Lab 4 (Enzyme Activity in Yeast Fermentation)

Enzyme Activity in Yeast Fermentation

Yeast performs alcoholic fermentation:

Glucose → Ethanol + CO₂ + ATP

The carbon dioxide (CO₂) produced during fermentation inflates a balloon attached to the flask. The size of the balloon reflects the amount of fermentation occurring.

Enzymes involved in fermentation are affected by:

• Temperature – influences molecular movement and can cause denaturation.

• pH – affects ionic bonds and protein structure.

Independent variables + Dependent variable + Controlled variables

Independent variables = Temperature and pH

Dependent variable = Balloon circumference (cm) - indicates CO₂ production

Controlled variables = Yeast volume, glucose concentration, total volume, incubation time, flask size, balloon size

Predict which temperature (4°C, 25°C, or 37°C) will produce the most CO₂ and explain why.

I predict that the flask at 37°C will produce the most CO₂. Enzyme activity increases with temperature up to an optimum because molecules gain kinetic energy, so yeast enzymes and glucose collide more frequently and eventually accelerate to generate energy to react.

Predict which pH (4 or 8) will produce more CO₂ and explain why.

I predict that pH 4 will produce more CO₂ than pH 8 and yeast naturally lives in mildly acidic environments, and at pH 8 the excess of OH⁻ ions alters the charges, distorting the active site so glucose binds less effectively, which might lower the rate of fermentation.

Which temperature produced the most CO₂? Explain why.

• 37°C, Higher temperature gives molecules more kinetic energy, so enzyme and substrate molecules collide more often and with enough energy to form enzyme-substrate complexes.

• 37°C is also closest to the optimal range for yeast fermentation enzymes (roughly 30–35°C), so the enzymes were working near their maximum reaction rate.

Why does a very cold temperature (4°C) reduce enzyme activity?

• Cold molecules move slowly and have low kinetic energy.

• Enzymes and substrate collide far less frequently, and fewer collisions have the correct orientation and energy to react.

Why does elevated temperature (37°C) affect enzyme activity compared to 25°C?

37°C is nearer the enzyme's optimum, so the rate increases: more frequent, and more energetic collisions.

How did pH 8 compare to pH 4?

pH 8 produced much less CO₂, than pH4. Yeast enzymes favor mildly acidic conditions like (pH4), so the basic environment like (pH8) suppressed activity.

How does pH affect enzyme structure and function?

pH alters charges on amino acid R groups which breaks ionic and hydrogen bonds which changes tertiary structure.

Which variable had the stronger effect: temperature or pH?

Temperature had the stronger effect

Was your hypothesis supported?

My hypothesis that fermentation would be fastest at 37°C and at acidic pH was supported. One unexpected result was that the 4°C flask still produced a small amount of gas, showing enzymes are slowed rather than stopped by cold. Possible sources of error include balloons not being sealed tightly, thickness of the ballon, uneven yeast distribution,.

Conclusion
• The effect of temperature on enzyme activity
• The effect of pH on enzyme activity
• The practical applications of understanding enzyme activity in fermentation

In conclusion the temperature had a strong, effect on enzyme activity in this experiment, CO₂ production increased from 4 cm at 4°C to 22 cm at 37°C, because higher kinetic energy increases the frequency of successful enzyme substrate collisions. Acidic pH 4 flask producing roughly twice the CO₂ of the pH 8 flask. The practical applications of understanding enzymes can be applied in brewing and baking, like food preservation, where refrigeration slows microbial enzymes to prevent spoilage.

Enzymes: how they work

• Enzyme = a biological catalyst (usually a protein) that speeds up a chemical reaction by lowering the energy needed to start it.

• The substrate binds to the active site, forming an enzyme–substrate complex. The enzyme is not used up; it is reused again and again.

What is an enzyme, and why can a small amount do a lot of work?

A protein catalyst that speeds up reactions. It isn’t used up, so each enzyme molecule is reused on many substrate molecules.

What is denaturation?

Loss of the enzyme’s 3-D shape when the bonds holding it break (too much heat or extreme pH). The active site changes, so the substrate can’t bind and the enzyme stops working.

The Fermentation Experiment: setup and variables

Yeast fermentation (no oxygen needed):

• glucose (C6H12O6) → 2 ethanol + 2 CO2 (+ a little ATP).

• Yeast enzymes run this reaction.

• The CO2 gas inflates the balloon,

• a bigger balloon = more CO2 = more enzyme activity.

Identify the independent, dependent, and controlled variables, and the control group

• Independent (what YOU Change)

  • temperature (4, 25, 37 °C) and pH (4, 8)

• Dependent (what YOU Measure)

  • balloon circumference (cm), which indicates CO2 production

• Controlled (kept the SAME)

  • yeast volume, glucose concentration, total volume, incubation time, flask size, balloon size

Which two flasks would you compare to see ONLY the effect of pH at room temperature?

T2 vs P1: both at 25 °C, so pH is the only difference

What gas inflates the balloon, and why does balloon size show enzyme activity?

CO2 from fermentation. Faster enzymes → more CO2 → larger circumference.

Why does a very cold temperature (4 °C) reduce enzyme activity?

• Molecules have low kinetic energy, so enzymes and glucose collide less often and with less energy.

• The enzyme is not destroyed, just slowed.

• Warming it restores activity (reversible).

