often leads to a 90% decrease in activity, 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 spectrophotometers IV-VISA assays were completed on a Thermo Fisher Scientific Geneses 20 (Model 400114). A Boucher funnel was used to filter immobilizers gel. Synthesis The enzyme used in this study is horseradish peroxides. To entrap the enzyme in a polysaccharide gel the following must be synthesized in this exact order: (1) 3. Ml of potassium phosphate buffer; (2) 2. 7 ml of creamily and methyl ibis- creamily solution; (3) 1. 0 ml of 0. 1 MGM/ml peroxides; (4) lull of 10% ammonium resurface; (5) lull of TEEMED. TEEMED is the catalyst used to aid in popularization of the polysaccharide gel and enzyme. Between the addition of each reagent the solution should be mixed well, and excess exposure to oxygen should be minimalists. As this solution polymerases the enzyme peroxides becomes entrapped in the polysaccharide cross-linked gel and the enzyme is effectively immobilizers. Figure l) Once the gel has popularized it is transferred to a beaker, ml of downsized water is added and the gel is then filtered through a Boucher funnel to wash away any enzyme that was not entrapped. Once the water has been filtered from the gel the amount of immobilizers enzyme recovered can be found by analytically weighing the product. Assay of Enzyme Activity The immobilizers enzyme’s activity is analyzed via a IV-VISA spectrophotometer in three different analyses. The gel should be separated into 0. 05, 0. 10, . 20 grams. Then 2. Ml of 4-mountaineering and phenol reagent, along with 2. Ml of H2O. This mixture activates the immobilizers enzyme providing a reaction that can be analyzed via XIV-VISA (Figure II). Each mass of immobilizers enzyme should be analyzed at a zero ND three minute mark at Mann. Note that each analysis should be inverted for either zero or three minutes accordingly and should be filtered through a syringe before analyzing. This same procedure should be used when analyzing the free enzyme, substituting the immobilizers mass with the free enzyme volumes: 10, 20, lull. The free enzyme stock solution used is . MGM/ml with a label activity of 300 manipulation.
Thermal Stabilization Assay In order to complete this assay the stock enzyme solution must be diluted to 1 :300 with downsized water. 1 ml of the diluted enzyme is left in a heat bath at approximately 70;C for four minutes. It is then removed from the heat bath and allowed to cool to room temperature. Once again 2. Ml of the 4-mountaineering and phenol reagent is added to the thermally tested enzyme. The absorbency should then be recorded at zero and three minutes. A control group should also be analyzed using the same procedure but left at room temperature.
After the free enzyme has been tested the immobilizers enzyme must also be thermally analyzed. Exactly 0. 1 Grams of immobilizers enzyme should be weighed out and 0. Ml of phosphate buffer should be added. The solution should sit in the hot bath described above for four minutes, be allowed to cool to room temperature and analyzed using the same procedure described above. A control for the immobilizers enzyme should also be analyzed at room temperature. The results of the thermal studies should yield proof that the immobilizers enzyme is now stabilized during heating while the free enzyme declines in activity level.
Mathematical Methods of Analysis To analyze the amount of activity present in free and immobilizers enzymes a graph should be presented comparing the change in absorbency versus the MGM or ml analyzed (AAA= (Abstain – Abstain). This plot should yield a linear trend line with an equation along the lines of y=mix+b where m=slope. The activity of immobilizers enzyme can be analyzed using the equation slope/6. 58=units/MGM. The activity of free enzyme can be found using the equation slope/6. 58*. Mall. To analyze thermal stability of immobilizers and free enzymes the percent activity remaining should be found.
The following equation should be used: (Deadheaded/Control)*100%. These analyses should then be interpreted to yield actual results. RESULTS The analysis of results of this study began with the calculations of the change in absorbency which were graphed accordingly as shown (Figure III/IV). It must be noted that in the analysis of the immobilizers enzyme there was an error while weighing out the 0. 1 Gram mass causing and error in analysis resulting in omission from results. The slopes from these graphs were then used to calculate the activity of each immobilizers and free enzymes using the aforementioned equations.
The results are displayed in the tables shown. (Figure V, Mathematical Analysis) The activity of the immobilizers enzyme is much smaller than that of the free enzyme. This is expected and can be explained via the method of manipulation. The polysaccharide cross- linked gel matrix is an intermolecular cross-linking of enzymes by multifunctional polymer cage. However, small substrate molecules can diffuse in and small product molecules can diffuse outlet . Therefore the amount of substrate that is reaching the immobilizers enzyme is far less than the amount that is reaching the free enzyme.
This results in less product formation and therefore less activity. However, while this type of manipulation provides less activity, it also provides more control over reactions, and helps to maintain the purity of an enzyme. The remaining activity after thermal analysis shows how monopolizing an enzyme helps to stabilize it during heating. Both immobilizers and free enzymes were treated the same way, however manipulation gave a 135% increase in activity while the free only gave 91%. Mathematical Analysis Data Description Activity (units/MGM) Remaining Activity Immobilizers . 00547 135. 71% Free 245. 51 91 . 07% Caution! Phenol is a chronic poison it may be fatal is inhaled, swallowed, or absorbed through skin DISCUSSIONS/CONCLUSIONS In accordance with acceptance developed by research, enzymes have increased stability and decreased activity during manipulation via entrapment by polysaccharide gel matrix. Immobilizers enzymes have a decreased cinematic activity due to the cross linked polymer cage that surrounds the peroxides enzyme that only lets in small amounts substrate and lets only relatively small products. According to Roger A.
Sheldon “The use of cross linking inevitably leads to dilution of activity owing o the introduction of non-catalytic ballast ranging from 90% to which results in lower space time yields and production activities. ” This is demonstrated by the . Intuitions/MGM activity given off by the immobilizers enzyme compared to the 245. 51 units/MGM from the free enzyme found in this study. Thermal studies also went in accordance with the research conducted by previous scholars on immobilizers enzymes. The findings of]. Feline Ditz and Kenneth J. Baulks Junior show that when allows for the immobilizers enzyme to maintain its activity.