Just Accepted NHase manuscripts

• Enantioselective biocatalytic hydrolysis of beta-aminonitriles to beta-amino-amides using Rhodococcus rhodochrous ATCC BAA-870, by Varsha Chhiba, Moira L. Bode, Kgama Mathiba, Wendy Kwezi, Dean Brady in Journal of Molecular Catalysis B: Enzymatic [doi:10.1016/j.molcatb.2011.12.005]

“A range of β-aminonitriles (3-amino-3-phenylpropanenitrile and derivatives) were synthesised by reaction of various benzonitriles with acetonitrile and subsequent reduction of the resulting acrylonitrile products. These compounds were hydrolysed to the corresponding amides using the nitrile biocatalytic activity of Rhodococccus rhodocrous ATCC BAA-870. Results showed that the nitrile hydratase enzyme was enantioselective for these compounds, in particular 3-amino-3-p-tolylpropanenitrile and 3-amino-3-(4-methoxyphenyl)propanenitrile and the corresponding amides (up to 85% in one case).” They found interesting levels of steric hindrance (probably) when the amino group was functionalized, and they had most success when the biotransformations were run at pH9 to minimize as much as possible protonation of the amine if it wasn’t derivatized. Obviously it being a Rhodococccus, this is an example of enantioselectivity with an iron-centred NHase.

• Biotransformation of the Neonicotinoid Insecticide Thiacloprid by Bacterium Variovorax boronicumulans Strain J1 and Mediation of the Major Metabolic Pathway by Nitrile Hydratase by Hui-Juan Zhang , Qian-Wen Zhou , Guang-Can Zhou , Yu-Min Cao , Yi-Jun Dai , Wei-Wei Ji , Guang-Dong Shang , and Sheng Yuan in Journal of Agricultural and Food Chemistry [DOI: 10.1021/jf203232u]

“A neonicotinoid insecticide thiacloprid-degrading bacterium strain J1 was isolated from soil and identified as Variovorax boronicumulans by 16S rRNA gene sequence analysis… A 2.6-kb gene cluster from V. boronicumulans J1 that includes the full length of the nitrile hydratase gene was cloned and investigated by degenerate primer polymerase chain reaction (PCR) and inverse PCR. The nitrile hydratase gene has a length of 1304 bp and codes a cobalt-type nitrile hydratase with an alpha-subunit of 213 amino acids and a beta-subunit of 221 amino acids.” This protein was then expressed in an active form in E. coli BL21. This bacterium is a boron-accumulating microbe isolated from soil.

Aligning cobalt centre NHases

I have been using the FATCAT protocol to look at structural alignments of differing cobalt centred NHases. This is available as a tool here.
Here is a picture of the alpha chains of 1V29 and 1 IRE aligned.

And here is a picture of the alignment of the beta chains. A bit more variation there.

A new review: Industrial Biotechnology- the future of green chemistry

The most recent issue of Green Chemistry has a 41 page review (DOI: 10.1039/C1GC15579B) by Udo Kragl and his co-workers Stefanie Wenda, Sabine Illner and Annett Mell, entitled  "Industrial Biotechnology- the future for green chemistry" which is a useful overview of where biocatalysis is now. A nice feature it has that I havent seen before is the use of little boxes which are labelled "critical remarks" to discuss highlight problems sometimes in the perception and sometimes in the reality of using biocatalytic process industrially.
As is necessary in this sort of review, there is a discussion (p. 3011) on how good nitrile hydratase is for bulk synthesis of acrylamide though it is interesting to see the scale of the use is "more than 50,000 tons per year" referring back to a viewpoint paper in ChemCatChem authored by Yuryev and Liese which actually says the Mitsubishi Rayon process "runs on scales up to 50,000 tons per year"- whatever that actually means! So it would appear that my suspicion that no one actually knows how successful (in terms of level of adoption) one of the most successfully implemented biocatalytic processes is, has not been contradicted!
Directly following the discussion of acrylamide manufacture is a discussion comparing the chemical and the Lonza chemoenzymatic routes to nicotinamide, with some excellent leading references.

Polymers and nitrile hydratase activity

There have been a few papers over the years looking at the possibility that nitrile-active enzymes might be able to attack nitrile groups on the surface of nitrile-containing polymers such as polyacrylonitrile.  An early example of this is the paper by Gübitz and co-workers [Nitrile Hydratase and Amidase from Rhodococcus rhodochrous Hydrolyze Acrylic Fibers and Granular Polyacrylonitriles, from Appl Environ Microbiol (2000)] which uses a cell free extract from Rhodococcus rhodochrous NCIMB 11216 to create pendant carboxylate groups on the fibres.

More recently there has been another paper looking at this topic using a different cell-free extract

Vikash Babu, Bijan Choudhury, Competitive adsorptions of nitrile hydratase and amidase on polyacrylonitrile and its effect on surface modification, Colloids and Surfaces B: Biointerfaces, Volume 89, 1 January 2012, Pages 277-282, 10.1016/j.colsurfb.2011.08.025.

This uses an extract from Amycolatopsis, and they do show conversion of the surface to carboxylate, by functionally tracking a NHase activity and an amidase activity. I am not sure how they know there isnt a nitrilase in there helping along too (NCBI records currently an Amycolatopsis species nitrilase).

In the light of the congested active site entrance which is generally found with NHase, it is quite interesting this works.

When nitrilases act like nitrile hydratases...

There were other posters than mine at Biotrans2011 which looked at nitrile active enzymes (from the groups of Norbert Klempier and Ludmila Martinkova). One of the issues that came up more than once was how sometimes nitrilases can sometimes stop being nitrile hydrolyzers and become nitrile hydraters (like NHases). This is an interesting topic in itself because, in terms of synthetic chemistry utility, nitrilases are probably more widely applicable than nitrile hydratases, and predicting when a particular nitrilase isnt quite up to the full hydrolysis could be a useful timesaver. In my experience, some IMAC purified nitrilases that we have worked with have upset commercial collaborators in stopping at the primary amide and not producing any carboxylic acid product, and the effect has been reported in the literature before, summarized in Martinkova and Kren 's recent nitrilase review.

Ludmila Martínková, Vladimír Křen, Biotransformations with nitrilases, Current Opinion in Chemical Biology, Volume 14, Issue 2, April 2010, Pages 130-137, 10.1016/j.cbpa.2009.11.018.

Obviously this leads me to the reverse query... Is it possible to get a nitrile hydratase to hydrate a nitrile under extreme/altered conditions?

My poster from Biotrans2011

Here is my poster from Biotrans2011. As you can see quite a lot of what I wrote has appeared on this blog previously.

Biotrans 2011

I am off to Biotrans 2011 next week. I am presenting a poster on nitrile hydratases which I will put up here on my return.