Nitrilases, Nitrile Hydratases, and Amidases

Nitrilases are quite rare in bacterial genomes and less than 20 were reported prior to the application of metagenomics for their detection in environmental DNA [81]. Two studies targeting environmental genomes report the detection of more than 337 novel nitrilases. This has dramatically increased the amount of information about nitrilases, and the newly discovered diversity can be applied for the enantioselective production of hydroxy carboxylic add derivatives [81]. 

Alcohol oxidoreductases capable of oxidizing short chain polyols are useful biocatalysts in industrial production of chiral hydroxy esters, hydroxy adds, amino adds, and alcohols [83]. In a metagenomic study without enrichment, a total of 24 positive clones were obtained and tested for their substrate specifidty. To improve the detedion frequency, enrichment was performed using glycerol or 1,2-propanediol and further 24 positive clones were deteded in this study. 

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Nitrilases, Nitrile Hydratases, and Amidases 5.03.8.1 The Reactions and the Enzymes... 

Nitrilases and amidases belong to the class of hydrolases and nitrile hydratase belongs to the class of lyase. Nitrilases are an important class of nitrilase superfamily that convert nitrile to the corresponding carboxylic acids and ammonia, whereas nitrile hydratase first converts into the corresponding amide and then this amide is transformed by amidase. There are very few reports for the surface modification of PAN and PA for increasing its hydrophilicity using nitrilases, nitrile hydratases, and amidases. 

The hydrolysis of aliphatic and aromatic nitriles into amides and/or carboxylic acids is an area of rapidly increasing interest. Already the Nitto Chemical Industry Co. Ltd. has developed a large-scale process for the conversion of acrylonitrile into acrylamide using Rhodococcus cells (see Section 6.7.2). Many other organisms, as well as the nitrilases, nitrile hydratases and amidases derived from them, can effect similar conversions. It is interesting to note that for dinitriles, only one of the nitrile groups... 

Rhodococcus rhodochrous was used for the hydrolysis of both granular PAN and acrylic fibers by nitrile hydratase and amidase (Tauber et al., 2000). Similarly, Agrobacterium tumefaciens (BST05) was found to convert polyacylonitrile into polyacrylic acid by nitrile hydratase and amidase (Fischer-Colbrie et al., 2006). Nitrilase was also used for the surface hydrolysis of PAN from Micrococcus luteus BST20 (Fischer-Colbrie et al., 2007). However, polyamidase from Nocardia farci-nica leads to an increase of polar groups on the surface of PA, which was measured by tensiometry (Heumann et al., 2009). 

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. 

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]. 

Our original studies with unprotected P-hydroxynitriles showed that these compounds were hydrolyzed by R. rhodochrous ATCC BAA-870, expressing a benzamide-induced cobalt type nitrile hydratase, to the corresponding amides and acids [11]. The formation of the amide implies a nitrile hydratase and amidase system (although sometimes nitrilases can release partially hydrolyzed substrates as amides [63]). Further studies in our laboratories demonstrated that the system was indeed a nitrile hydratase and amidase cascade reaction functioning via a two... 

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]. 

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]. 

Very few reports are available for the enzymatic surface modification of synthetic fibers. Peroxidase, lipase, cutinase, nitrilase, nitrile hydratase, amidase, protease, and hydrolase have been reported for the surface modification of synthetic polymers (Table 4.1). 

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]. 

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

The first pathway via the amide uses two different enzymes a nitrile hydratase and an amidase, and in the second one, the nitrile is directly hydrolysed into ammonium carboxylate by a nitrilase. When the enzymes are not pure, we often discriminate between the two routes by using the amide, a substrate which is present only in the first system. 

Glycolic acid can be produced via fermentation process [6] from glycolo-nitrile hydrolysis by mineral acid, such as sulfuric acid [7,8]. Both processes produce a multi-component solution with the acid concentration typically less than 10 wt% for fermentation technology, and less than 40 wt% for glycolo-nitrile hydrolysate. The acid can be produced by the enzymatic conversion (typically the enzyme catalyst used is nitrilase or a combination of a nitrile hydratase and an amidase) of glycolonitrile which results in the production of an aqueous solution of ammonium glycolate [9]. 

Since Rhodococcus sp. SP 361 lacks a nitrilase but no amide intermediates were detected, a slow, pro-(S)-selective nitrile hydratase and a fast, nonselective amidase were proposed to be active. This contrasts with most other cases bearing a fast nitrile hydratase without or with low enantioselectivity and a slow, stereospecific amidase. 

L-Amino adds could be produced from D,L-aminonitriles with 50% conversion using Pseudomonas putida and Brembacterium sp respectively, the remainder being the corresponding D-amino add amide. However, this does not prove the presence of a stereoselective nitrilase. It is more likely that the nitrile hydratase converts the D,L-nitrile into the D,L-amino add amide, where upon a L-spedfic amidase converts the amide further into 50% L-amino add and 50% D-amino add amide. In this respect the method has no real advantage over the process of using a stereospecific L-aminopeptidase (vide supra). 

The discovery and exploitation of enzymes in aldoxime-nitrile pathway nitrile hydratase, amidase, nitrilase, aldoxime dehydratase, etc., are shown along with the use of methodologies, such as organic chemistry, microbial screening by enrichment and acclimation culture techniques, enzyme purification, gene cloning, molecular screening by polymerase chain reaction (PCR). 

There are two pathways for the degradation of nitriles (a) direct formation of carboxylic acids by the activity of a nitrilase, for example, in Bacillus sp. strain OxB-1 and P. syringae B728a (b) hydration to amides followed by hydrolysis, for example, in P. chlororaphis (Oinuma et al. 2003). The monomer acrylonitrile occurs in wastewater from the production of polyacrylonitrile (PAN), and is hydrolyzed by bacteria to acrylate by the combined activity of a nitrilase (hydratase) and an amidase. Acrylate is then degraded by hydration to either lactate or P-hydroxypropionate. The nitrilase or amidase is also capable of hydrolyzing the nitrile group in a number of other nitriles (Robertson et al. 2004) including PAN (Tauber et al. 2000). 

Nitriles are interesting precursors of both amides and carboxylic acids. In vivo there are two pathways for the bioconversion of nitriles to carboxylic acids (Scheme 6.19). In the first method a nitrilase catalyzes the enantioselechve hydrolysis of a racemic or prochiral nitrile. The second pathway involves a two-enzyme cascade in which an aselective nitrile hydratase (NHase) catalyzes the hydration of the racemic nitrile to the racemic amide followed by an amidase-catalyzed enantioselechve hydrolysis to the carboxylic acid. The amidase is generally, but not always, (S)-selechve, resulting in the formahon of a 1 1 mixture of the (S)-acid... 

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],... 

Figure 5.5 Degradation routes for nitriles. The first route is a two-step reaction involving a nitrile hydratase, which converts the nitrile to the amide, and an amidase, which converts the amide to the corresponding acid. The second pathway involves direct hydrolysis of the nitrile to the carboxylic acid and ammonia by a nitrilase.

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. 

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]. 

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