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?
>> Thanks
>> John Collins (jc at johnsc.demon.co.uk at home)
I'm not familiar with fungal cultures, but aprotinin (a serine protease
inhibitor) is often added to mammalian cell cultures to inhibit the
degradation of recombinant proteins. We have added aprotinin to our
Chinese hamster ovary (CHO) cell cultures under some circumstances, and
found that this technique works very well. Of course if the protease in
question is not a serine protease this will not work.
Hope this helps.
Roy Kimura
Dept. of Chemical Engineering
Northwestern University
