The Effect of Fructose as an Inhibitor in the Hydrolysis of Lactose

Suriyamas 1 The Effect of Fructose as an Inhibitor in the Hydrolysis of Lactose Research Question: How do varying concentrations of fructose (0.00 mM, 20.00 mM, 40.00 mM, 60.00 mM, 80.00 mM, and 100.00 mM) inhibit o-Nitrophenyl-β-D-galactopyranoside (ONPG) hydrolysis by lactase enzyme at 37°C for 20 minutes, as measured by absorbance at 420 nm using a spectrophotometer? Introduction and Background Information: Lactase is an essential digestive enzyme produced in the brush border of the small intestine that hydrolyses lactose into its absorbable monosaccharides, glucose and galactose (Goosenberg and Afzal). Lactose is a common disaccharide found in dairy products such as milk and cheese. However, some individuals develop lactose intolerance over their lifetimes due to reduced lactase production, often resulting from genetic mutations that downregulate lactase-encoding gene expression (MedlinePlus). This deficiency impairs lactose digestion, leading to gastrointestinal symptoms such as bloating, abdominal pain, and diarrhea (Goosenberg and Afzal). Another commonly consumed sugar is fructose, found in fruits, honey, and certain vegetables. Notably, studies have identified fructose as a competitive inhibitor of lactase (Song et al.), as its structural similarity to lactose allows it to bind to the enzyme’s active site. As a result, fructose directly competes with lactose for binding to lactase, reducing lactose binding and thereby decreasing the rate of lactose hydrolysis (VanDee et al.). Moreover, lactase functions optimally under conditions resembling the human small intestine: a slightly acidic to neutral pH of 6.0-7.0 and a temperature of 37°C (Verducci). These conditions will be maintained as controlled variables throughout this investigation to isolate the effect of fructose concentration on enzyme activity. This investigation aims to examine the effect of increasing fructose concentration on lactose digestion by lactase. To enable quantitative analysis of this enzyme activity, this investigation will use o-nitrophenyl-β-D-galactopyranoside (ONPG). ONPG is a synthetic compound similar to lactose that can be hydrolyzed by lactase and is widely used in scientific assays (Leksmono et al.). When ONPG is hydrolyzed by lactase, it produces D-galactose and ortho-nitrophenol (See Fig. 1), a yellow compound that absorbs strongly at 420 nm (Leksmono et al.). Fig. 1- Hydrolysis of ortho-nitrophenol-beta-D-galactopyranoside (Leksmono et al.) Therefore, the rate of ONPG hydrolysis can be quantified by measuring changes in absorbance at 420 nm using a spectrophotometer, where a higher absorbance reading corresponds to greater production of ortho-nitrophenol and higher lactase activity, while a lower absorbance reading indicates reduced ortho-nitrophenol formation and decreased enzyme activity.

Suriyamas 2 Hypotheses: Null Hypothesis (H0): Increasing the concentration of fructose in the solution will have no effect on the ONPG hydrolysis by the lactase enzyme, because fructose does not influence lactase activity under the experimental conditions. Alternative Hypothesis (Ha): If the concentration of fructose in the solution increases, then ONPG hydrolysis by lactase will decrease. This is because fructose acts as a competitive inhibitor, competing with ONPG for the lactase active site and reducing the formation of ONPG-lactase complexes, thereby lowering product formation (VanDee et al.). Thus, the highest fructose concentration (100.00 mM) will produce the lowest absorbance values. Variables: Range Manipulation Independent Variable: Concentration of Fructose 0.00 mM, 20.00 mM, 40.00 mM, 60.00 mM, 80.00 mM, and 100.00 mM These concentrations represent the concentration of fructose in the final reaction solution. Refer to the Method section, and under Preparation of Solutions, refer to “Fructose Solutions”. Dependent Variable: Absorbance (AU) of ortho-nitrophenol at 420 nm Measurement: Absorbance was measured using a Vernier Go Direct SpectroVis Plus Spectrophotometer calibrated at 420 nm. Control Variables What Why How Concentration and volume of ONPG and lactase Maintaining constant ONPG and lactase concentrations ensures changes in absorbance reflect fructose inhibition, not variations in substrate or enzyme. Refer to “0.01% Lactase Solution” and “ONPG Solution (5mM)” under Preparation of Solutions in the Method pH of the solution Maintaining pH at 7 ensures lactase activity is unaffected by pH changes, isolating the effect of fructose on ONPG hydrolysis (Popescu et al.). All stock solutions were prepared using a pH 7 buffer. Temperature of the solution Lactase functions optimally at 37 °C; deviations from this temperature affect enzyme efficiency, thereby altering the amount of ortho-nitrophenol produced and, consequently, the absorbance values. A double-boiler setup was used to maintain solutions at 37 °C (as a water bath was unavailable), with thermometers monitoring temperature throughout the experiment. Time allowed for reaction Longer or shorter times would change the amount of o-nitrophenol produced, thereby altering the measured absorbance values. All trials were allowed to react for a fixed 20-minute period using a digital stopwatch before absorbance was measured.

