A nitrile hydratase for cyanopyridines... and it's a bit more stable than usual.

There is a paper in Process Biochemistry which describes a new NHase from Aurantimonas manganoxydans which shows improved stability than you can normally expect from a NHase. It is entitled “Efficient cloning and expression of a thermostable nitrile hydratase in Escherichia coli using an auto-induction fed-batch strategy”, and it is by Xiaolin Peia, Hongyu Zhang, Lijun Meng, Gang Xu, Lirong Yang and Jianping Wu. This NHase is four times more rapid at converting 3-cyanopyridine to its corresponding amide as valeronitrile, and the authors emphasize their enzyme's stability though in the world of NHases where nothing is what you might describe as thermophilic, please don't get too expectant! They have a great table of NHase thermostability which I reproduce with their enzyme's data inserted.

 

A nitrile hydratase for cyanopyridines... (but it prefers aliphatic nitriles)

There has been a recent paper in Journal of Molecular Catalysis B on a nitrilase that converted cyanopyridines. This enzyme came from a strain of Pseudomonas putida. There is also a recent paper in the same journal entitled “Discovery of a new Fe-type nitrile hydratase efficiently hydrating aliphatic and aromatic nitriles by genome mining” by Xiaolin Peia, Lirong Yang, Gang Xu, Qiuyan Wang and Jianping Wu.which describes a nitrile hydratase, this time, from a Pseudomonas putida strain (F1) which they have shown to be able to turn over 3-cyanopyridine in a 1L fed batch reactor. Their activity data suggests it actually prefers aliphatic nitriles: acrylonitrile rates as 941 U/mg and valeronitrile as 535 U/mg as compared to 3-cyanopyridine at 26 U/mg. They describe how it was cloned into E. coli, needing the inclusion of an activator protein to get activity. They also include a nice phylogenetic tree showing the spread of known iron type NHases... plenty of examples in the Rhodococcus but spreading outwards into Pseudomonas.

Scaling up a nitrilase based hydrolysis

Efficient Production of (R)-o-Chloromandelic Acid by Recombinant Escherichia coli Cells Harboring Nitrilase from Burkholderia cenocepacia J2315 by Dongzhi Wei and co-workers is published in Organic Process Research and Development at  DOI:10.1021/op400174a.  It being OPRD and from a group working in a Laboratory of Bioreactor Engineering, it’s going to be an interesting read on doing a nitrilase reaction on the larger scale.  We are currently working on scaling up a different nitrilase-based reaction currently from bench scale to the “too heavy to use normal glassware” scale so it’s good to see how others do it.

 

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A nitrilase for cyanopyridines...

Characterization and functional cloning of an aromatic nitrilase from Pseudomonas putida CGMCC3830 with high conversion efficiency toward cyanopyridine by Hong Xu and co-workers in Journal of Molecular Catalysis B: Enzymatic is the first report of cloning of an aromatic nitrilase from Pseudomonas genus. It has a particular penchant for 3-cyanopyridine.

Zaparucha nitrilase activities

One of the joys of the Zaparucha nitrilase paper recently published in Advanced Synthesis and Catalysis is that their search for nitrilase activity is not confined to those enzymes which have already been automated annotated as "nitrilase". Within their enzyme set are proteins annotated as amidases, cyanide hydrolases, ureidopropionases, hydrolases (nice and broad that!) and even glycosyl. Here is my analysis of their protein types done by text searching their supplementary materials.

Text search strings are across the top and obviously "nitrilase" will also find those in "nitrilase/cyanide", and "hydrolase" occurs all over the shop. The second line indicates the totals after my removal of those proteins which didnt show any nitrilase activity for them. (You can take the chemist stance "something wrong with the protein" or the molecular biologists stance "you havent found the right substrate yet" at your own whim!). A very interesting set of annotations!

 

Selectivity in nitrilases

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.

Nitrile active enzyme posters

We are at Biotrans 2013 in Manchester this week. Whilst it has to be said that transaminases is the most frequently described enzyme type described here, there are a few posters describing nitrile active enzymes.
Nicola D'Antona has a poster entitled "Biotransformations of nitrile ferrocenes catalyzed by the bienzymatic system of nitrile hydratase/amidase: first examples of molecular recognition and enantioselectivity of chiral planar substrates". This poster describes the use of a couple of Rhodococcus species to hydrate and hydrolyze either one or two cyano groups appended to the double decker ferrocene sandwich. Considering NHases' dislike of bulky substrates that is a neat discovery. They even get some enantioselectivity especially in the acids where the amidase in the whole cell is strongly selective in determining the sterics of the outcome.
Birgit Wilding has a poster entitled "Enantioselective synthesis of a taxol side chain precursor with bacterial and fungal nitrilases". Obviously finding a biocatalytic way to build even a portion of a pharmaceutical is a great demonstration of using this technology to provide a green and inherently stereoselective route for the commercial arena. An interesting finding reported on this poster is the tendency of the bacterial nitrilases (two of which came from our Nzomics panel) in her panel to serve up not just the acid but the primary amide of her side chain precursors too. The ratio of amide to acid is sensitive to substrate concentration... Intriguing- I had one person previously complain about getting amide when they wanted acid with one of these enzymes but I don't know what their substrate was!
Cintia Milagre is displaying a poster called "Synthesis of alkyl and aryl substituted amides by nitrile hydratases" summarizing work on nitrile hydratase discovery from Brazilian soil samples and their utility at hydrating alpha substituted nitriles.
Carine Vergne-Vaxelaire is displaying a poster entitled "Biocatalytic tools for organic chemists: new nitrilases for synthesis of building blocks" which outlines the high throughput screening of 125 nitrilases against 25 structurally diverse nitriles, and is linked to a lecture being given later in the week by Anne Zaparucha.
Sander van Pelt has a poster called "Nitrile Hydratases: from genes to immobilized biocatalyst" highlighting the extra utility that a covalently linked enzyme aggregate preparation from CLEA Technologies has in terms of stability. It is nice to see that the NHases from AJ270 and CGA009 that we have worked on together are still useful examples in this field.
Finally hiding undercover at P387 is our poster on nitrilases. Graeme Turnbull has written up his semi-quantitative colorimetric methodology for assaying the activity of nitrilases as cell-free extracts (which screw up a pH driven colorimetric method) and then showing how he has applied it to a panel of 14 enzymes and 40 diverse substrates.