Lactase Supplement at Refrigerator Temperature
A student investigates whether a lactase supplement hydrolyzes lactose at refrigerator temperature. She prepares 5% lactose solution in pH 6.0, 7.0, and 8.0 buffers. For each pH she adds 0.10 g crushed tablet to 10 mL substrate at 4 °C, 25 °C, and 37 °C and measures glucose with a handheld meter at 0, 10, 20, and 30 min. One 37 °C trial uses whole milk instead of lactose solution because she ran out of substrate. The meter reads ‘HI’ for the 37 °C pH 7 trial at 20 and 30 min. She reports: ‘The enzyme is heat-loving and works best at pH 7; it will not work in a refrigerator.’ Her graph shows glucose rising fastest at 37 °C, pH 7, but the 4 °C tubes also rise from 0.2 to 1.1 mmol/L over 30 min. The teacher notes the meter’s detection limit is 0.1–33.3 mmol/L and that milk contains lactose but also its own sugars and proteins.
1. Evaluate the student’s claim that the enzyme is ‘heat-loving’ and ‘will not work in a refrigerator.’ Use the data and the meter’s limits.
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The claim is not supported as stated. The 4 °C tubes rise from 0.2 to 1.1 mmol/L, so activity is reduced, not absent; ‘will not work’ is false. The 37 °C pH 7 reading of ‘HI’ means glucose exceeded 33.3 mmol/L, so the rate cannot be quantified or compared with other trials. ‘Fastest’ is plausible but not measured. ‘Heat-loving’ overgeneralizes from only 4, 25, and 37 °C; the optimum could be 37 °C or higher, but no 50–60 °C data exist, and the enzyme may denature above 40 °C. The milk trial adds lactose but also milk sugars and proteins, so it is not a clean temperature trial. A defensible conclusion would say activity is present at 4 °C and higher at 37 °C, with the caveat that the 37 °C rate was off-scale.
2. Identify two confounding variables or missing controls and explain how each weakens the conclusion.
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First, whole milk versus buffered lactose solution is a confound: milk has its own sugars, proteins, pH, and buffering capacity, any of which can affect the meter reading or enzyme stability. A difference in that trial cannot be attributed to temperature alone. Second, there is no boiled-enzyme control, so any glucose increase could come from nonenzymatic hydrolysis, microbial contamination, or glucose in the tablet’s fillers. Third, there is no replication; each pH-temperature combination appears to be a single tube, so one bubble or timing error can drive the result. These weaknesses mean the student has observed glucose production under several conditions, but she has not isolated the enzyme’s temperature and pH response.
3. Propose a revised experiment that would allow a defensible comparison of pH and temperature effects, including at least two controls.
Read the worked answer
Use one substrate lot-5% lactose in 50 mM buffer at pH 6, 7, and 8-and pre-incubate substrate and enzyme separately at 4, 25, 37, and 50 °C. Add a fixed mass of crushed tablet, then stop 1-mL aliquots at 0, 5, 10, 20, and 30 min by boiling or acidification. Include a boiled-enzyme control at each temperature, a substrate-only blank, and a vehicle control with inactive tablet filler. Run glucose standards and dilute samples into the meter’s 0.1–33.3 mmol/L range. Use three replicates and randomize tube order. Calculate initial rates from the linear phase rather than relying on a single endpoint. This design separates enzymatic hydrolysis from nonenzymatic glucose release and keeps the 37 °C samples on scale.