Suriyamas 3 Materials: Name of equipment Quantity Uncertainty o-Nitrophenyl-β-D-galactopyranoside (ONPG) 0.250g - Lactase enzyme (CAS # 9031-11-2) 0.200g - Fructose (CAS # 57-48-7) 20.000g - Buffer solution pH 7 250 mL - Distilled water 1 L - 50 mL beaker 5 - 250 mL beaker 2 - 600 mL beaker 1 - 2000 mL beaker 1 - 25 mL test tube 9 - Thermometer 1 ± 0.5°C Stirring rods 1 - 10 mL volumetric pipette 2 ± 0.04 mL Pipette filler 2 - 100 mL graduated cylinder 1 ± 0.5 mL 10 mL graduated cylinder 1 ± 0.05 mL Digital balance (rated 120 g) 1 ± 0.001 g Digital stopwatch 1 ± 0.01 s Micropipette (100 - 1000μL) 1 ± 2.5 μL Micropipette tips 8 - Vernier Go Direct SpectroVis Plus Spectrophotometer 1 ± 0.001 AU Laptop with Vernier Spectral Analysis Software 1 - Hot plate 1 - Aluminium foil 30×40 cm - Weigh boat 1 - Scoopula 1 - Masking tape 1 roll - Permanent marker 1 -

Suriyamas 4 Method: Labelling materials 1. Label two, seperate 10 mL volumetric pipettes “Buffer” and “Distilled Water”. Preparation of Solutions Fructose Solutions 1. Place a clean weighing boat on a digital balance and tare to zero. 2. Measure 1.440 g of fructose using a scoopula, then transfer it into a 50 mL beaker. 3. Add 20 mL of pH 7 buffer by using the “Buffer” 10 mL volumetric pipette twice. 4. Stir with a glass stirring rod until all visible particles have dissolved. 5. Add 20 mL of distilled water using a 10 mL volumetric pipette labeled “Distilled Water” in successive transfers, and stir for 30 seconds using the glass stirring rod. 6. Using masking tape and a marker, label the beaker “Fructose 20.00 mM”. 7. Wash and dry the stirring rod, scoopula, and weighboat using distilled water and a paper towel. 8. Repeat steps 2-6 for the remaining concentrations: 2.880 g for 40.00 mM, 4.320 g for 60.00 mM, 5.770 g for 80.00 mM, and 7.210 g for 100.00 mM. Label each beaker accordingly. (See Fig. 2) Fig. 2 - Prepared fructose solutions (by Author) Lactase Solution (0.01%) 1. Place a clean weighing boat on a digital balance and tare to zero. 2. Measure 0.100 g of lactase powder using a scoopula and transfer it into a 250 mL beaker. 3. Using the “Distilled Water” 10 mL volumetric pipette, rinse the weighing boat with distilled water to collect any remaining powder into the beaker. 4. Using the “Distilled Water” 10 mL volumetric pipette, add 50 mL of distilled water in successive transfers. Stir with a glass rod until fully dissolved. 5. Using the “Buffer” 10 mL volumetric pipette, add 50 mL of pH 7 buffer solution in successive transfers. Stir with the glass rod for 10 seconds. 6. Label the beaker “Lactase Solution” 7. Wash and dry the stirring rod, scoopula, and weighboat using distilled water and a paper towel.

