One of the things we are interested in looking at with our increasingly wide panel of nitrilases is how substrate selectivity works with classic Group 1 nitrilases.
Our standard panel of substrates we are assaying our nitrilases against has fifty substrates so we span quite a range of different structural types and traits. Here is an excerpt from our posters' table of data.
Colour coding mirrors the response that we see with
our assay and carries handy semi-quantitative values as well which we get from UV/vis spectroscopy. What we have here is a table with different substrates along the top and different nitrilases down the side.
The top row here shows a nitrilase which hydrolyzes arylaliphatic substrates with great gusto (showing a 10 against 2-phenylacetonitrile) but has not a lot of interest in alkyl or aryl nitriles generally. The fifth row shows a nitrilase which is an enthusiast for aromatic nitriles peaking at para-substituted aromatics but with some interest in almost everything else!
To some extent the other patterns of activties are also interesting... we see some nitrilases which show activities which are active against only a few compounds which are quite structurally diverse. This may be a low expression level only letting us see the most active members of wider groupings (because this data table comes out of a screening programme of cell-free extracts), and this is something we will be investigating. It would be nice to hope that there was something a bit deeper going on though.
Finally we have some enzymes like one in the fourth line here which appear to be oblivious to everything. These show up a difference of response within our team. I, as the chemist, say "perhaps it just isn't active at all"; our molecular biologists say "nonsense- with that sequence it's just got to be a nitrilase, you haven't found the right substrate yet"!
We'll keep looking... for new nitrilases and new substrates.
