arsphys at cc.usu.edu
Mon Mar 10 15:37:23 EST 2003
I have been using a preparation of alpha-amylase (Clarase) for several
years. We use it to digest starch from plant tissues prior to measurement
of carbohydrates. The Clarase has a significant level of carbohydrate in
it, giving us too high a background, so we have to remove the carbohydrate
before we can use the enzyme prep.
We have been cleaning up the enzyme by dissolving it in water, dialyzing,
then precipitating with 75% acetone. This is repeated 3-4 times, followed
by another dialysis, then freeze drying. In the end, we get a nice, light,
fluffy powder. The only problems with this procedure are:
1. It is very time consuming
2. I suspect we lose some activity due to the acetone tmt.
3. It generates acetone which we have to redistill or dispose of.
Recently I tried cleaning up a test batch on a BioGel P6-DG column. I got
excellent separation of the carbohydrates from the protein, but when I
freeze-dried the pooled enzyme fractions, I got a gooey, syrupy mass in the
bottom of the flask, not a nice fluffy powder. I have seen this remedied
by diluting the sample more before freeze drying, but that didn't seem to
Now for the question (you probably thought I'd never get there!) Any
suggestions on how to freeze-dry this sample and get a powder instead of goo?
USDA-Agricultural Research Service,
Forage and Range Research Lab
Utah State University, UMC 6300
Logan, UT 84322-6300
e-mail: arsphys at cc.usu.edu
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