Two separate variables were hypothesized and tested within the confines of the experiment on their effects on the productivity of lactase enzyme. The measurement of light absorbance levels of different solutions based on both the concentration of o-nitrophenol (ONP) as well as the pH of the solution tested. To begin with, differing concentrations of ONP were tested to find the absorbance levels of ONP. Three different pH values, 5, 6, and 7, in solution, were tested over a period of 8 minutes and their absorbance’s recorded. Both plots of data were then used to find at which concentrations of ONP absorption levels, and thus enzyme activity rates, were at their highest across each of the three pH solutions. Based on the results of both experiments, it was found that a pH of 5 is the optimal pH for the productivity of lactase enzyme.
Lactase enzyme is used throughout the food industry to break lactose, found in milk, down into the sugars glucose and galactose. A deficiency in lactase prevents the breakdown of lactose and results in what is called lactose intolerance, which can lead to digestion problems, cramps, and possibly diarrhea upon ingestion of dairy products (Phillips). Through the use of lactase outside of the body, milk is separated into its more viscous and creamy form for the production of dessert products such as ice cream.
Many enzymes are proteins that lower the activation energy needed to start chemical reactions that are crucial to the survival of life. Alike to other proteins enzymes undergo changes in shape based on the surrounding environment, and thus their productivity changes as well. Many factors affect the productivity of enzyme including temperature, concentration of enzyme in a reaction, and pH (Gundlach 441).
In this experiment the factors of concentration and pH were tested. The tests performed were done so in order to investigate the effects of pH and concentration difference on the overall productivity of lactase enzyme by measuring light absorbance levels of enzyme while changing each of the two variables over time. It was hypothesized that as concentration of enzyme
precipitate in the form of ONP was increased so would the absorption levels. It was also hypothesized that if the lactase enzyme reaction took place at a pH of 5 it would have the highest absorbance level as reactions with other pH levels would change the enzyme’s shape and thus decrease overall absorbance (Fankhauser).
MATERIALS & METHODS:
Absorbance levels of a 40% ONP concentration in 5 mL were measured at wavelengths ranging from 400-600 nm by 20 nm intervals to find the optimal wavelength for testing. Absorbance levels of a pH 5 buffer and ONP solution were measured at amounts of ONP ranging from 0 to .40 μ mol. A blank was made containing 20% lactase enzyme in 5 mL. In a small beaker 15 mL of pH 5 buffer and 6 mL of enzyme were mixed. To each of 5 cuvettes, initially 1 mL of Na2CO3 was added. As soon as 3 mL of ONPG was added to the small beaker, creating a 0.05 μ mole concentration of ONP, a timer was started and 4 mL of solution was added to the cuvette labeled 0. Four mL of solution was transported from the beaker to each of the 4 remaining cuvettes at the appropriate times of 2, 4, 6, and 8 minutes. After the final 8 minute cuvette was filled the absorbance levels of each cuvette were measured at 415 nm. This experiment was completed 3 times and then repeated 3 times again using both a pH 6 and 7 buffer.
Figure 1 shows the changes in the absorbance level as the concentration of ONP was increased. Figure 2 shows the absorbance levels of 0.050, 0.017, and 0.016 μ mol ONP at the different pH levels of 5, 6, and 7 respectively over a period of 8 minutes. Figure 3 depicts the combined results of both figure 1 and 2, and shows how the concentration of ONP changed over a time period of 8 minutes at the different pH levels.
Figure 1: Standard Concentration Curve of ONP
As the concentration of ONP increased, so did the level of absorbance. y=0.8613x R2=0.9995 n=3
Figure 2: Absorbance Curve for ONP
Diamonds are pH 5 (y=0.1044x + 0.0615, R2=0.9926), Squares are pH 6 (y=0.0138x + 0.0161, R2=0.9968), and Triangles are pH 7 (y=0.0037x + 0.0147, R2=0.993). As time passed the level of absorbance increased.
Figure 3: Concentration of ONP vs. Time
Diamonds are pH 5 (y= 0.1212x + 0.0714, R2=0.9926), Squares are pH 6 (y= 0.016x + 0.0714, R2=0.9962), and Triangles are pH 7 (y= 0.0044x + 0.0167, R2=0.9952). As time passed the concentration of ONP rose.
In the experiment, the two variables tested were the change in concentration of lactase enzyme precipitate in solution and the different pH values. The first hypothesis stated that as the concentration increased so would the absorption level. According to Figure 1 such is the case where as the total concentration of ONP increased, the absorption increased steadily in an all but linear fashion. This shows that an increased concentration of enzyme within a reaction, whether lactase or not, could mean increased activity and thus a better catalysis of reactants, such as lactose, into more usable product, such as glucose and galactose (Phillips). The second hypothesis stated that the maximum absorption levels from the reaction of ONPG to produce ONP precipitate would be highest at a pH of 5 and decrease as a solution got farther from that optimal pH value (Fankhauser). As shown in Figure 2 the tests acted exactly as was originally predicted. Figure 2 mainly shows that lactase enzyme has a very small window of for optimal pH, centering around 5 and decreasing drastically at a pH of 6. This shows that the body’s ability to keep pH values in the digestive tract, as well as other areas, mostly constant, is crucial to the survivability of life. If pH values are not within the optimum for an enzyme to function, it not only lowers production but also begins to unfold the proteins and thus denatures the enzyme and eventually halts the catalysis of the reaction if the pH is too far to either side of the optimum. This relates back to the rate of enzyme catalysis of lactose in the human digestive tract and how fluctuations in pH can cause issues such as lactose intolerance (Phillips). While the optimum pH range of lactase enzyme is shown to be very small, it is not the case for all enzymes. Every protein reacts differently when
introduced to new surrounding environments. In some cases proteins and thus enzymes can have a large tolerance to changes in pH; such is the case with acid phosphatase, an enzyme found throughout the body as well as in blood. Acid phosphatase is shown to have an optimum pH ranging anywhere from 4.9 to 6.0 (Gundlach 444). This reiterates the profound and necessary ability to regulate pH levels throughout the different systems of the body. While both concentration of ONP and the optimum pH ranges are discussed and tested in this lab, one major factor that was not observed in detail was time. The lactase enzyme catalyzed the reaction and each experiment was set to run over the course of 8 minutes, one possible change that could be made would be to run the experiment for a longer period of time to see its effects on the rate of production of ONP. In both Figure 2 and 3 the reaction was tested at a final time of 8 minutes, but in each of the tests it was observed that the absorption level as well as the concentration level of ONP was still climbing. It is possible that at letting the reaction run for a longer period of time could have shown different results as well as show an optimum time for the reaction to run to go along with the optimum pH level of 5. The conclusions drawn by these experiments, greatly help in the understanding of how lactase, as well as other enzymes, help in catalyzing reactions throughout the body such as the breakdown of the lactose carbohydrate found in milk in the digestive tract, and how these enzymes react to changes in their surrounding environment.
Fankhauser, David. “LACTASE pH OPTIMUM.” . University of Cincinnati Clermont College,, 21 Nov 2009. Web. 1 May 2013. . Gundlach, G., and E. Luttermann-Semmer. “The Effect of pH and Temperature on the Stability and Enzymatic Activity of Prostatic Acid Phosphatase Studies on the Optimization of a Continuous Monitored Determination of Acid Phosphatase, II.” Biochemisches Institut am Klinikum der Justus Liebig Universität Gießen. 25. (1987): 441-446. Print. Phillips, Theresa. “Enzymes Used in the Dairy Industry .” Biotech / Biomedical. About.com, n.d. Web. 1 May 2013. .