Nitrilase nitrile hydratase activity

Mateo, C., Chmura, A., Rustler, S. et al. (2006) Synthesis of enantiomerically pure (S)-mandelic acid using an oxynitrilase-nitrilase bienzymatic cascade a nitrilase surprisingly shows nitrile hydratase activity. Tetrahedron Asymmetry, 17, 320-323. 

Fernandes, B.C.M., Mateo, C., Kiziak, C., Chmura, A., Wacker, J., van Rantwijk, R, Stolz, A., and Sheldon. R.A. 2006. Nitrile hydratase activity of a recombinant nitrilase. Advanced Synthesis and Catalysis, 348 2597-2603. 

Not surprisingly, some nitrilase reactions were accompanied by the formation of the corresponding amides, such as pipecolic amide 12b (up to 10%) and pyrrolidine-3-carbo3amide 10b (for a discussion of nitrile hydratase activity of nitrilases see Section 15.3.3). 

However, the rise of more sophisticated analytical techniques has triggered communications on this subject substantially in recent times. This nitrile hydratase activity of nitrilases was frequently observed when substrates activated in the a-position to the nitrile group were used [45, 46]. Most recently, a study appeared on this subject suggesting a mechanistic rationale of amide formation [47]. 

In recent years, the enantioselective hydrolysis of nitriles has been studied in more detail. Whereas in the past only whole cell catalysts had been investigated, it is now possible to assign the activities to specific enzymes occurring in the cell. These enzymes are nitrilases, nitrile hydratases and/or amidases. 

SP 361 SP 409 Immobilized enzyme mixture from Rhodococcus sp. containing nitrilase, nitril hydratase,esterase, epoxide hydrolase and amidase activity discontinued... 

Compilation of the biotransformation information has led to the postulation that the cloned Pseudomonas gene has both nitrilase and nitrile hydratase activities. This novel nitrile hydrolyzing activity is summarized in Figure 8. 

R.A. (2006) Nitrile hydratase activity of a recombinant nitrilase. Adv. Synth. Catal., 348, 2597-2603. 

Further, E. coli cells expressing the cyanide hydratases genes from A. niger and P, chrysogenum were found to exhibit activities for both HCN and some nitrile compounds, preferably 2-cyanopyridine (1-3.6% relative activity compared to HCN) [6]. Dual nitrilase/cyanide hydratase activities were also described for the enzymes in Fusarium oxysporum and Fusarium lateritium [36, 37]. It is possible that this dual activity is a general feature of cyanide hydratases but has largely gone unnoticed. 

Besides an (5)-specific nitrilase and an (5)-specific amidase, Rhodococcus rlwdo-chrous ATCC 21197 was supposed to contain a partially selective nitrile hydratase activity toward (i ,5)-2-arylpropionitriles [43] (Fig. 16). 

The biocatalytic differentiation of enantiotopic nitrile groups in prochiral or meso substrates has been studied by several research groups. For instance, the nitrilase-catalyzed desymmetrization of 3-hydroxyglutaronitrile [92,93] followed by an esterification provided ethyl-(Jl)-4-cyano-3-hydroxybutyrate, a useful intermediate in the synthesis of cholesterol-lowering dmg statins (Figure 6.32) [94,95]. The hydrolysis of prochiral a,a-disubstituted malononitriles by a Rhodococcus strain expressing nitrile hydratase/amidase activity resulted in the formation of (R)-a,a-disubstituted malo-namic acids (Figure 6.33) [96]. 

In summary, the formation of optically active compounds through hydrolysis reactions is dominated by biocatalysis mainly due to the availability and ease of use of a wide variety of esterases, lipases and (to a lesser extent) acylases. Epoxide ring-opening (and related reactions) is likely to be dominated by salen-metal catalysts while enzyme-catalysed nitrile hydrolysis seems destined to remain under-exploited until nitrilases or nitrile hydratases become commercially available. 

The nitrile group is a versatile building block, in particular since it can be converted into acids or amides. It undergoes hydrolysis but requires relatively harsh reaction conditions. Nature provides two enzymatic pathways for the hydrolysis of nitriles. The nitrilases convert nitriles directly into acids, while the nitrile hy-dratases release amides. These amides can then be hydrolysed by amidases (see also above). Often nitrile hydratases are combined with amidases in one host and a nitrile hydratase plus amidase activity can therefore be mistaken as the activity of a nitrilase (Scheme 6.32). A large variety of different nitrilases and nitrile hydratases are available [100, 101] and both types of enzyme have been used in industry [34, 38, 94]. 

Cyanide hydratase and cyanide dihydratase belongs to the nitrilase branch of nitrilase superfamily, using HCN as the only efficient substrate and producing amide and acid products, respectively. Microorganisms appear in fact to have evolved separate metabolic pathways for the hydrolysis of inorganic cyanide. Thus, most nitrilases (as well as nitrile hydratases) till now investigated do not display activity... 

The enzymatic hydrolysis of a broad range of nitriles to the corresponding amides and acids is documented [35]. These conversions are effected directly by nitrilases or by successive action of a nitrile hydratase and an amidase. Most of these enzymes are usually unstable and whole-cell preparations are preferred. However, recently a purified nitrile hydratase preparation without amidase activity was shown to convert several 2-arylpropionitriles enantioselectively to the corresponding optically active amides (eq. (3)) [36]. 

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]. 

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. 

Nitrilases have been studied less than the nitrile hydratases. The enzymes appear as homomultimers, exhibiting a wide range of molecular masses. The reaction mechanism depicted in Fig. 12.1-2 has been proposed recently by Kobayashi et al. 2S. Several nitrilases have been found to be inhibited by reagents which bind to thiol groups, indicating that sulfhydryl groups are essential for the catalytic activity of... 

Analysis of the reaction products showed that during conversion of succinonitrile into 3-cyanopropionic acid, succinamic acid (H2NOC-CH2-CH2-COOH) was detected as a free intermediate in the reaction mixture, suggesting that enzymatic activities other than nitrilases (i.e. nitrile hydratase) were present in the cell... 

However, production of 2,6-difluorobenzamide (Scheme 12.1-18) was effected in 99.5% n-heptane using the nitrile hydratase from Rhodococcus sp. NCIMB 12 21 81841. The enzymatic reaction was found to be activated by light (see 12.1.3.4). More recently, Layh and Willetts have studied nitrile transformations in various organic solvents and biphasic mixtures using a nitrilase from Pseudomonas sp. DSM 11387 and a nitrile hydratase from Rhodococcus sp. DSM 113971 51. The enzymes exhibited good stabilities in biphasic mixtures with hydrophobic solvents when dispersed in... 

Apart from the nitrilase, two other enzymes were purified, a nitrile hydratase acting on ammonium 5-cyanovalerate (ref. 19) and an amidase acting on adipamate (ref. 20).These enzymes were separated on the Q sepharose fast flow column. Consequently, the specific activity after the second step was due to the nitrilase alone and would be higher than 9300 U/mg of pure nitrilase. 

Recently we determined that two R. rhodochrous strains (A29 and A99) expressed nitrilase activity after induction. These strains were capable of enantioselectively hydrolyzing racemic 3-amino-3-phenylpropanenitrile directly to the corresponding (R)-3-amino-3-phenylpropanoic acid with >95% ee (Table 14.1) in a kinetic resolution. Various inhibitors were used, that indicated the observed hydrolytic activity was due to the presence of a nitrilase rather than a nitrile hydratase and amidase pair [47]. 

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