Why did 37 °C produce more CO2 than 25 °C? What would happen at 60 °C?

• 37 °C is near the optimum: more frequent, more energetic collisions.

• At 60 °C, heat breaks the hydrogen bonds holding the enzyme’s shape → denatured → activity drops sharply and doesn’t return.

How does pH affect enzyme structure and function?

• pH changes the H+ concentration → charges on R-groups change → the ionic and hydrogen bonds that hold the shape break or rearrange → the active site changes shape → substrate binds poorly. Extreme pH = permanent denaturation.

How did pH 8 compare to pH 4 in your data?

Almost the same: 25 °C → 13.3 vs 13.4 cm (0.1 cm apart). At 37 °C, pH 8 was slightly higher (16.2 vs 15.3 cm). pH 8 did not reduce fermentation. Physiology · Lab 4 Study Guide: Enzyme Activity in Yeast Fermentation

Decision tree: why did enzyme activity drop?

Which flask produced the most CO2? Which temperature?

• P2 (37 °C, pH 8): +6.2 cm.

• At pH 4 the winner was T3 (37 °C): +5.3 cm.

• Either way, 37 °C produced the most.

Which variable had the stronger effect, temperature or pH?

Temperature. 25 → 37 °C added 1.9 cm (pH 4) and 2.9 cm (pH 8). pH 4 → 8 changed only 0.1 cm (25 °C) and 0.9 cm (37 °C).

Why does refrigeration keep food from spoiling?

• Cold slows the enzymes of bacteria and fungi (low kinetic energy → fewer collisions),

• so they grow and break down food more slowly. It slows them; it doesn’t kill them.

Self-Test (exam format: MC · True/False · Fill-in)

Part A · Multiple choice (1–15) 1. Enzymes are best described as: a) biological catalysts that speed up reactions b) sugars that store energy c) fats that make up membranes d) molecules used up in every reaction 2. The part of the enzyme where the substrate binds is the: a) R-group b) cofactor c) active site d) cell membrane 3. In this lab, the dependent variable was: a) temperature b) pH c) yeast volume d) balloon circumference (CO2) 4. The gas that inflated the balloons was: a) oxygen b) carbon dioxide c) nitrogen d) hydrogen 5. The products of yeast fermentation are: a) ethanol + CO2 b) water + oxygen c) glucose + ATP only d) lactic acid only 6. A very cold temperature (4 °C) slows enzymes because: a) the enzymes are denatured b) molecules move slowly, so there are fewer collisions c) the pH changes d) the glucose freezes solid 7. Above an enzyme’s optimum temperature: a) the enzyme keeps working faster forever b) molecules slow down c) bonds break and the active site loses its shape (denaturation) d) glucose turns into protein 8. Which of these is REVERSIBLE? a) heat denaturation b) denaturation by extreme pH c) boiling the yeast d) slowing caused by cold 9. pH affects an enzyme by changing: a) the amount of glucose b) the charges on amino-acid R-groups c) the size of the flask d) the color of the yeast 10. Which flask was the control? a) T2 (25 °C, pH 4) b) T1 (4 °C, pH 4) c) T3 (37 °C, pH 4) d) P2 (37 °C, pH 8) 11. To see ONLY the effect of pH at 25 °C, compare T2 with: a) T1 b) T3 c) P1 d) P2 12. Which flask had the largest change in circumference? a) T1 b) T3 c) P1 d) P2 13. Which is a CONTROLLED variable? a) temperature b) glucose concentration c) balloon circumference d) pH 14. Yeast fermentation enzymes work best at about: a) pH 1–2 b) pH 8–10 c) pH 12–14 d) pH 4–6 15. Why repeat each condition three times? a) to make results more reliable b) to use up the yeast c) to change the independent variable d) to heat the flasks Part B · True / False (16–25) 16. T / F Enzymes are used up during the reaction. 17. T / F Most enzymes are proteins whose shape is held by hydrogen and ionic bonds. 18. T / F Every enzyme works best at pH 7. 19. T / F Refrigeration slows food spoilage by slowing microbial enzymes. 20. T / F A bigger balloon means more CO2 was produced, so there was more enzyme activity. 21. T / F In this experiment, pH 8 greatly reduced CO2 production. 22. T / F Cold temperatures permanently destroy enzymes. 23. T / F In your data, the 4 °C flask produced slightly more gas than the 25 °C flask. 24. T / F In this experiment, temperature had a stronger effect than pH. 25. T / F The independent variable is the thing you measure.

Part C · Fill in the blank (26–40) 26. Temperature at which an enzyme works fastest = its ______ temperature 27. Permanent loss of an enzyme’s shape = ______ 28. The molecule an enzyme acts on = the ______ 29. Enzyme + substrate bound together = the enzyme–______ complex 30. The sugar the yeast used = ______ 31. The gas measured with the balloon = ______ 32. T3: 10 → 15.3 cm. Change = ______ cm 33. P2: 10 → 16.2 cm. Change = ______ cm 34. pH 4, 25 → 37 °C: 13.4 → 15.3 cm. Difference = ______ cm 35. (new numbers) 10.0 → 12.7 cm. Change = ______ cm 36. Human enzymes work best near ______ °C 37. Lower pH = more ______ ions 38. pH 8 is acidic or basic? ______ 39. Bonds broken by heat that hold enzyme shape: ______ bonds 40. Making CO2 and ethanol from glucose without oxygen = ______