Two publications on the nitrilase from Sphingomonas wittichii RW1

Sphingomonas wittichii RW1 is a bacterium which has been shown to have the ability to degrade polychlorinated dioxins, and hence has had its genome fully sequenced. It has two different nitrilases in its genome.

YP_001264656.1

YP_001261492.1

Two papers just released with the corresponding authors Er-Zheng Su and Dong-Zhi Wei report on the nitrilase with the accession code YP_001264656. These authors and enzyme have previously been mentioned on this blog- I complained there was no information on the enzyme discovery... here it is!

The first entitled “Cloning, Overexpression, andCharacterization of a High Enantioselective Nitrilase from Sphingomonaswittichii RW1 for Asymmetric Synthesis of (R)-Phenylglycine” and published in Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology describes the construction of a mini-library of nitrilase enzymes chosen by the similarity to a nitrilase from Pseudomonas fluorescens EBC191 (GenBank accession no. AAW79573) which has been shown to be highly activity toward phenylglycinonitrile. The nitrilase from Sphingomonas wittichii RW1 was shown to be most promising nitrilase to achieve chiral synthesis of R-phenylglycine in a kinetic resolution mode.

The second entitled “Efficient asymmetric synthesis ofD-N-formyl-phenylglycine via cross-linked nitrilase aggregates catalyzeddynamic kinetic resolution” and published in Catalysis Communications describes the preparation of cross-linked enzyme aggregates (CLEAs) of nitrilase from Sphingomonas wittichii RW1, and then its use to perform dynamic kinetic resolution to make D-N-formyl-phenylglycine. The paper also demonstrates that the CLEAs have improved stability over non-immobilized enzyme and are reuseable up to six times whilst retaining 70% initial activity.

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).