In article <3qdak4$la3 at imp.demon.co.uk>, jc at johnsc.demon.co.uk (John
Collins) wrote:
> I'm looking at the factors governing the production of an
> extracellular enzyme by a filamentous fungus. There is a lot of
> evidence that extracellular proteases are degrading the enzyme and it
> is possible that some of the factors which apparently enhance enzyme
> synthesis are merely reducing protease levels. Has anyone any
> experience of incorporating protease inhibitors into growth media?
> This would seem to be the most direct method of addressing the
> protease problem. We currently use PMSF during enzyme purification but
> it is toxic and not particularly stable in aqueous solution so it does
> not look a good candidate for adding to media. I've heard that AEBSF
> is non-toxic and more stable. Has anyone used it in a similar
> application? Finally, what are the prospects of using inhibitors like
> pepstatin and leupeptin?
Isn't it likely that introducing protease inhibitors into your growth media
is going to mess up the cell growth somewhat? Anyway, pepstatin is a good
inhibitor of aspartate-class proteases; these are unlikely to be a problem
in your system unless you are working with appreciably acidic media.
Leupeptin
is reasonably stable in aqueous solution at room temperature with a half-life
of a few hours. E-64 is an excellent inhibitor of cysteine proteases and is
stable at room temp. The downside of using it is the price of course which may
be prohibitive if you're looking at large volumes (i.e. litres) of growth media.
EDTA is the obvious candidate for inhibiting metalloproteases, but, again, I'm
not sure how much your fungi will like having it in solution with them :) It
may be worth investigating the possibility of using alpha-2 macroglobulin as a
broad specificity inhibitor. I'm afraid I can't comment on the stability of this
protein as I've never used it...it's not at all cheap to buy either. You might
also like to try BPTI as a serine protease inhibitor as this is a remarkably
stable protein. Hope this is of some use to you
Nick Fisher
Biochemistry Dept.
Cambridge University
England