The effects of changing temperature and pH conditions on the activity of the β-galactosidase enzyme and its affinity for ONPG were studied. A standard curve was developed as a control, and was used to obtain the assays for the 6 different reaction rates studied, corresponding with the 4 temperatures and 4 pH’s. The reaction results were measured for its absorbance, which was converted into mM of ONP present using the standard curve. Using conversions in a form similar to stoichiometry,
Introduction
β-galactosidase is neither a new nor untested enzyme. It is very well-known and commonly used in many experiments in many different aspects of Biology. It can be found in a number of species, including yeast, fungi and E.coli bacteria, and has often been used in the hydrolysis of the disaccharide lactose (Zhao and Chen, 2001). Another main functions includes catalyzing the transgalactosylation of lactose, as well as cleaving the resulting allolactose into monosaccharides (D. H. Juers et. al., 2012). When acting as an enzymatic catalyst, it is very useful in this role. In fact, when β-galactosidase is used as a catalyst in the transgalactosylation of lactose, bifidobacteria and lactobacilli can be produced from that reaction, and they are proven to be good for human health (D.F.M Neri et al., 2009).
In this study, the catalytic activity of β-galactosidase will be tested in different temperature conditions. It will be acting on a substrate ONPG that bears many similarities to lactose, and the rate at which the enzyme can cleave the disaccharide substrate and produce ONP will be measured to determine the efficiency of the enzyme in these conditions. It is important to observe what condition might produce the most efficient reaction, so β-galactosidase may be used in the best possible way in the future.
The purpose of the study will be to find if room temperature is the best environment for a reaction catalyzed by β-galactosidase, or if altering the temperature will affect the enzyme in a more positive or negative way. It is expected that room temperature will not be the most efficient condition for the catalytic enzyme; one of the altered temperature environments will produce stronger results. It is expected that at 37 °C and pH 7.5, the most molecules of ONPG would be hydrolyzed due to the conditions being similar to that of the human stomach.
Materials and Methods
In order to determine the effects of temperature and pH on the reaction rate of the hydrolysis of ONPG, the following method was used.
Standard Curve- This first portion of the experiment was used to set a control in the form of a standard curve and find an equation for the curve. 8 test tubes, each containing increasing concentrations of ONP (0.025 mM, 0.050 mM, 0.075 mM, 0.100 mM, 0.125 mM, 0.150 mM, 0.200 mM, 0.250 mM) were used, with 1.5 mL of ONP solution in each tube. 1.5 mL of 0.1M NA2CO3 stop solution was then added to each tube. A spectrophotometer, which was set to 420 nm, was used to measure the data. The spectrophotometer was then calibrated with a blank (a 1:1 ratio of stop solution and 0.1M Sodium Phosphate buffer at pH 7.6). Finally, the absorbance of each solution of ONP and stop solution was measured in the spectrophotometer, then recorded into a table.
Reaction Assay- This second portion of the experiment was used to observe the various changes in the reaction velocity, using different concentrations of ONPG and changing the controlled element. First, the effect of temperature on the enzyme, β-galactosidase, was measured. Label 3 sets of 3 test tubes, each of them with times at minute intervals of 1, 3, and 5. Fill each test tube with 2.0 mL of 0.1 M Na2CO3, which are also our “stop” solution. This means that the Na2CO3 will stop the reaction of the enzyme. Next, prepare a blank solution (2.0 ml of 0.1 M Na2CO3, 1.6 ml of pH 7.5 Sodium Phosphate buffer, 0.20 ml ONPG, and 0.20 ml distilled water. This blank will calibrate the spectrophotometer. Prepare the Large Reaction tube with a 50 ml conical tube with a lid. This will include 20.0 ml of 7.5 Sodium Phosphate Buffer and 2.5 ml of 50 mM ONPG. Then, starting a timer, immediately start the reaction by adding 2.5 ml of enzyme stock solution to the Large Reaction tube and mix gently for few seconds. At the time intervals mentioned above (1, 3, and 5), remove 6.0 ml of the reaction mixture and immediately add the 2.0 ml stop solution, to stop the reaction at exactly the right time. All of these must be removed into each of the corresponding labeled test tubes. Then repeat this exact step for the other 2 time intervals. After the 5 minutes, the same steps are repeated for 3 other temperatures with a total of 4 different temperatures of 4 °C, 20 °C, 37 °C, and 51 °C. Next, the effect of pH on the enzyme, β-galactosidase, was measured. The same procedure as the case for the temperature was followed, with a few exceptions. This time, the controlled element is the temperature of 37 °C, instead of the pH 7.5. This time the range of the differing pH’s are 5.5, 6.5, 7.5, and 8.5. This is done by changing the pH of the Sodium Phosphate buffer, but conducting the above experiment at 37 °C. Once all of the test tubes are filled with the corresponding samples, read the absorbance of each of the test tubes at 420 nm after calibrating the spectrophotometer using the blank solution that was created in the earlier steps. Record the absorbance into an organized table clearly labeled for each of its correct treatments.
Statistical Calculations- To process all sets of data involved, Microsoft excel was used. First calculate the concentration of ONP in each of the test tubes by using the standard curve equation determined initially. The raw data for each set of trials (ONPG concentration, absorbance) was documented in each of its corresponding tables. Then, for every concentration of ONPG, the 3 absorbance values measured were averaged. This was repeated for each temperature and pH, and those averages were used to find the amount of mM of ONP produced per minute by using the linear slope equation (y =2.5927x-0.0238), which was derived from the standard curve. This reaction rate, ONP produced per minute, was graphed on the y-axis, and the corresponding ONPG concentration was placed on the x-axis to produce the assay graph. This was performed for all 4 temperatures and all 4 pH’s. The formula (mmol/liter/min * 0.001 liter/ml * #ml * 6.023*1020 molecules/mmol) was used to calculate the rate of hydrolysis of ONPG in molecules per minute. To make understanding this long equation easier, it was divided into several steps. Part A (mmol/liter/min * 0.001 liter/ml) was used to find the rate of reaction, which is equal to mM/min. Part A converts mmol/liter/min to mmol/ml/min. In Part B, the total number of molecules per minute is calculated by multiplying the total volume of material in the Large Reaction tube (ml) and the mmol/ml/min that we attained in Part A, which leaves (mmol/min * #ml). In Part C, the value found in Part B is converted from mmol to molecules. This is done by multiplying the value from Part B by (6.023*1020 molecules/mmol), which will convert the units from mmol/min to molecules/min. Before moving on to the last part of the calculations, the quantity of enzyme molecules present during the reaction must be found. Because 2 ml of enzyme was used in the reaction and the enzyme stock solution for the experiment had 1.4*1012, the number of molecules present in the reaction can be found with the equation (#ml enzyme * molecules/ml). Part C solves for the number of molecules of ONP produced per minute and Part D gives the number of enzyme molecules present. Finally by putting both of these together with [(molecules ONP/min)/(#molecules Enzyme)], we can find the number of molecules of ONPG hydrolyzed per minute per molecule of enzyme.
Discussion
After solving for the amount of ONP hydrolyzed per minute, the most optimal working condition for β-galactosidase is at pH 7.5. For temperature data shows that the best condition is at 51 °C, which is the highest temperature that this experiment was performed at. The very first thing that should be kept in mind before discussing the results is that the enzyme, β-galactosidase, is found in the E. coli, which are bacteria found in the human stomach. The human body operates at roughly around 37 °C and at pH 7.5.
References
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