Nitrile hydratase-amidase

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]. 

The ability of an amidase to catalyze the acyl transfer to acceptors other than water (e.g., hydroxylamine) has been utilized in the laboratory synthesis of potential 

Nitrilase is a useful alternative to the NHase/amidase cascade, the benefits of nitrilase often being its higher enantioselectivity or stability. However, a drawback of nitrilase is its tendency to form mixtures of carboxylic acids and amides, this phenomenon being caused by an untypical cleavage of the reaction intermediate leading to the amide as the side product [55]. 

Therefore, in order to obtain the carboxylic acid as the final product, it was proposed to transform the amide using an amidase. The reactions can proceed in a single reactor or in two reactors connected in series. The utility of this method was exemplified by the hydrolysis of 4-cyanopyridine into isonicotinic acid [56], an intermediate in the synthesis of the tuberculostatic drug isoniazid. Nitrilases from Aspergillus niger or Fusarium solani and an amidase from R. erythropolis were chosen for this process, as they exhibited compatible substrate specificities. If the cascade reactions were carried out in two separate reactors, a crude extract from R. erythropolis A4 cells could serve as the amidase source. In a one-pot reaction, the NHase present in this extract would compete with the nitrilase and increase 

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Enantioselective transformations of several cyclopropane or oxirane-containing nitriles were studied using nitrile-transforming enzymes [78]. Microbial Rhodococcus sp. whole cells containing a nitrile hydratase/amidase system hydrolyzed a number... 

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). 

We also found that the occurrence of aldoxime dehydratase is as wide as that for nitrile-degrading enzymes such as nitrile hydratase, amidase and/or nitri-lase. All of the nitrile degraders hitherto isolated contained aldoxime dehydratase activities. The author would like to propose that the pathway in which aldoximes are successively degraded via nitrile could be named as the aldoxime-nitrile pathway (Fig. 1). 

Desymmetrization is not restricted to a single class of enzyme. For example, Madrell et reported the gram-scale preparation of a key intermediate of the lovastatin lactone through the desymmetrization of 3-(benzyloxy)glutaronitrile using whole cells from Brevibacterium R312. The transformation occurs via a dual nitrile hydratase/amidase-catalysed hydrolysis to afford acid in 65 % yield and 88 % ee (Scheme 1.49). 

Bacteria, Aberdeen, UK), was found to have a highly active nitrile hydratase/amidase enzyme system, based on whole-cell biotransformation experiments. Subsequently, individual enzymes (nitrile hydratase and amidase) from this strain were cloned and expressed separately in E. coli However, distribution of some strains or other materials from these public collections may be limited, usually as a result of the restrictions on their commercial use imposed by intellectual property rights. 

A potential versatile route into a-amino acids and their derivatives is via a combination of (i) nitrile hydratase/amidase-mediated conversion of substituted malo-nonitriles to the corresponding amide/acid followed by (ii) stereospecific Hofmann rearrangement of the amide group to the corresponding amine. Using a series of a,a-disubstituted malononitriles 14, cyanocarboxamides 15 and bis-carboxamides 16, the substrate specificity of the nitrile hydratase and amidase from Rhodococcus rhodochrous IF015564 was initially examined (Scheme 2.7). The amidase hydrolyzed the diamide 16 to produce (R)-17 with 95% conversion and 98%e.e. Amide 17 was then chemically converted to a precursor of (S)-a-methyldopa. It was found... 

PAN was for a long time thought to be resistant to microbial attack. However, various bacteria that produced nitrile-converting enzymes were isolated from waste-waters of factories producing PAN fibre. Eor example, a nitrile hydratase/amidase enzyme system was studied from Mesorhizobium sp. E28 [68]. Also, bacteria (namely Ralstonia solanacearum and Acidovorax avenae) were used for the removal of acrylic acid from such waste-waters [69]. Later, on the basis of NMR... 

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],... 

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). 

FIGURE 4.4 Mechanism of nitrilase, nitrile hydratase, amidase on PAN. 

Hydrolysis is the most common pathway for the microbial metabolism of nitriles. It can proceed by one of the two distinct mechanisms. The enzymes involved are nitri-lases, or a nitrile hydratase-amidase system (Figure 17.1) ... 

Strategic importance of biocatalyzed synthetic transformations in terms of eco-compatibility and cheaper processes has been widely stressed previously. Among the developed biotransformations catalyzed by nitrilases or nitrile hydratases/ amidases systems, a special interest is focused toward stereoselective reactions able to give access to molecules otherwise impossible to obtain by classical chemical routes. Hereby, selected examples aim to offer an overview of research in this direction. Examples of industrial processes using nitrile hydrolyzing biocatalysts are also illustrated. 

Nitrile Hydratase/Amidase Biocatalyzed Transformation of 3-Hydroxy-4-Aryloxybutanenitriles and 3-Hydroxy-3-Arylpropanenitriles... 

FIGURE 17.10 Nitrile hydratase/amidase catalyzed biotransformation of O-protected P-hydroxy nitriles. 

Nitrile Hydratase/Amidase Catalyzed Stereoselective Transformation of cis- and frans-N-Protected-P-amino-cyclopentane/ hexane Nitriles... 

Rhodococcus erythropolis AJ270 (nitrile hydratase/amidase)... 

Rhodococcus erythropolis AJ270 (nitrile hydratase/amidase) ------------------------ ... 

During our longstanding interest in the biohydrolysis of nitriles, we found that whole cell preparations of certain Rhodococci, such as R. erythropolis A4 (formerly R. equi A4), R. sp. R312, and R. erythropolis NCIMB 11540, containing the nitrile hydratase/amidase enzyme system, are efficient catalysts for stereoselective microbial hydrolysis of N-protected carbocyclic P-amino nitriles ( )-la-( )-4a, to P-amino acids lc-4c and amides lb-4b, respectively (Scheme 15.1) [33, 34]. 

Nitrile Hydratase/Amidase Biotran ormations 251 Table 15.1 Rhodococcus erythropolis A4 whole cell biohydrolysis isolated products. 

UF-Membrane Bioreactors for Kinetics Characterization of Nitrile Hydratase-Amidase-catalyzed Reactions a Short Survey... 

Yeast strains belonging to the species of Candida fahianii, Candida guilliermondii, Candida tropicalis, Deharyomyces hansenii, Saccharomyces cerevisiae, Torulopsis Candida CandidaJumata), and Williopsis satumus also exhibit the nitrile hydratase/ amidase system able to use the series of aliphatic mono- and dinitriles as well as their matching amides as the sole N-source [20]. 

Temperature Dependence of the Nitrile Hydratase-Amidase Cascade System 275... 

This was performed for each enzyme independently, feeding the reactor with the appropriate substrate (nitrile for the cascade reaction, amide for the sole amidase). The activation energies of both catalysed reactions were evaluated together with those of the inactivation process that inevitably takes place even under the most suitable operational conditions. In the nitrile hydratase/amidase cascade system nitrile hydratase is the more labile enzyme that imposes process temperature choice. These findings make accessible the complete kinetic expression of the dependence from temperature of reaction rate, allowing accurate prediction on reactor performances for process scale-up. 

Enantioselective hydrolysis of nitriles by the nitrile hydratase/amidase system has often been attributed to the combination of a non-selective nitrile hydratase and a selective amidase. However, more recently several enantioselective nitrile hydratases have also been identified and studied in detail. For example, a nitrile hydratase from... 

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