Hi -
I will answer this question in two parts, first from experience (for
which there are no references - I do have about 10 years experience
with protein purification and enzyme characterization, for whatever
that's worth, and a couple of degrees too) and secondly from what
literature I could dig up.
I have worked with many an enzyme and have found that immobilization
often prolongs the life of the protein. So, empirically, I agree with
your boss, that immobilization can prolong the active lifespan of
enzymes (although it can also kill an enzyme with certain
linkages...). Why is this so? Well, I've noticed the following
things with various of the enzymes that I've used (I think that I
can't mention the specific enzymes, for probably the same reason that
you might not be able to, confidentiallity agreements and what-not).
Aggregation is often a problem with proteins in solution; the higher
the concentration of enzyme, the quicker the aggregation, and usually,
the faster the enzyme dies. This can be further increased if redox
sites are involved, at least in part due to cysteine reactivity and
divalent bonds forming between enzymes leading to inactive sludge
(often why DTT or other reducing agents are included in enzyme storage
buffers). Additionally, enzymes which undergoe conformational changes
during their catalysis also can become more prone to denature in a
purified state (they're probably in some sort of complex naturally,
and, perhaps being anchored helps prevent them from unfolding?) -
denatured proteins also tend to glom up more readily, rendering dead
enzyme quite quickly. Certain enzymes (those designed to chew up
other molecules) also will exhibit some activity against themselves
(even if low, this adds up quickly in the high concentration, low
other-substrate type environment of storage). Immobilization solves
several of these problems - enzymes are at a relatively low
concentration for aggregation and inter-enzyme reactions with each
other, while they can still be at a high relative concentration for
reaction with substrate flowed through the beads (or over them, your
description sounds like Poros-type beads though).
So, now the literature.
A nice discussion of one groups attempts at increasing stability by
immobilization (conformation change argument)
http://www.icp.csic.es/biocatalisis/web3/eng/lineas.html
A report of extending enzyme length by immobilization:
http://www.carleton.ca/~kbstorey/kbs24.htm
Another report of enzyme storage:
http://ift.confex.com/ift/2001/techprogram/paper_7649.htm
This report suggests that immobilization improves thermal stability:
http://bab.portlandpress.com/bab/036/bab0360007.htm
An industry page that briefly mentions the issue:
http://www.sigmaaldrich.com/Brands/Fluka___Riedel_Home/Organic___Synthetic/Enzymes/in_Organic_Chemistry/Immobilization.html
A report on trypsin stability and immobilization:
http://ift.confex.com/ift/2002/techprogram/paper_13916.htm
Chymotrypsin immobilized with the suppostion of decreased
denaturation:
http://www.ornl.gov/schcg/enzyme_stabilization.htm
I hope that this helps answer your question - feel free to ask if you
have further questions.
synarchy |
