The experiment investigated the effects of two separate variables on the productivity of lactase enzyme. The variables tested were the concentration of o-nitrophenol (ONP) and the pH of the solution. The experiment involved measuring the light absorbance levels of different solutions with varying ONP concentrations. Additionally, three different pH values (5, 6, and 7) were tested, and their absorbance levels were recorded over an 8-minute period. The data obtained from both experiments were then used to determine the highest enzyme activity rates at specific ONP concentrations and pH levels. The results revealed that a pH of 5 is optimal for lactase enzyme productivity.
Lactase enzyme is utilized in the food industry to hydrolyze lactose, a milk sugar, into glucose and galactose. Lactase deficiency hinders the digestion of lactose, causing lactose intolerance, characterized by digestive issues, cramps, and potentially diarrhea after consuming dairy products (Phillips). By utilizing extrinsic lactase, milk can be processed into a denser and smoother consistency for creating dessert items like ice cream.
Enzymes, which are proteins, reduce the energy needed for essential chemical reactions crucial to survival. Similar to other proteins, enzymes change shape based on their environment, affecting productivity. Various factors like temperature, enzyme concentration, and pH influence enzyme productivity (Gundlach 441).
In this experiment, the effects of concentration and pH on lactase enzyme productivity were investigated. The absorbance levels of the enzyme were measured while changing the concentration and pH over time. The hypothesis was that increasing the concentration of enzyme (as ONP precipitate) would increase the absorption levels. It was also hypothesized that the reaction would have the highest absorbance at a pH of 5, as other pH levels would alter the enzyme’s shape and decrease overall absorbance (Fankhauser).
MATERIALS & METHODS:
Absorbance levels of a 40% ONP concentration in 5 mL were measured at various wavelengths from 400-600 nm with intervals of 20 nm. Absorbance levels of an ONP solution and a pH 5 buffer were measured using ONP amounts ranging from 0 to .40 μmol. A blank solution was created by combining 20% lactase enzyme with 5 mL. In a small beaker, 15 mL of pH 5 buffer and 6 mL of enzyme were mixed. Each of the 5 cuvettes initially contained 1 mL of Na2CO3. As soon as 3 mL of ONPG was added to the small beaker to create a concentration of 0.05 μmol of ONP, a timer was started. At appropriate times (2, 4, 6, and 8 minutes), 4 mL of the solution from the beaker were added to each remaining cuvette. After filling the final cuvette at the 8-minute mark, the absorbance levels of all cuvettes were measured at a wavelength of 415 nm. This entire experiment was repeated three times and then three more times using both a pH 6 and 7 buffer.
Figure 1 presents the variations in absorbance level with increasing concentration of ONP. Figure 2 represents the absorbance levels of 0.050, 0.017, and 0.016 μ mol ONP at pH levels 5, 6, and 7 respectively, during an 8-minute timeframe. Figure 3 displays the consolidated outcomes from both figure 1 and 2, illustrating the changes in ONP concentration at different pH levels over an 8-minute period.
Figure 1: Standard Concentration Curve of ONP
As the concentration of ONP increased, the level of absorbance also increased. y=0.8613x R2=0.9995 n=3
Figure 2: Absorbance Curve for ONP
Diamonds represent pH 5 (y=0.1044x + 0.0615, R2=0.9926), squares represent pH 6 (y=0.0138x + 0.0161, R2=0.9968), and triangles represent pH 7 (y=0.0037x + 0.0147, R2=0.993). As time progressed, the absorbance level increased.
Figure 3: Concentration of ONP vs. Time
Diamonds: pH 5 (y= 0.1212x + 0.0714, R2=0.9926)
Squares: pH 6 (y= 0.016x + 0.0714, R2=0.9962)
Triangles: pH 7 (y= 0.0044x + 0.0167, R2=0.9952)
As time passed the concentration of ONP rose.
The experiment examined lactase enzyme concentration and pH values as variables. The first hypothesis proposed that increasing lactase concentration would lead to higher absorption levels. Figure 1 supports this hypothesis, showing a steady increase in absorption with higher total ONP concentration. This indicates that greater enzyme concentration results in increased activity and improved catalysis of reactants.
The second hypothesis stated that maximum absorption levels would be highest at pH 5 and decrease as the solution moved away from this optimal pH value. Figure 2 validates this hypothesis, demonstrating that lactase enzyme has a narrow optimal pH range centered around 5 and significantly decreases at pH 6. This emphasizes the significance of maintaining consistent pH values in the digestive tract and other areas for life survival (Phillips, Fankhauser). If the pH values are not within the enzyme’s optimum range, it not only reduces production but also causes protein unfolding, denaturing the enzyme, and ultimately halting reaction catalysis if the pH deviates too much from ideal conditions.
This finding is relevant to understanding lactose digestion in the human digestive tract and how fluctuations in pH can result in issues like lactose intolerance (Phillips). While the optimum pH range for lactase is narrow, this is not true for all enzymes as each protein reacts differently when introduced to new environments.In certain cases, proteins and enzymes can endure significant pH changes. Acid phosphatase is an example of such an enzyme, which is present in the body and blood. Its ideal pH ranges from 4.9 to 6.0 (Gundlach 444). This highlights the crucial need to regulate pH levels in different bodily systems. While this lab examines both ONP concentration and optimal pH ranges, it fails to thoroughly investigate time as a factor. The catalyzed reaction by lactase enzyme lasted for 8 minutes in each experiment. One potential modification could be extending the duration of the experiment to observe its impact on ONP production rate. Both Figure 2 and Figure 3 test the reaction at the 8-minute mark, but in each case, ONP absorption and concentration levels continue to rise. If the reaction had been allowed to progress for a longer period, it may have yielded different outcomes indicating an optimal reaction time alongside the optimal pH level of 5.The findings from these experiments greatly enhance our understanding of how lactase and other enzymes facilitate reactions in the body by breaking down lactose in milk within the digestive tract.Furthermore, these findings shed light on how these enzymes respond 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. .