Identification of Disaccharide Hydrolases in Yeast
This exercise would follow nicely after the fermentation lab. Students discovered that yeast are able to ferment sucrose and usually maltose, but not lactose. Since hydrolysis of all three would yield glucose, which is fermented, yeast must lack lactase and, possibly, maltase activity. The sucrase (invertase) of yeast is well known. In this exercise, students test yeast or yeast extract for sucrase, maltase, and lactase activity, using the DNSA reaction (see below) to detect reducing ends produced by hydrolysis.
Students first prepared a standard curve for the DNSA assay with 0, 5, 10, 15, and 20 mM glucose.
For the hydrolysis reactions, I tested both whole yeast and yeast extract (below). A 1% suspension of whole yeast gave pretty much the same results as the extract with sucrose and, oddly enough, gave no noticeable cloudiness in the spectrophotometer. The extract was fairly tedious to prepare and a yeast suspension might work just as well for this exercise. It might need to be poisoned with iodoacetate or N-ethylmaleimide to prevent metabolism of any hydrolysis products. It's something to play around with. Alternatively, preparation of an extract would probably be a good learning exercise (as well as physical exercise) for the students.
Students mixed 10 mL of 20 mM lactose, maltose, or sucrose with 1 mL of yeast extract in a small flask and immediately removed a 0.1-mL sample for analysis. The flasks were incubated in a 40 degree water bath and students took samples at 20, 40, and 60 minutes. I had the students use flasks so they could more easily remove samples with the 1-mL pipetters they were using. Other possible arrangements would be to set up the four (or fewer) time points in separate tubes, remove them at appropriate times, and carry out the DNSA reaction directly in the tube. I made no effort here to control pH, and there isn't anything special about 40 degrees.
At the end of the exercise, I remind students with appropriate questions that sucrose hydrolysis yields two reducing carbons, whereas hydrolysis of lactose or maltose yields only one. Aware of this, they can use the standard curve and their reaction progress curves to calculate an initial velocity of hydrolysis in micromoles per unit time. (If I were to include a protein assay, they could calculate specific activities.)
Hydrolysis of sucrose was evident, the more so because unhydrolyzed sucrose (not a reducing sugar) gives no color in the test. Lactose and maltose are expected to give a high baseline because of their reducing carbons, but a significant increase in color from hydrolysis should have been evident. Students' results with those sugars were more variable, and I don't recall any consistent trends. I should have had them do duplicates of each point. I don't think the conditions of the color reaction cause hydrolysis. I need to run through this a few more times to optimize it, but I think it's a good exercise.
DNSA reaction: The procedure used here was to mix 0.1 mL of sample with 0.3 mL of DNSA reagent and heat the mixture for 15 minutes in a boiling water bath. After the tube is cool, dilute with 8 mL of water and determine the absorbance at 540 nm.
Yeast extract: I shook 6 g of dry yeast and 14 g of sand in 30 mL of water vigorously for 15 minutes in a plastic 50-mL centrifuge tube. I decanted the liquid above the sand, then used a glass Pasteur pipet reaching though the bed of sand to remove the rest of the liquid. Centrifuging at 8,000 rpm for 15 minutes (SS-34 Sorvall rotor) gave 17 mL of a clear yellow-brown supernatant.
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