Suriyamas 5 ONPG solution (5 mM) 1. Place a clean weighing boat on a digital balance and tare to zero. 2. Measure 0.250 g of ONPG powder and transfer to a 250 mL beaker. 3. Measure 83 mL of pH 7 buffer solution using a 100 mL graduated cylinder and add it to a beaker. 4. Stir with a glass stirring rod until fully dissolved. 5. Thoroughly rinse the graduated cylinder with distilled water. 6. Using the same graduated cylinder, measure 83 mL of distilled water and add it to the beaker. Stir to mix. 7. Label the beaker “5 mM ONPG Solution” Double Boiler Setup 1. Scrunch a 30×40 cm piece of aluminium foil into a 6×2 cm disk to serve as a prop (See Fig. 3). 2. Fill a 2000 mL beaker with 200 mL of tap water and place the foil prop in the center. 3. Place a 600 mL beaker containing 400 mL of tap water on the prop inside the 2000 mL beaker. 4. Using a 10 mL graduated cylinder twice, transfer 15 mL (10 + 5) of each solution (ONPG, lactase, all five fructose solutions, distilled water, and tap water) into separate, labelled 25 mL test tubes. Place all the test tubes in the 600 mL beaker. 5. Insert a thermometer into the tap water test tube as a reference. 6. Place the 2000 mL beaker on a hot plate and set it to the lowest setting. Once the thermometer in the reference test tube reads 37 °C, all solutions are ready for use. (See Fig. 4) Fig. 3 - Aluminium foil prop (by Author) Fig. 4 - Double Boiler Setup (by Author)

Suriyamas 6 Set up to Prevent Mix-up and Cross-contamination 1. Place a 30 cm strip of masking tape on the bench and label according to Fig. 5. 2. Lay a clean tissue next to the tape and place one clean micropipette tip in front of each label. For all subsequent procedural steps, micropipette tips must be swapped to the designated tip for the corresponding solution before each transfer, and returned to their place afterwards. Fig. 5 - Setup for micropipette tips for each solution (by Author) Calibrating the Spectrophotometer 1. Using a micropipette, successively transfer 1760 μL of ONPG solution (1000 μL + 760 μL) and 200 μL of distilled water from the double boiler into a cuvette. 2. Cap the cuvette and gently invert it 6 times, ensuring no bubbles form. 3. Turn on the Vernier Go Direct SpectroVis Plus Spectrophotometer. 4. Using a laptop and the Vernier Spectral Analysis software, connect to the spectrophotometer via Bluetooth and select “Absorbance vs. Time (Kinetics)”. 5. Following the program instructions, allow the spectrophotometer to warm up, insert the prepared cuvette, and press calibrate. Set the wavelength to 420 nm and confirm the baseline reads 0 AU. Control Trials 1. After calibration, successively transfer 1760 μL of ONPG solution (1000 μL + 760 μL) and 200 μL of distilled water from the double boiler into a cuvette using a micropipette. 2. Micropipette 40 μL of lactase solution into the cuvette and start a stopwatch simultaneously. 3. Cap the cuvette and gently invert it 6 times to prevent bubbles from forming. 4. Prepare another cuvette by using a micropipette to transfer 1760 μL of ONPG solution (1000 μL + 760 μL) and 200 μL of distilled water from the double boiler into a cuvette. 5. After 2 minutes, micropipette 40 μL of lactase solution into the cuvette. 6. Cap the cuvette and gently invert it 6 times to prevent bubbles from forming. 7. Repeat steps 4-6 for three more trials, staggering each trial by 2 minutes. 8. After each trial has reacted for 20 minutes, insert the cuvetter into the spectrophotometer, and record the absorbance at 420nm. 9. Rinse all cuvettes and caps with distilled water.

