Nitrilase-amidase

Hydrolases Hydrolytic reactions in H O Lipase, protease, esterase, nitrilase, amidase, glycosi-dase, phosphatase... 

Figure 11.11 Biotransformation of 4-cyanopyridine into isonicotinic acid by a nitrilase-amidase cascade in a single reactor and a two-reactor system [56].

Hydrolysis of the nitriles 36a and Shh" by an immobilized enzyme system (nitrilase/ amidase activity) prepared from Rhodococcus sp. SP409 provided the corresponding acids 37 in 40-50% yields. However, the more bulky substrate 36c was unreactive under these conditions (Figure 12.18). 

Several classes of enzymes have been used to separate stereoisomers of a-H-and a-disubstituted amino acids, eg amidases, nitrilases, hydantoinases, acylases and esterases. 

As illustrated in Figure A8.3 nitrilases catalyse conversions of nitriles directly into the corresponding carboxylic adds (route A), while other nitrile converting enzymes, die nitrile hydratases, catalyse the conversion of nitriles into amides (route B) which, by the action of amidases usually present in the whole cell preparations, are readily transformed into carboxylic adds (route C). 

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 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]. 

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). 

Similarly, the selective herbicides, bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) and ioxynil (3,5-diiodo-4-hydroxybenzonitrile) are degraded by soil bacteria to their corresponding amide products 3,5-dibromo-4-hydroxybenzamide (BrAM) and 3,5-diiodo-4-hydroxybenzamide (IAM) but are not further degraded to the corresponding acids. The identification of amidases or nitrilases able to effect these transformations, in a soil bacterium, would be of value as a bioremediation agent [48],... 

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

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]. 

FIGURE 19.1 General reactions of hydrolase enzymes. In the same group as nitrilase enzymes are the amidases. This includes amino acid amidase ... 

Wong, C.-H., Whitesides, G. M. Chapter 2 Use of Hydrolytic Enzymes Amidases, Proteases, Esterases, Lipases, Nitrilases, Phosphatases, Epoxide Hydrolases. In Enzymes in Synthetic Organic Chemistry, Elsevier New York, 1994, p. 41. 

Recently a number of enzymatic systems have been developed at several chemical companies including Upases (synthesis of enantiotrope alcohols, R-amid, S-amin), nitrilases (R-mandehc acid), amidases (non-proteinogenic L-amino acids), aspartic acid ammonia lyase (L-aspartic add), penicilin acylase (6-Aminopenicilanic acid), acylases (semisynthetic penicillins), etc.( Koeller and Wong, 2001 and references therin). 

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). 

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. 

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