This is another enzyme-isolation lab like the lysozyme exercise described elsewhere. I have had variable success with it; one year the students recovered no activity. I ascribed that to the fact that we used yams rather than sweet potatoes, but another time students tried it with yams it worked okay.

The isolation procedure is based roughly on Methods in Enzymology, vol I, pp.154-156. Students grind two sweet potatoes in a meat grinder and then mix in a blender for two minutes with a quantity of water equal to half the mass of pulp. It seems that the meat grinder can be omitted if the sweet potatoes are frozen and thawed before use; this allows a large batch of sweet potatoes to be used for more than one year. Insoluble debris is removed by centrifugation for 15 minutes in a Sorvall SS-34 rotor at 12,000 rpm. The enzyme is separated from the supernatant by precipitation using ammonium sulfate. The original paper describes tweaking ammonium sulfate concentrations up and down to optimize recovery and purity. Because our results differed from those reported, I simplified the procedure. Students add ammonium sulfate in the amount of 0.47 g per mL of supernatant and harvest the precipitate by centrifugation at 12,000 rpm for 10 minutes. Pellets were suspended with 1 mL of water for each 8 mL of original pulp and recentrifuged (12,000 rpm, 10 minutes). Supernatants were saved cold and should contain active enzyme.

I tried to get the students to think their way through the rest of the exercise. I told them they could determine the amount of amylase activity by mixing 0.1 mL of their preparation with 0.1 mL of 1% starch and 0.1 mL of pH 5 buffer (around 0.1 M acetate/acetic acid), incubating at 37 degrees for 15 minutes, adding 0.3 mL of DNSA reagent, heating in a boiling water bath for 10 minutes, diluting with 4 mL of water, and determining absorbance at 540 nm. I asked them to formulate and carry out the appropriate controls to be sure any color formed in the reaction reflected actual hydrolysis catalyzed by enzyme.

I then instructed them to prepare a maltose standard curve in order to determine the reaction rate in micromoles maltose formed per minute. I gave them open ended instructions to look at the reaction's dependence on pH (I prepared buffers at pH 3, 5, 7, 9, and 11), starch concentration, and amount of enzyme, calculating all rates as micromoles maltose formed per minute, and to comment on the various dependences in light of what they should expect based on typical kinetic predictions.

This is a pretty nice exercise, and there are lots of other things one could do with it; the enzyme from sweet potato is the same beta-amylase I use in other exercises above. Since beta-amylase is a pretty large protein (206,000), gel filtration chromatography would be a useful purification step.

Revised 6/14/07