Nitrile amidase system

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... 

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. 

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]. 

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... 

In addition to the outstanding chemoselectivity, high regioselectivity can also be found in the hydrolysis of dinitriles and has been used for example in the chemo-enzymatic production of lactams from aliphatic a,co-dinitriles. Using a nitrilase from Acidovomxfacilis or a nitrile hydratase/amidase system from Comamonas testosteroni high yields of the lactams have been achieved (see Scheme 12.1-14)164. ... 

Cantarella, M., Cantarella, L., Gallifiioco, A., and Spera, A. (2006) Use of a UF-membrane reactor for controlling selectively the nitrile hydratase - amidase system in Microbacterium imperiale CBS 498-74 resting cells. Case study ben-zonitrile conversion. Enzyme Microb. Technol., 38, 126 134. 

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... 

Stereospecific nitrilases were used for the conversion of a-arninonitiiles to optically active L-amino acids (Fig. 4). In an early investigation, L-alanine was formed by an l-specific nitrilase from alginate-immobilized cells of Acinetobacter sp. APN [25]. A decrease of the enantioselectivity with the time was supposed to be caused by a racemase forming d- from L-alanine. The stereoinversion of racemic a-aminopropionitrile led to a conversion yield above 50%. Similar L-a-amino acid preparations showed no stereoinversion and additionally accumulated the D-amide due to the presence of a nitrile hydratase/ amidase system [26,27]. Additionally, a number of L-a-amino acids were synthesized by a 45-kDa monomeric nitrilase from R. rhodochrous PA-34 [28]. Remarkable in this case was the preferential hydrolysis of a-aminopropionitrile to D-alanine in contrast to the l-alanine formation by the Acinetobacter nitrilase (Fig. 4). 

Figure 17 Racemic resolution of (/ ,5)-2-arylpropionitriles by stereoselective nitrile hydratase/ amidase systems from Rhodococcus sp. SP 361 and Agrobacterium tumefaciens d3.

Figure 18 Stereospecific formation of the anti-inflammatory agent (S)-naproxen by the stereoselective amidase/nitrile hydratase system from two Rhodococcus strains.

Predominantly Rhodococcus species have been isolated as biocatalysts bearing stereoselective nitrile-converting enzymes. It has been assumed that rhodococci generally contain a nitrile hydratase/amidase system [128], but to regard this as a rule might be... 

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. 

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],... 

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]. 

Figure 17.1 Nitrile conversion catalyzed by nitrile hydratase (NHase)-amidase (AMase) system from Microbacterium imperiale CBS 498-74 here illustrated.

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

The operational thermal stability of enzymes can be easily evaluated in experiments carried out in a CSMR fed with a saturating substrate concentration, while varying the temperature but maintaining all the other parameters constant. Each enzyme of the cascade system was tested by feeding the CSMR with the appropriate substrate. The kinetic characterization of amidase-catalyzed reactions in runs fed with a nitrile was hampered by the fact that the intracellular enzyme works in cascade with nitrile hydratase. The concentration of amide, produced in situ in the first step, varied with the time and reaction conditions and did not assure the differential conditions needed for an accurate analysis, the amide being completely converted by amidase in some runs. Hence, amidase activity was characterized independently by feeding the reactor with amide as the substrate [35]. 

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. 

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. 

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