Nitrilases EC

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. 

Nitrilase [EC 3.5.5.1], also known as nitrile aminohy-drolase and nitrile hydratase, catalyzes the hydrolysis of a nitrile to produce a carboxylate and ammonia. The enzyme acts on a wide range of aromatic nitriles. Nitrile hydratase [EC 4.2.1.84], also known as nitrilase, catalyzes the hydrolysis of a nitrile to produce an aliphatic amide. The enzyme acts on short-chain aliphatic nitriles, converting them into the corresponding acid amides. However, this particular enzyme does not further hydrolyze these amide products nor does the enzyme act on aromatic nitriles. 

There are two distinct classes of enzymes that hydrolyze nitriles. Nitrilases (EC. 3.5.5.1) hydrolyze nitriles directly to corresponding acids and ammonia without forming the amide. In fact, amides are not substrates for these enzymes. Nitriles also may be first hydrated by nitrile hydratases to yield amides which are then converted to carboxylic acid with amidases. This is u two-enzyme process, in which enanlioselectivity is generally exhibited by the amidase. rather than the hydratase. 

The microbial degradation of nitriles can occur via two different enzymatic pathways [39], different from cyanogenesis [40] (i) nitrilase (EC 3.5.5.1) catalyzes the direct hydrolysis of nitriles to the corresponding carboxylic acids and ammonia (Eq. 1) [41] and (ii) nitriles are catabolized in two stages - they are first converted to the corresponding amides by nitrile hydratase (EC 4.2.1.84) (Eq. 2), and then to the acids and ammonia by amidase (Eq. 3). The microbial degradation of nitriles has also been reviewed elsewhere [42-45]. 

Alternatively, enantiopure 2-hydroxycarboxylic acids can be obtained via a dynamic kinetic resolution of the (chemically synthesized) cyanohydrin in the presence of an enantioselective nitrilase (EC 3.5.5.1) (see Figure 16.1, route b). Racemization of the cyanohydrin, via reversible dehydrocyanation, takes place readily at pH 7 or above. The methodology [1] is attractive on account of the mild reaction conditions and is industrially applied in the multiton-scale synthesis of (R)-mandehc acid [2]. 

Nitrile biodegradation is performed by a variety of microorganisms and proceeds through two different enzymatic pathways direct transformation to carboxylic acids and ammonia, with some exceptions, catalyzed by a nitrilase (EC 3.5.5.1) [1-3] or a two-step reaction, the first catalyzed by nitrile hydratase (EC 4.2.1.84) that produces an amide intermediate, which is further hydrolyzed to the acid and ammonia by an amidase (EC 3.5.1.4) [4, 5],... 

The degradation of nitriles by nitrilases (EC 3.5.5.1) has been the subject of intense study, especially as it relates to the preparation of the commodity chemical acrylamide. Nitrilases catalyze the hydrolysis of nitriles to the corresponding acid plus ammonia (Figure 1 reaction 5), whereas nitrile hydratases (EC 4.2.1.84) add water to form the amide. Strains such as Rhodococcus rhodo-chrous Jl, Brevibacterium sp., and Pseudomonas chlororaphis have been used to prepare acrylamide from acrylonitrile, which contain the hydratase and not nitrilase activity [12]. A comparison of these strains has been discussed elsewhere [98]. Other uses of nitrilases, however, have primarily been directed at resolution processes to stereoselectively hydrolyze one enantiomer over another or regiose-lectively hydrolyze dinitriles [99-101]. 

Over the past, about 30 years, more than 100 studies have been published on the use of nitrilases (EC 3.5.5.x) as catalysts for the hydrolysis of nitriles, the focus being on enzymes suitable for the synthesis of industrially important products such as mandelic acid and other hydroxy acids, mandelamide or unnatural amino acids, often enantiopure compounds. In these works, several strategies were applied in order to get new nitrilases (Figure 12.1). 

Nitrilases (EC 3.5.5.1) catalyze the direct hydrolysis of organic cyanides or nitriles into the corresponding carboxylic adds without the release of intermediate amides. These enzymes are generally believed to contain a conserved Glu-Lys-Cys catalytic motif [20]. Although yet unproven due to a lack of crystal structures, the... 

The nitrilase mediated DKR route to enantiomerically pure 2-hydroxycarboxylic acids is restricted to the (R)-enantiomers because, to our knowledge, no (S)-selec-tive nitrilases for cyanohydrin substrates are commonly available [11]. We reasoned that a fully enzymatic route to the (S)-acids should be possible by combining an (S)-selective oxynitrilase (hydroxynitrile lyase, EC 4.1.2.10, (S)-hydroxynitrile lyase) and a non-selective nitrilase in a bienzymatic cascade (see Figure 16.3). Besides being more environmentally acceptable than chemical hydrolysis, the mild reaction conditions of the combined enzymatic reaction would be compatible with a wide range of hydrolysable groups. 

In the same group as nitrilase enzymes are the amidases. This includes amino acid amidase (EC 3.5.1.4), used to prepare amino acids, usually through resolution, and also penicillin G acylase (penicillin G amidohydrolase) (EC 3.5.1.11), used in the manufacture of semisynthetic penicillins [102,103]. Immobilized penicillin G acylase has most recently been used to catalyze the formation of A-a-phenylacetyl amino acids, which can then be used in peptide coupling reactions (see Section 13.2.3.2) [104]. 

The two enzyme classes nitrile hydratases (RCN + H20 — RCONH2) and nitrilases (RCN + 2H20 —y RCOOH + NH3) actually belong to two distant groups in the EC system, with the hydratases being classified as lyases (EC 4.2.1.84) and nitrilases as hydrolases (EC 3.5.5.1). Microorganisms that produce a nitrile hydratase also seem to produce amidases, which enable them to convert nitriles into carboxylic acids in a two-step reaction. Actually, amidase side-activity can be a problem with commercial nitrile hydratase preparations (if the target structure is the amide). Nitrilases, however, hydrolyze the nitrile without the formation of a free amide intermediate. 

Hydrations of nitriles amides C=C Rhodococci, microbial nitrilases, nitrile hydratases microbial amido hydrolases BY, Ec, Prm, fumarase... 

There are many examples of nitrilase-catalyzed reactions in which amides form a considerable amount of the reaction products, such as the transformations of acrylonitrile analogs and a-fluoroarylacetonitriles by nitrilase 1 from Arabidopsis thaliana [17], the conversion of p-cyano-L-alanine into a mixture of L-asparagine and L-aspartic acid by nitrilase 4 from the same organism [18] or the transformations of mandelonitrile by nitrilase from Pseudomonas jhiorescens [19] or some fungi [8], Moreover, formamide is the only product of the cyanide transformation by cyanide hydratase. Therefore, this enzyme was classified as a lyase (EC 4.2.1.66), although it is closely related to nitrilases, as far as its aa sequence and reaction mechanism are concerned [3]. 

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