Nitrile-forming enzymes

The role of ferrous ions in modifying the enzymatic degradation of glucolimnanthin is not completely understood. It is known that species of Brassica produce the so-called nitrile-forming enzymes, such as the epispecifier protein, that require ferrous ions at micromolar concentrations [18]. When we incubated... 

Besides the use of stereoselective nitrile-converting enzymes as described above, useful chiral building blocks have also been obtained by stereoselective nitrile-forming enzymes. The main product class of nitrile-forming enzymes are cyanohydrins (a-hydroxynitriles, 1-cyanoalkanols), which are versatile synthons in organic synthesis that are readily convertible to a-hydroxy acids [90], a-hydroxy aldehydes [91], ethanolamines [92], amino alcohols, pyrethroid insecticides [93], imidazoles, and heterocycles [94]. Examples of valuable bioactive products derived from chiral cyanohydrins are (i )-adrenaline, L-ephedrin, and (5)-amphetamines [95]. For the synthesis of chiral cyanohydrins, stereoselective enzymes from both plant and bacterial sources have been used. 

The dehydration reaction of aldoxime to form nitriles using the resting cells of Rhodococcus sp. YH3-3 was optimized. We found that the enzyme was induced by aldoxime and catalyzed the stoichiometric synthesis of nitriles from aldoximes at pH 7.0 and 30°C. Phenylacetonitrile once synthesized from phenylacetaldoxime was hydrolyzed to phenylacetic acid, since the strain has nitrile degradation enzymes such as nitrile hydratase and amidase. We have been successful in synthesizing phenylacetonitrile and other nitriles stoichiometrically by a selective inactivation of nitrile hydratase by heating the cells at 40°C for 1 h. Various nitriles were synthesized under optimized conditions from aldoximes in good yields. 

Leflunomide (Arava) is an anti-proliferative drug that inhibits dihydroorotate dehydrogenase, an enzyme essential for the synthesis of pyrimidines such as uracil, via aromatisation of dihydroorotic acid. It also has some immunomodulatory effects and is useful for intractable cases of rheumatoid arthritis, despite being liable to cause a number of serious side-effects. It is actually a prodrug, being converted into the active compound, a nitrile formed by cleavage of the isoxazole ring (cf. 25.5.1). 

In a full account of preliminarily published results, detailed evidence is presented for the above conclusions, reached through experiments with linen flax seedlings, producing linamarin (62 R = R = Me), and cherry laurel or peach shoots, synthesizing prunasin (62 R = H, R = Ph). A notable incorporation of 2-oximinoisovaIeric acid and 2-oximino-3-phenylpropionic acid into linamarin and prunasin, respectively, may conceivably reflect the role of the oximino-acids as sources of nitriles, formed by a long-known, non-enzymic conversion. Experiments aimed at inducing accumulation of intermediates... 

Siace the late 1980s, there has been considerable activity ia the development of fermentative approaches for the preparation of the amide from the nitrile. Organisms such as A.chromohacter (35), A.grohactenum (36), Streptomjces (37), Phodococcus (38,39), and Comebacterium (40) have been described. Purified enzymes ia either free (41) or immobilized form (42,43) have also been used ia this appHcation. 

There are two distinct classes of enzymes that hydrolyze nitriles. Nittilases (EC 3.5.5. /) hydrolyze nittiles directiy to corresponding acids and ammonia without forming the amide. In fact, amides are not substrates for these enzymes. Nittiles also may be first hydrated by nittile hydratases to yield amides which are then converted to carboxyUc acid with amidases. This is a two-enzyme process, in which enantioselectivity is generally exhibited by the amidase, rather than the hydratase. 

On the other hand, 3-phenylpropionitrile was synthesized from Z-3-phenyl-propionaldoxime (0.75 M) in a quantitative yield (98gP ) by the use of cells of E. coli JM 109/pOxD-9OF, a transformant harboring a gene for a new enzyme, phenylacetaldoxime dehydratase, from Bacillus sp. strain OxB-1. Other arylalkyl- and alkyl-nitriles were also synthesized in high yields from the corresponding aldoximes. Moreover, 3-phenylpropionitrile was successfully synthesized by the recombinant cells in 70 and 100% yields from 0.1 M unpurified P/Z-3-phenylpropionaldoxime, which is spontaneously formed from 3-phenylpropionaldehyde and hydroxylamine in a butyl acetate/water biphasic system and aqueous phase, respectively. 

Bacterial enzymes have been reported to catalyze the hydrolysis of nitriles [118][121], A nitrilase (EC 3.5.5.1) acts to hydrolyze aromatic nitriles directly to the carboxylic acid. A nitrile hydratase (a lyase, EC 4.2. E84) acts on short-chain aliphatic nitriles to form the amide. As discussed below, the hydrolysis of nitriles to amides is also documented in mammals, but little appears known about the enzymes involved. 

Generally, enzymatic hydrolysis of nitriles to the corresponding acids can either proceed stepwise, which is the case for catalysis by the nitrile hydratase/amidase enzyme system, or in one step in the case of nitrilases. Both systems have been investigated for surface hydrolysis of PAN [10], Complete hydrolysis with either system was monitored by quantification of ammonia and/or polyacrylic acid formed as a consequence of hydrolysis of nitrile groups [70-72], As a result, considerable increases in colour levels (e.g. 156% for commercial nitrilase) were found upon dyeing [72],... 

---