Nitrilases aromatic nitriles

Thermally stable nitrilase from Streptomyces sp. MTCC 7546 was induced by benzonitrile enrichment. While discovered by induction with aromatic nitrile, the enzyme was shown to exhibit a strong preference for aliphatic nitriles, with as high as 30-fold greater activity with aliphatic substrates compared with benzonitrile. The enzyme displays optimal activity at pH 7 and 50 °C [56]. 

A nitrilase from the hyperthermophile Pyrococcus abyssi, which exhibits optimal growth at 100 °C, was cloned and overexpressed. Characterization of this nitrilase revealed that it is operational as a dimer (rather than the more common multimeric structure for nitrilases), with optimal pH at 7.4 and optimal apparent activity at 80 °C with Tm (DSC) at 112.7 °C. The substrate specificity of the nitrilase is narrow and it does not accept aromatic nitriles. The nitrilase converts the dinitriles fumaronitrile and malononitrile to their corresponding mononitriles [58],... 

Bacterial enzymes have been reported to catalyze the hydrolysis of nitriles [118][121], A nitrilase (EC 3.5.5.1) acts to hydrolyze aromatic nitriles directly to the carboxylic acid. A nitrile hydratase (a lyase, EC 4.2. E84) acts on short-chain aliphatic nitriles to form the amide. As discussed below, the hydrolysis of nitriles to amides is also documented in mammals, but little appears known about the enzymes involved. 

Nitrilase [EC 3.5.5.1], also known as nitrile aminohy-drolase and nitrile hydratase, catalyzes the hydrolysis of a nitrile to produce a carboxylate and ammonia. The enzyme acts on a wide range of aromatic nitriles. Nitrile hydratase [EC 4.2.1.84], also known as nitrilase, catalyzes the hydrolysis of a nitrile to produce an aliphatic amide. The enzyme acts on short-chain aliphatic nitriles, converting them into the corresponding acid amides. However, this particular enzyme does not further hydrolyze these amide products nor does the enzyme act on aromatic nitriles. 

Dhillon, J.K., Chhatre, S., Shanker, R., and Shivaraman, N. 1999. Transformation of aliphatic and aromatic nitriles by a nitrilase from Pseudomonas sp. Canadian Journal of Microbiology, 45 811-5. 

Among aliphatic nitriles, unbranched saturatured or unsaturated compounds with a medium chain length (for instance, propionitrile, butyronitrile, hexaneni-trile, and acrylonitrile) are hydrolyzed with the highest relative rate (about 20-35% of that for benzonitrile) by aromatic nitrilases. Branched nitriles are in general poor substrates of these enzymes but, for instance, isobutyronitrile is one of the best substrates of the aliphatic nitrilase from R. rhodochrous K22 [28]. 

Microbial degradation of nitrile compounds was performed by different microorganisms, capable of growing on various aliphatic and aromatic nitriles [107-109], They are degraded through two pathways (Figure 7) one is the direct hydrolysis of nitriles to carboxylic acid and ammonia, catalyzed by nitrilases. Nitrilases, that utilize benzonitrile and related aromatic nitriles as substrates, have been purified from Pseudomonas sp. [110.111], Nocardia sp. strains NCIB 11215 [112] and NCIB 11216... 

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... 

Aromatic nitrilases, which mainly act on (hetero-)aromatic nitriles, for example, benzonitrile... 

Nitrilases catalyze the direct hydrolysis of nitriles to the corresponding acids. Compared to nitrile hydratases, more nitrilases bearing a high stereoselectivity have been described. Nitrilases generally comprise of one type of subunit in multiple association (ae-ao) with a few monomeric (a) and dimeric (aj) exceptions. As a rough rule, they are mostly active with aromatic nitriles but exhibit high activity toward aliphatic nitriles less often [20-24]. 

A crude mixture of enzymes isolated from Rhodococcus sp. is used for selective hydrolysis of aromatic and aliphatic nitriles and dinitriles (117). Nitrilase accepts a wide range of substrates (Table 8). Even though many of them have low solubility in water, such as (88), the yields are in the range of 90%. Carboxylic esters are not susceptible to the hydrolysis by the enzyme so that only the cyano group of (89) is hydrolyzed. This mode of selectivity is opposite to that observed upon the chemical hydrolysis at alkaline pH, esters are more labile than nitriles. Dinitriles (90,91) can be hydrolyzed regioselectively resulting in cyanoacids in 71—91% yield. Hydrolysis of (92) proceeds via the formation of racemic amide which is then hydrolyzed to the acid in 95% ee (118). Prochiral 3-substituted glutaronitriles (93) are hydrolyzed by Phodococcus butanica in up to 71% yield with excellent selectivity (119). 