Suriyamas 7 Experimental Trials 1. Follow the steps from “Calibrating the Spectrophotometer” using the 20.00mM fructose solution in place of distilled water. 2. Conduct steps 1-9 from “Control Trials” using the 20.00mM fructose solution in place of distilled water. 3. Repeat steps 1-2 with the 40.00 mM, 60.00 mM, 80.00 mM, and 100.00 mM fructose solutions. Safety: - Glassware can break into sharp fragments, causing serious injury. Therefore, gloves, a lab coat, and goggles must be worn, and any broken glass must be collected using a broom and dustpan and disposed of in a designated glass container. - ONPG and ortho-nitrophenol are harmful chemical irritants if ingested or come into contact with the skin or eyes (National Center for Biotechnology Information); PPE must be worn at all times, and any affected areas should be rinsed immediately with running water. - The hot plate must operate above 37 °C to maintain the required temperature in the double-boiler setup, posing a burn hazard. Experimental trials should be conducted on the opposite side of the lab bench while it is in use to avoid accidental contact. Environmental Concerns: ONPG is toxic to aquatic life, so all waste will be collected and disposed of in accordance with the school’s chemical waste guidelines (Ibrahim et al.; Edvotek). Ethical Concerns: No ethical concerns arise, as the experiment only uses commercially sourced enzymes and involves no human or animal subjects. Raw Data: Qualitative Data: ● As fructose concentration increased, the intensity of the yellow color produced after 20 minutes became progressively lighter, indicating reduced ortho-nitrophenol formation. ● The control solution turned deep yellow, whereas higher fructose concentrations appeared pale yellow to nearly transparent. ● At higher fructose concentrations (80-100 mM), color development appeared slower, with less visible change during the early stages of reaction. ● Absorbance values were automatically recorded every other second. Minor inconsistencies in the timing of staggered trials may have caused slight variations in the data.

Suriyamas 8 Quantitative Data: Table 1 - Raw Data Table of Absorbance Measured at 420nm for each Concentration of Fructose Concentration of Fructose (mM) Increase in Absorbance AU (± 0.001 AU) Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 0 0.145 0.150 0.142 0.147 0.144 20 0.123 0.126 0.131 0.124 0.122 40 0.109 0.112 0.108 0.110 0.107 60 0.090 0.093 0.099 0.091 0.088 80 0.081 0.082 0.078 0.081 0.084 100 0.070 0.066 0.062 0.064 0.061 Processed Data: Table 2 - Processed Data Table of Average Increase in Absorbance, Standard Deviation, and Total % Uncertainty of Absorbance Concentration of Fructose (mM) Average Increase in Absorbance (± 0.001 AU) Standard Deviation Total % Uncertainty of Absorbance (%) 0 0.147 0.00305 8.46 20 0.125 0.00356 8.58 40 0.109 0.00192 8.70 60 0.092 0.00421 8.87 80 0.081 0.00217 9.02 100 0.065 0.00358 9.32 Sample Calculations for 40 mM Fructose Concentration: Table 3 - Sample Calculation for the Mean Absorbance of 40 mM Fructose Concentration Mean 𝑥( ) 𝑥 = 𝑖=1 𝑛 ∑ 𝑥𝑖 𝑛 𝑥 = 0.109 + 0.112 + 0.108 + 0.110 + 0.107 5 AU𝑥 ≈ 0. 109

