Chiral intermediate for cilastatin by nitrilase hydratase/amidase combo

There is a new accepted manuscript in the Journal of Molecular Catalysis B from Yu-Guo Zheng and co-workers entitled "Industrial production of chiral intermediate of cilastatin by nitrile hydratase and amidase catalyzed one-pot, two-step biotransformation" which describes their research into using a chiral nitrile to carboxylic acid conversion which results from a cascade of NHase from (Rhodococcus boritolerans FW815) then amidase (from Delftia tsuruhatensis ZJB-05174). The compound they are aiming to hydrolyze is rac 2,2-dimethylcyclopropanecarbonitrile to the S-2,2-dimethylcyclopropanecarboxamide which is an intermediate in the synthesis of cilastatin (a booster component to some antibiotics which prolongs their activity by blocking the kidney’s dehydropeptidase activity). This is achieved using a NHase which is notably active but not chirally selective, and an amidase which is only avid for the R enantiomer of the amide. The R acid and S amide are separated using macroporous resin adsorption chromatography and the R acid is converted to the acid chloride. This can than have its chirality scrambled with a bit of heat such that its conversion to amide by addition of ammonia gives a fresh racemic batch of amide to challenge the R selective amidase with. The conditions described are to get it to work on the 100kg scale. Considering that this is a report on a process which has been undergoing continuous optimization since 2005, I guess the choice of amidase to do the chiral resolution was “baked in” before there was literature evidence of significantly stereoselective nitrile hydratases which might have prompted an enzyme discovery effort in that direction. Having said that racemization using the wrong acid chloride and heat rather than on the wrong isomer of the nitrile is a neat touch.

Nitrilase from sequence to 50 litre scale

We have been working on developing a specific nitrilase reaction in a consortium with Chemoxy and Biocatalysts Ltd with funding assistance from the UK Technology Strategy Board. In nine months, we have gone from the selection of amino acid sequences to expressed active proteins to assessment of substrate preference using a novel greener assay method which works with cell-free extracts to chemistry on the one litre scale, and finally at the end of last month to doing the reaction in the 50 litre plant at CPI's National Industrial Biotechnology Facility. Just to finish the boasting, the outcome of that reaction was over a kilo of product with great conversion.

We are now going into a TSB-funded collaborative research and development project with the same partners to really scale this process and bring it nearer to market by optimizing the enzyme, its use and reuse. It's going to be fun.

Dynamic Kinetic Resolution in alpha aminonitrile hydrolysis

The use of an equilibrium between enantiomers of your starting material to enable hydrolysis of more than 50% of a racemic mixture has been a point of keen interest in nitrilase research. The hurdle to it becoming widespread  has tended to be that the pH at which racemization occurs at a useful rate tends not to be one that your standard biocatalyst is happy operating at. A newly accepted manuscript into Tetrahedron Letters called “High yield synthesis of D-phenylglycine and its derivatives bynitrilase mediated dynamic kinetic resolution in aqueous-1-octanol biphasicsystem” by Jian Qiua, Erzheng Su, Wei Wang, and Dongzhi Wei is a useful addition to this literature. They use a nitrilase (after citing unpublished data on its enantioselectivity… why unpublished? It looks an interesting nitrilase!) from Sphingomonas wittichii RW1 to get DKR in a biphasic system (buffer/octanol).

How does the nitrile hydratase activator protein work?

This is a question which is still up for debate. It would appear to be involved with incorporation of the cobalt ion in those NHases which are cobalt-centred. In a newly accepted manuscript of FEMS Microbiology Letters entitled “The effect of flexibility and positive charge of the C‐terminal domain on the activator P14K function for nitrile hydratase in Pseudomonas putida” by Zhemin Zhou and co-workers, mutants of the relevant proteins were modelled and made, and then tested in the hydration of 3-cyanopyridine.

 

Supporting a nitrile hydratase for better performance

There is a new accepted manuscript for the Journal of Molecular Catalysis B: Enzymatic which adds to the literature on the support of a nitrile hydratase to increase this notoriously sensitive enzyme’s robustness.  PVA/Chitosan-glutaraldehyde cross-linkednitrile hydratase as reusable biocatalyst for conversion of nitriles to amides by SV Pawar and GD Yadav  describes how to prepare a cross linked assembly containing the NHase from Rhodococcus rhodochrous ATCC BAA-870. When they test this immobilized enzyme in the hydration of 3-cyanopyridine, they find it is able to withstand a higher temperature and more basic conditions. They also show that their preparation is a reusable format which retains 50% of activity after 8 batches.

The figure below from the paper shows relative activity of free and immobilized NHase over time at 45 degrees C in 0.1M phosphate buffer. The upper line is the immobilized form.

 

Degradation of nitrile-containing ionic liquids but how?

It is well known that soil bacteria and fungi bioremediate nitrile containing compounds to get at their nitrogen, and that nitrilase/nitrile hydratase pathway enzymes in a range of bacteria/fungi are amenable to such a transformation. A colleague of mine, Steve Cummings, reviewed this area a few years back in a paper entitled "The current and future applications of microorganisms in the bioremediation of cyanide contamination". One possible source of environmental cyanide contamination that Steve didn’t foresee was that from ionic liquids. These have been the subject of a lot of enthusiasm in green chemistry circles as they offer immensely useful solvation properties with no appreciable vapour pressure. Their use on anything other than a laboratory bench scale has met some resistance because of concerns about their environmental toxity. A recent paper entitled “Biodegradation potential of cyano-based ionic liquid anions in a culture of Cupriavidus spp. and their in vitro enzymatic hydrolysisby nitrile hydratase” by Stefan Stolte and co-workers in Environmental Science and Pollution Research has looked at biological hydration/hydrolysis of ionic liquids containing cyano groups.

After trying an organism, they have used a couple of Sigma enzymes to see if these will process their choice of ILs. They discover that the Sigma nitrilase isn’t interested but the Sigma NHase is quite happy to turnover. But I do wish that this information went further. Sigma does not tell you what the source organism(s) for their Nase and NHase enzymes is/are, merely that they are recombinant from E. coli. There is so much variation in substrate selectivity for both classes (for instance, glance at the slice of Steve’s Table 1 below) that getting a positive or a negative hit from a single example of unknown origin tells you not much at all in general.

Sol-gel encapsulation of Cobalt centred nitrile hydratase

The nitrile hydratase from Pseudonocardia thermophila is one of the most stable NHases currently described. It is cobalt centred, and one strain of it has an entry in the PDB. Holz and co-workers have just published a paper entitled "Acrylamide Production using Encapsulated Nitrile Hydratase from Pseudonocardia thermophila in a Sol-gel matrix" in the Journal of Molecular Catalysis A. They show that their system is able to convert acrylonitrile to acrylamide neatly, demonstrating the advantages of their enzyme support system in terms of increasing the robustness of these still rather sensitive enzymes. Interesting to me is that their orthosilicate sol-gels boost this enzyme's stability in methanol to take as much as a70% v/v mix happily.