thermolysin advice needed

adwright at iastate.edu adwright at iastate.edu
Mon Mar 21 15:35:00 EST 1994


I have been using Pronase E to hydrolyze protein in grain samples for
amino acid determination by microbiological bioassay.  It is cheap
and reliable and good enough for plant breeding purposes :)
For example, finding the high methionine corn in a segregating 
population.

I have sometimes gotten high backgrounds from the pronase and not
always very complete hydrolysis. 

I saw in a SIGMA ctalog that thermolysin works out to be cheaper per 
unit activity than pronase and have been considering using it since
i will be adding less protein (enzyme and contaminating protein)
 to my sample, which ought to cut down on the background.
 But  since only Pronase is called "unusually non-specific" i am a little
concerned about how complete the hydrolysis would be using thermolysin
alone.

I am considering trying thermolysin followed by pronase. The background
contribution of thermolysin that has been hydrolyzed by pronase, if any
ought to not be a problem.  


I have heard that thermolysin is available now as an expression product
of E. Coli and is considerably cheaper than it was.  I have been unable to 
locate a commercial source, however. Can anyone help?

BTW does anyone know how specific or nonspecific thermolysin is regarding
what bonds it will break?

ANy help or experiences would be appreciated.
I'll post or E-mail my findings if anyone wants to know.
Thanks in advance
Allen


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