Nitrilases regioselective

The regioselectivity of a Rhodococcus rhodochrous nitrilase has been demonstrated for the conversion of 5-fluoro-l,3-dicyanobenzene to 5-fluoro-3-cyano-benzoic acid [62]. The nitrilase was expressed in an Escherichia coli transformant, and a cell-free extract was employed as catalyst (0.14wt% cell-free extract) in 0.1m sodium phosphate buffer (pH 7.2) at 25 °C containing 0.18 m 5-fluoro-l,3-dicyanobenzene. After 72 h, the conversion was >98% and the reaction was stopped by addition of phosphoric acid (pH 2.4) to yield 5-fluoro-3-cyano-benzoic acid as a crystalline product (97% isolated yield). 

Hydrolysis of Nitriles. The chemical hydrolysis of nitriles to acids takes place only under strong acidic or basic conditions and may be accompanied by formation of unwanted and sometimes toxic by-products. Enzymatic hydrolysis of nitriles by nitrile hydratases, nitrilases, and amidases is often advantageous since amides or acids can be produced under very mild conditions and in a stereo- or regioselective manner. 

A crude mixture of enzymes isolated from Rhodococcus sp. is used for selective hydrolysis of aromatic and aliphatic nitriles and dinitriles (117). Nitrilase accepts a wide range of substrates (Table 8). Even though many of them have low solubility in water, such as (88), the yields are in the range of 90%. Carboxylic esters are not susceptible to the hydrolysis by the enzyme so that only the cyano group of (89) is hydrolyzed. This mode of selectivity is opposite to that observed upon the chemical hydrolysis at alkaline pH, esters are more labile than nitriles. Dinitriles (90,91) can be hydrolyzed regioselectively resulting in cyanoacids in 71—91% yield. Hydrolysis of (92) proceeds via the formation of racemic amide which is then hydrolyzed to the acid in 95% ee (118). Prochiral 3-substituted glutaronitriles (93) are hydrolyzed by Phodococcus butanica in up to 71% yield with excellent selectivity (119). 

R. D. Fallon, J. E. Gavagan, R. DiCosimo, and M. S. Payne, Purification, cloning, sequendng and over-expression in Escherichia coli of a regioselective aliphatic nitrilase from Acidovorax facilis 72W, Appl. Microbiol. Biotechnol. 2003, 61, 118-122. 

In addition to the outstanding chemoselectivity, high regioselectivity can also be found in the hydrolysis of dinitriles and has been used for example in the chemo-enzymatic production of lactams from aliphatic a,co-dinitriles. Using a nitrilase from Acidovomxfacilis or a nitrile hydratase/amidase system from Comamonas testosteroni high yields of the lactams have been achieved (see Scheme 12.1-14)164. ... 

Nitrilase, nitrile hydratase Nitrile hydrolysis Chemo- and regioselectivity Enantioselectivity, enzyme stability State of the art, stable (commercial) nitrilases ... 

Bayer, S., Birkemeyer, C., and BaUschmiter, M. (2011). A nitrilase from a metagenomic library acts regioselectively on aliphatic dinitriles. Applied and Environmental Microbiology, 89,91-98. 

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