BIOL2010 Biochemistry:Effect In Yeast and its Culture

BIOL2010 Biochemistry:Effect In Yeast and its Culture

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BIOL2010 Biochemistry

Question:

Effect In Yeast and its Culture

Practical Exercise 5 –The Pasteur Effect In Yeast

The ‘Pasteur effect’ refers to the observation made by Louis Pasteur that alcoholic fermentation is suppressed in the presence of oxygen. At the same time, glucose consumption is greatly decreased relative to growth of the organism and generation of energy. It occurs as the metabolism of an organism switches from anaerobic to aerobic pathways (see Fig. 5.1). 

Figure 5.1. 

Alternative metabolic pathways used by organisms in the presence/absence of oxygen. In anaerobic conditions, pyruvate is reduced to either lactic acid or ethanol and CO2 in order to regenerate NAD+ for glycolysis to continue. No additional energy is obtained from this process. In aerobic conditions, however, NAD+ is regenerated in the electron transport chain during oxidative phosphorylation, converting pyruvate to CO2, reducing O2 to H2O, and generating a substantial amount of energy in the process. This experiment is designed to demonstrate the Pasteur Effect in yeast. In anaerobic conditions, yeast cells produce ethanol by fermentation, however they switch to aerobic respiration in the presence of oxygen. You should refer to your text for a more detailed explanation of the Pasteur Effect and the molecular mechanisms that are thought to be responsible for this example of metabolic control.

Exercise 5.1. Preparation of yeast cultures

You will be provided with a yeast suspension that has been prepared by the technical staff as follows: Baker’s yeast was washed three times in 0.66 M KH2PO4, pH 4.8, and made up to a 1 % wet weight suspension. Glucose (11 mL, 0.25 M) was added to 100 mL of this yeast suspension and mixed well. Immediately after mixing, a sample of this yeast suspension was removed and centrifuged, and the supernatant stored on ice as the “time = 0” sample for both the glucose and ethanol assays. Once you have collected your yeast suspension, you will perform the following:

1. Pipette 4 mL of the yeast suspension with glucose into each of two 50 mL Erlenmeyer flasks which have been placed in an ice bath. One flask should be labelled with ‘O2’ and the other flask ‘N2’.

 2. Keeping both flasks on ice, gas one flask with N2 for 5 minutes, then immediately stopper both flasks with glass stoppers wrapped in parafilm, making sure that they seal well (see figure 5.2 below).

3. Place both flasks in a shaker incubator at 25 °C for 45 minutes.

4. After incubation, transfer the contents to 15 mL tubes and centrifuge at 4000×g. After centrifugation, carefully remove the supernatant for the glucose and ethanol assays, being careful not to disturb the cells at the bottom of the tube, as they will interfere with absorbance measurements.

Ethanol assay

Ethanol can be quantitatively oxidised to acetaldehyde by NAD+ and alcohol dehydrogenase (ADH) provided that the acetaldehyde is removed from the reaction to pull the equilibrium in the right direction. This can be achieved by chemically trapping it with semicarbazide. Eq. 1 CH3CH2OH + NAD+ → 3CHO + NADH + H+ Eq. 2 CH3CHO + NH2NHCONH2 → 3CH=N-NHCONH2 + H2O Under these conditions, the amount of NADH formed is equal to the amount of ethanol originally present (see Eq. 1). You will estimate the amount of ethanol produced by the yeast cells in each of your fractions using this reaction. You will convert the ethanol to acetaldehyde according to Eq 1, and measure the NADH produced by spectrophotometry (1 ethanol molecule leads to formation of 1 NADH).

1. Prepare seven spectrophotometer tubes (duplicate tubes for each fraction, plus one for a blank), each containing 2.9 mL of Ethanol Assay Buffer (1.1 % Na pyrophosphate, 0.5 % semicarbazide, 0.22 % glycine, pH 9.0).

2. Add 20 μL aliquots of the appropriate supernatant fraction to each tube (duplicate tubes for t = 0, O2 and N2, add water in the case of the blank).

3. Add 80 μL of Alcohol Dehydrogenase/NAD+ solution (300 U/mL ADH, 30 mM NAD+ , pH 7.0) to every tube and mix well

4. Incubate all tubes in a 30 °C water bath for 30 minutes.

5. Read absorbances for each tube at λ = 340 nm

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