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 They use a nitrilase (after citing unpublished data on its enantioselectivity… why unpublished?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.