Suriyamas 9 Table 4 - Sample Calculation for the Standard Deviation of 40 mM Fructose Concentration Standard Deviation σ( ) σ = 𝑖=1 𝑛 ∑ (𝑥𝑖−𝑥)2 𝑛 σ = (0.109 − 0.109)2 + (0.112 − 0.109)2+ (0.108 − 0.109)2+ (0.110 − 0.109)2+ (0.107 − 0.109)2 5 σ ≈ 0. 00192 Table 5 - Sample Calculation for the Total % Uncertainty in the Increase of Absorbance of 40 mM Fructose Concentration Calculation for Increase in Absorbance % Uncertainty Propagation (Total % Uncertainty) ∆𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 = ∆𝑉𝑂𝑁𝑃𝐺 𝑉𝑂𝑁𝑃𝐺 + ∆𝑉𝐹𝑟𝑢𝑐𝑡𝑜𝑠𝑒 𝑉𝐹𝑟𝑢𝑐𝑡𝑜𝑠𝑒 + ∆𝑉𝐿𝑎𝑐𝑡𝑎𝑠𝑒 𝑉𝐿𝑎𝑐𝑡𝑎𝑠𝑒 + ∆𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒𝑆𝑝𝑒𝑐𝑡𝑟𝑜𝑝ℎ𝑜𝑡𝑜𝑚𝑒𝑡𝑒𝑟 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒𝐴𝑣𝑒𝑟𝑎𝑔𝑒 ∆𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 = ∆𝑚𝑖𝑐𝑟𝑜𝑝𝑖𝑝𝑒𝑡𝑡𝑒 ×2 1760 + ∆𝑚𝑖𝑐𝑟𝑜𝑝𝑖𝑝𝑒𝑡𝑡𝑒 200 + ∆𝑚𝑖𝑐𝑟𝑜𝑝𝑖𝑝𝑒𝑡𝑡𝑒 40 + ∆𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒𝑆𝑝𝑒𝑐𝑡𝑟𝑜𝑝ℎ𝑜𝑡𝑜𝑚𝑒𝑡𝑒𝑟 0.109 ∆𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 = 2.5×2 1760 + 2.5 200 + 2.5 40 + 0.001 0.109 ∆𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 = 0. 00284090909 + 0. 0125 + 0. 0625 + 0. 00917431192 ∆𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 = 0. 08701522101 ≈ 8. 70% Table 6 - Sample Calculation of the Lower and Upper Thresholds for Outliers in the Raw Data for 40 mM Fructose Concentration Calculation of Threshold(s) for Outliers The first and third quartiles (Q1) and (Q3) were calculated using the 1-Var Stats function on a TI-84 graphing calculator after entering the full ordered dataset from Table 1. ,𝑄1 = 0. 081 𝑄3 = 0. 124 𝐼𝑄𝑅 = 𝑄3 − 𝑄1 𝐼𝑄𝑅 = 0. 124 − 0. 081 𝐼𝑄𝑅 = 0. 043

Suriyamas 10 Calculating Lower Threshold: 𝐿𝑜𝑤𝑒𝑟 𝑇ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 = 𝑄1 − (1. 5 × 𝐼𝑄𝑅) 𝐿𝑜𝑤𝑒𝑟 𝑇ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 = 0. 081 − (1. 5 × 0. 043) AU𝐿𝑜𝑤𝑒𝑟 𝑇ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 = 0. 017 Calculating Upper Threshold: 𝑈𝑝𝑝𝑒𝑟 𝑇ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 = 𝑄3 + (1. 5 × 𝐼𝑄𝑅) 𝑈𝑝𝑝𝑒𝑟 𝑇ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 = 0. 124 + (1. 5 × 0. 043) AU𝑈𝑝𝑝𝑒𝑟 𝑇ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 = 0. 189 Test for Statistical Significance: A one-way ANOVA from Social Science Statistics was conducted to compare the means of six groups, including the control, using a significance level of 0.05. The analysis yielded p < 0.001, indicating the differences are statistically significant and unlikely due to chance, suggesting a causal relationship between fructose concentration and absorbance, reflecting lactose digestion (Stangroom). Graph: Fig. 6 - Graph of Concentrations of Fructose (mM) vs the Average Increase in Absorbance (AU) (by Author using Logger Pro) The graph indicates a strong negative linear relationship between fructose concentration (mM) and the average increase in light absorbance (AU). A linear regression revealed a very strong negative correlation (R² = −0.9955), supporting the hypothesis that higher fructose concentrations reduce ONPG digestion.

