"Phil Harrison" <arsphys at cc.usu.edu> wrote in message
news:5.1.0.14.0.20030310132614.00a17e50 at cc.usu.edu...
> 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
> help any.
>> 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?
>> Thanks,
>> Phil
>> Phil Harrison
>> USDA-Agricultural Research Service,
> Forage and Range Research Lab
> Utah State University, UMC 6300
> Logan, UT 84322-6300
> Phone: 435-797-3209
> FAX: 435-797-3075
> e-mail: arsphys at cc.usu.edu>> ---
>From what you described it seems that 75% acetone is critical for
"fluffiness" of the final powder. It is conceivable that mixing your eluted
protein with another organic solvent (e.g. ethanol if it is considered to be
environment friendly of course) could solve you problem. Otherwise, I would
try to concentrate the protein and store it frozen.
-Emir