Nitrilase was initially discovered in plants as an enzyme involved in the biosynthesis of the plant hormone indole-3-acetic acid (IAA) [74,75], Recently, four genes of nitrilases (belonging to arylacetonitrilase) involved in the IAA biosynthesis have been cloned and characterized from Arabidopsis thaliana [76-78], After the discovery of the plant nitrilase in 1964, various nitrilases were purified and characterized [41], Nitrilases are roughly classified into three major categories according to substrate specificity (i) aromatic nitrilase, which acts on aromatic or heterocyclic nitriles (ii) aliphatic nitrilase, which acts on aliphatic nitriles (iii) arylacetonitrilase, which acts on arylacetonitriles. These three types... 

The application of nitrilases is broad. A purified nitrilase from Bacillus pallidus was employed to hydrolyse a wide variety of aliphatic, aromatic and heteroaromatic nitriles and dinitriles (Scheme 6.33) [102]. Nitrilases have also been patented for the hydrolysis of a-substituted 4-methylthio-butyronitriles, however, no stereoselectivity was reported [103]. 

All known fungal nitrilases exhibit high relative activities towards benzonitrile and its m- and p-substituted derivatives. Therefore, according to the nitrilase classification [46] they belong to aromatic nitrilases. However, almost all fungal nitrilases also hydrolyze aUphatic nitriles, albeit at lower relative rates. 

The substrate specificities of all four biochemically characterized fungal nitrilases and two enzymes from rhodococci (an aromatic and an aUphatic nitrilase) are compared in Table 14.3. Only a few nitriles were examined as substrates of aU these enzymes. Moreover, the comparison of substrate specificity is difficult in some cases as different activity assays have been used for different enzymes. For instance, the determination of activity by ammonia measurement did not reflect potential amide formation. Nevertheless, common patterns can be found in substrate preferences of aromatic nitrilases. 

Recently, the potential of bacterial enzymes for the synthesis of aromatic, optically active amides, and carboxylic acids firom racemic nitriles was evaluated. An enantiomer-selective amidase, active on several 2-aryl and 2-aryloxy propionamides, was identifided and purified from Brevibacterium sp. strain R312 [145]. A nitrilase, found in Acinetobacter sp. strain AK226 and able to hydrolyze efihciently both aromatic and aliphatic nitriles, was reported to hydrolyze racemic nitriles to optically active 2-aryl propionic acids [146]. Enzyme system of Rhodococcus butanica could be successfully adapted for the kinetic resolution of a-arylpropionitriles resulting in the formation of (R)-... 

Aromatic, heterocyclic, and certain unsaturated aliphatic nitriles are often directly hydrolyzed to the corresponding acids without formation of the intermediate free amide by a so-called nitrilase enzyme. The nitrile hydratase and nitrilase enzyme use distinctively different mechanisms of action. 

Nitrilases are classified into branch 1 of the nitrilase superfamily, which is comprised of enzymes acting on various nonpeptide CN bonds [15]. All the proteins of this superfamily are characterized by a conserved catalytic triade (glu, lys, cys) and an additional conserved glu residue that seems to participate in the reaction mechanism [2]. Members of class 1 transform the CN bonds in nitriles and cyanides. The enzymes in which these activities were confirmed share in some cases levels of aa sequence identity as low as about 20%. This sequence diversity is reflected in different substrate specificities and different reaction products (carboxylic acids, amides) in various subtypes of these enzymes (aromatic nitrilases, aliphatic nitrilases, arylacetonitrilases, cyanide hydratases, cyanide dihydratases). 

Cyanopyridine (precursor of picolinic acid, intermediate of pharmaceuticals) is transformed by aromatic nitrilases at lower relative rates compared to arylace-tonitrilases. Surprisingly, the cyanide hydratase from A. niger KIO exhibited the highest specific activity for this substrate among the enzymes examined [18, 72]. This enzyme, as well as other cyanide hydratases (from the Fusarium genus), accepted a number of nitrile substrates, albeit at much lower relative rates than HCN [3, 72]. 

The majority of aromatic nitrilases hydrolyze not only (hetero) aromatic but also aliphatic saturated or unsaturated nitriles (Table 12.2). The ability of some of the enzymes to hydrolyze acrylonitrile has an industrial impact, as it enables the synthesis of acrylic acid (a polymer building block), the elimination of acrylonitrile from wastewaters [73], or the construction of acrylonitrile biosensors [74]. The nitrilase from R. rhodochrous J1 required activation by ammonium sulfate prior to use for the transformation of acrylonitrile. This activation resided in a rearrangement of the enzyme s quaternary structure, resulting in an increase in molecular weight from... 

The application of the immobilized nitrilase SP 409 of Rhodococcus sp. from Novo Industri (Denmark), which covers a wide substrate spectrum of aliphatic, aromatic, het-erocylic, and carbohydrate nitriles, proved to be synthetically viable for the mild and stereoselective transformation of base-sensitive carbohydrate nitriles [39]. Preferentially the P anomer of a diastereomeric glycosyl cyanide was hydrolyzed to the corresponding acid (Fig. 8). Using a C-7 alkoxylated glycosyl cyanide, amide intermediates were also detected, indicating the additional presence of a nitrile hydratase. 

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