Fermentation, respiration and enzyme specificity: a yeast model.

Introduction

The process of respiration evolved to utilize sugars for the production of usable chemical energy in the form of ATP. Depending on whether oxygen (O2) is present or not, respiration can be aerobic or anaerobic. Aerobic respiration in most eukaryotic cells is considerably more efficient since up to 36 molecules of ATP can be produced per molecule of glucose whereas anaerobic respiration may only produce 2 ATPs per glucose. ATP is used by the cells to power many biochemical reactions. This will be discussed in class in more detail in a few weeks.

Aerobic respiration: C6H1206 + 6 O2 => 6 H20 + 6 CO2

Anaerobic respiration: C6H1206 => 2 C2H5OH + 2 CO2

In these experiments, you will examine the process of anaerobic respiration (fermentation) in yeast cells using a simple system that measures the progress of the reaction by the accumulation of CO2 gas in a reaction vessel (an upturned tube). Each group will investigate a separate aspect of the process.

1. Effect of substrate concentration (glucose); kinetics of glucose use and CO2 production.

This group will examine a variety of glucose concentrations (from zero upwards) to determine the optimal concentration. About 10 concentrations should be used. In addition, in the minimum concentration found to give optimal CO2 production, this group will examine the use of glucose by yeast using glucose test strips, "Dextrostix" and relate this to CO2 production.

2. Effect of pH.

This group will examine fermentation (CO2 production) at pHs 2,4,6,8,10 and 12. What is the optimal pH? 5% glucose will be used.

3. Effect of temperature.

This group will determine the optimal temperature for fermentation at a variety of temperatures. 5% glucose will be used.

4. Substrate specificity (lactose and melibiose).

This group will examine the ability of yeast to utilize lactose (a beta-disaccharide of glucose and galactose) and melibiose (an alpha disaccharide of glucose and galactose) for fermentation. The effects of two dietary supplements on the ability of yeast cells to digest these sugars will be examined.

Reagents and materials available:

Yeast suspension 7% w/v in water.

Glucose solutions (5%) in water.

Glucose.

Buffers. Ph 2,4,6,8,10,12

Water baths

Beakers

Ice

Reaction tubes.

Thermometers

wax pencils

Basic Procedure.

1. Place 8ml of a 7% suspension of bakers yeast in a 15ml screw-capped vial. Fill the remainder of the tube with sugar solution. When filling, extend the fluid meniscus above the lip of the tube.

2. Screw on the cap. The cap was perforated with 3 or 4 small holes so a few drops of fluid will spurt from the holes. Ensure that no bubbles remain in the tube.

3. Invert the tube and place in a large beaker filled with 40oC water. Use a thermometer and hot water/ice to ensure that the temperature remains constant.

4. At appropriate time points, mark the tube with a wax pencil at the bottom of the CO2 bubble (include foam with the bubble). At the end of the experiment, empty the tube and record the ml of CO2 that correspond to the pencil markings.

Figure 1. Fermentation tube made from a 15-ml plastic centrifuge tube. Perforations were made in the cap with a needle.

Todays exercise.

1. Get experimental assignments and design experiments in detail. Produce typed protocol (by next week), and hand in detailed list of required reagents and equipment (today).

2. The typed protocol will be graded for completeness and feasibility. Group grades will be assigned.

Next week.

1. Hand in protocol

2. Perform experiments according to the protocol (though adjustments may be required as you conduct the investigation).

3. Present findings to the whole lab. Use the blackboard.

4. Each student will produce a <1000 word summary of all of the data. Notes should be taken during the group presentations of the data. Data must be referenced to each group. Lab reports will be due after spring break.