S -specific nitrilase

Figure 5.6 Production of S(+)-ibuprofen from rac. 2-(4 -isobutylphenyt)-propionitrile using an S-specific nitrilase irora. Acinetobacter (Yamamoto eta/., 1990).

Ibuprofen [(iS)-2-(4-isobutylphenyl)propionic acid] has been prepared by resting cells of Acinetobacter sp. AK 226 [34]. Evidence for a single-step nitiilase-catalyzed reaction was obtained by the lack of an intermediate amide formation, the remaining R) nitrile (Fig. 12), and the inability to transform the racemic amide. The (S)-specific nitrilase was purified and characterized as a typical high molecular mass enzyme of 580 kDa [45]. 

Figure 10 Synthesis of (S)-2-arylpropionic acids by an (S)-specific nitrilase from Rhodococcus rhodochrous ATCC 21197.

Figure 16.3 Bienzymatic procedure for the synthesis of (S)-2-hydroxycarboxylic acids, using an (S)-specific hydroxynitrile lyase and a non-specific nitrilase in tandem.

Figure 6 Synthesis of (J )-mandelic acid from (J ,S)-mandelonitrile by an (R)-specific nitrilase from Alcaligenes faecalis ATCC 8750.

Figure 8.10 Relative specific activity of Y296 mutations of Alcaligenes faecalis Nitl650 nitrilase for conversion of racemic 2-chloromandelonitrile Y296A (A), Y296F (F), Y296C (C), Y296G (G), Y296N (N), Y296T (T), and Y296S (S)...

The a-chloroacetamide group has features that are beneficial for undirected ABPP. Its small size does not bias binding elements towards a specific class of enzyme, and it possesses reactivity towards a broad variety of nucleophilic amino acid residues. A library of a-chloroacetamide-based probes were synthesized by Cravatt s group. The binding element in these probes was a dipeptide that was varied with small, large, hydrophobic, and charged side chains, and a biotin or rhodamine tag was appended as a reporter tag. Upon screening of eukaryotic proteomes with this library, many enzymes previously unaddressed by directed ABPP probes were uncovered. These included fatty acid synthase, hydro-xypyruvate reductase, malic enzyme, and the nitrilase superfamily [163, 164]. In contrast to the sulfonate esters, a-chloroacetamides react preferentially with cysteine residues in the proteome. 

The product of a NHase/amidase cascade reaction is an acid, which is the same as the single enzymatic reaction performed by a nitrilase. However, the NHases usually have different substrate specificities than nitrilases, making them more suitable for the production of certain compounds. Although most organisms have both NHase and amidase activity (see earlier text), it is sometimes preferable, in a synthetic application, to combine enzymes from different organisms. The reasons for this are the enantioselectivity of the amidase or specific activity or substrate specificity of either of the enzymes. In this way, products with different enantiomeric purity can be obtained. Recently, a coupling of a NHase with two different amidases with opposite enantiopreference together with an -amino-a-caprolactam racemase that allows the formation of small aliphatic almost enantiopure (R)- or (S)-amino acids via dynamic kinetic resolution processes has been described [52]. 

Together with R. rhodochrous ATCC 21197 [43] and Pseudomonas putida NRRL 18668 [51], also Rhodococcus sp. C3II md Rhodococcus erythropolis MP 50 were used for the enantiospecific preparation of (S)-naproxen [65]. Rhodococcus sp. C3II lacks a nitrilase but exhibits nitrile hydratase and amidase activities, both of which are constitutive and prefer the (5 )-enantiomers of naproxen derivatives. On the other hand, the enzymes from R. erythropolis MP 50 were induced by nitriles and its nitrile hydratase was (R)-specific [44]. Due to the presence of a strictly (5)-specific amidase, both strains finally formed (5)-naproxen with high enantioselectivity (Fig. 18). Evidence for the enantioselectivity of the nitrile hydratases of both strains was obtained by the formation of optically active amides in the presence of the amidase inhibitor diethyl phosphoramidate [63,65]. The nitrile hydratase of Rhodococcus sp. C3II whole cells was used for the sjmthesis of (>S)-naproxen amide with 94% e.e. after 30% conversion in the presence of the amidase inhibitor [63]. In addition, the highly stereoselective amidases of these two strains were used to prepare (5)-ketoprofen (Fig. 28), and the amidase from R. erythropolis MP 50 was used to prepare (5)-2-phenylpropionic acid with more than 99% e.e. and more than 49% conversion [66,67]. 

After acetylation with vinyl acetate, the Amano lipase PS was used for the (R)-specific ester cleavage of (R,S)-cyanohydrin acetate [113]. The chirality of acetylated (JS)-and nonacetylated (JR)-cyanohydrin products was enhanced using R. rhodochrous ATCC 21197 for the further conversion to optically active a-hydroxy acids (Fig. 35), which are useful starting materials in synthetic organic chemistry [90]. The Rhodococcus strain has been reported to contain a stereospecific nitrilase (Fig. 22), a stereospecific nitrile hydratase (Figs. 10 and 11), and a stereospecific amidase (Figs. 16 and 27). 

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