Suriyamas 11 Conclusion: The data obtained from my experiment displays a clear negative relationship between fructose concentration and ONPG hydrolysis by lactase. Average absorbance at 420 nm decreased progressively from 0.147 AU at 0 mM fructose to 0.065 AU at 100 mM fructose (See Table 2), representing a 55.8% reduction in ortho-nitrophenol production across the tested range. This trend is strongly supported by the linear regression, with an R2 value of −0.9955, indicating that 99.55% of the variance in absorbance is attributable to the changes in fructose concentration. The one-way ANOVA test also yielded a p-value = < 0.001, well below the critical threshold of p ≤ 0.05, confirming the collected data is statistically significant and unlikely to result from random variation. Additionally, standard deviations across all groups ranged from ±0.00192 to ±0.00421 AU, and total percentage uncertainties ranged from 8.46% to 9.32% (See Table 2). While these percentage uncertainties are relatively large, the consistent directional trend across all five fructose concentrations and the low standard deviations within each group suggest that the variation does not undermine the conclusion. Therefore, the null hypothesis is rejected, and the alternative is accepted. My findings are consistent with the established mechanism by which fructose acts as a competitive inhibitor of lactose digestion. For example, a 2013 study by Song et al. investigated the enzymatic synthesis of lactulose from whey lactose and the kinetic effects of various sugars on lactase activity. Their results demonstrated that fructose acts as a competitive inhibitor, as shown by Lineweaver-Burk plots, in which increasing fructose concentrations increased the Michaelis constant (Km) without altering the maximum reaction velocity (Vmax) (Song et al.). This kinetic pattern is characteristic of competitive inhibition and confirms that fructose directly competes with lactose for the enzyme’s active site (Strelow et al.). Another study, conducted by Alpers and Cote (1971), used an in vivo perfusion method in the rat intestine to measure lactose hydrolysis in the presence of other dietary sugars. Their results demonstrated that fructose is a potent inhibitor of lactose hydrolysis under physiological conditions, mirroring the competitive inhibition observed in laboratory settings (Alpers and Cote). This provides strong physiological validation for my experiment, as it confirms that the interference I observed is a documented reality of the digestive process, where the concentration of sugars like fructose at the intestinal surface directly impacts the efficiency of lactose breakdown. Evaluation: In this investigation, five distinct fructose concentrations, along with a control, were tested in 20.00 mM increments. While this range was sufficient to establish a clear inhibitory trend, increasing the resolution of the independent variable would provide a more detailed mapping of the inhibition kinetics. For example, by using 10.00 mM increments, or extending the range to 200.00 mM. This higher density of data points would enable more precise measurements and identification of the concentration at which the inhibitory effect plateaus, offering insight into its competitive nature. Furthermore, a wider range would enable more quantitative comparability with established scientific findings on Km and Vmax values. While no significant outliers were identified in the raw data (See Table 6), the percentage uncertainty for each data point was relatively high, ranging from 8.46% to 9.32% (See Table 2). This stems primarily from the 6.25% uncertainty associated with micropipetting the 40 μL lactase volume (See Table 5), which is the largest source of potential error in the experimental design.

Suriyamas 12 Sources of Error Error Significance Improvement Volume of lactase enzyme in the final solution (Significant systematic error) Pipetting 40 μL of lactase with an uncertainty of ±2.5 μL (~6.25%) may alter enzyme concentration, affecting ONPG hydrolysis and absorbance values. Dilute the lactase stock so a larger volume can be pipetted, and adjust fructose and ONPG concentrations accordingly to maintain the intended final reaction composition. Temperature fluctuations in the double-boiler setup (Moderate systematic error) Manually maintaining the double boiler at 37 °C (±0.5 °C) may cause small fluctuations that disproportionally affect enzyme activity (LibreTexts). Use a thermostatically controlled water bath to ensure a stable 37°C throughout incubation. Delay between incubation and absorbance measurement (Moderate random error) Delays in transferring cuvettes may allow the reaction to continue, altering absorbance. Quench the reaction immediately after 20 minutes of reacting using sodium carbonate. Inconsistent stirring during the preparation of reaction mixtures (Minor random error) Manual stirring may unevenly distribute lactase, ONPG, and fructose solutions, causing slight variations in local concentrations and affecting enzyme kinetics across trials. Use a magnetic stirrer for a fixed time (10s) to ensure uniform mixing and an even distribution of all solutions before using them in the reaction. Evaporation of liquids at 37 °C during incubation (Minor systematic error) Evaporation at 37 °C may slightly increase reactant concentrations and absorbance. Cover all test tubes with stoppers inside the double-boiler to minimize evaporation and maintain stable solution concentrations. Extension: Investigate how varying ONPG substrate concentrations against a fixed fructose concentration would influence reaction rates, extending the current method, which held ONPG constant at 5 mM. From this data, I could also calculate the Michaelis constant (Km) and maximum velocity (Vmax) through a Lineweaver-Burk plot to mathematically validate the competitive nature of the inhibition. Another investigation could explore how the inhibitory strength of fructose fluctuates under non-optimal environmental stressors, such as the varying pH and temperature levels found throughout the digestive tract, moving beyond the strictly controlled pH 7.0 and 37°C conditions maintained in this study.

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