Nitrilases transformations

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). 

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],... 

The regioselectivity of a Rhodococcus rhodochrous nitrilase has been demonstrated for the conversion of 5-fluoro-l,3-dicyanobenzene to 5-fluoro-3-cyano-benzoic acid [62]. The nitrilase was expressed in an Escherichia coli transformant, and a cell-free extract was employed as catalyst (0.14wt% cell-free extract) in 0.1m sodium phosphate buffer (pH 7.2) at 25 °C containing 0.18 m 5-fluoro-l,3-dicyanobenzene. After 72 h, the conversion was >98% and the reaction was stopped by addition of phosphoric acid (pH 2.4) to yield 5-fluoro-3-cyano-benzoic acid as a crystalline product (97% isolated yield). 

A prochiral bis(cyanomethyl) sulfoxide was converted into the corresponding mono-acid with enantiomeric excesses as high as 99% using a nitrilase-NHase biocatalyst. The whole-cell biocatalyst Rhodococcus erythropolis NCIMB 11540 and a series of commercially available nitrilases NIT-101 to NIT-107 were evaluated in this study. As outlined in Figure 8.18, the prochiral sulfoxide may be transformed into five different products (plus enantiomeric isoforms), of which, three are chiral (A, B, and C) and two achiral (D and E). Only products A, B, and E were observed with the biocatalysts employed in this investigation. Both enantiomerically enriched forms of both A and C could be obtained with one of the catalysts used. The best selectivities are as follows (S)-A 99% ee, (R)-A 33% ee, (S)-C 66% ee, and (R)-C 99% ee, using NIT-104, NIT-103, NIT-108, and NIT-107 respectively. Each of these catalysts produced more... 

Holtze, M.S., Sorensen, J., Christian, H. and Aamand, J. (2006) Transformation of the herbicide 2,6-dichlorobenzonitrile to the persistent metabolite 2,6-dichlorobenzamide (BAM) by soil bacteria known to harbor nitrile hydratase or nitrilase. Biodegradation, 17, 503—510. 

Transgenic plants containing a nitrilase specific for the herbicide bromoxynil (= 3,5-dibromo-4-hydroxybenzonitrile)have also been developed [93] the Cal-gene company transformed tobacco plants with the bacterial Klebsiella ozaenae gene encoding nitrilase [94] that detoxifies the herbicide by hydrolysis (conversion of bromoxynil to 3,5-dibromo-4-hydroxybenzoic acid), resulting in the establishment of the herbicide-resistant transgenic plants. 

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. 

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. 

FIG U RE 17.6 Nitrilase biocatalyzed transformation of racemic (i /5)-mandelonitrile for the production of (i )-mandelic acid. 

Nitrilase Catalyzed Stereoselective Transformation of cis- and trans-N-Protected Conformationally Constrained y-Amino Butyronitriles... 

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. 

During our work on enzymatic nitrile transformation, carbocyclic y-amino nitriles have emerged from a comprehensive screening among structurally diverse amino nitriles as being weU-suited substrates for nitrilase-mediated hydrolysis. In contrast, as stated in the previous section, the analogous carbocychc 5-amino nitriles are strictly non-substrates for nitrilases. 

Essentially, nitrilases NIT-106 and NIT-107 were the most efficient catalysts, whereas NIT-lOl and NIT-105 turned out to be less suitable for the synthesis of Y-amino adds. cts-3-Aminocyclopentanecarboxylic acid (-h)-13c was prepared by Nrr-104, whereas (-)-13c was produced by NIT-106 in an enantiocomplementary manner in a high e.e. (97%) dose to the theoretical yield of a kinetic resolution. The respective trans-isomer (-)-14c was obtained in only 55% e.e. by the same enzyme. All other nitrilases examined could not enhance this result NrT-106 revealed similar outstanding sdectivities in the transformation of six-membered aminonitrile cts-( )-15a to (-)-15c in almost optical purity (>99% e.e.) and in a 29% isolated yield [43]. 

The bienzymatic transformation of aldehydes and HCN in the presence of an oxynitrilase and a nitrilase is a useful addition to the synthetic repertoire, provided that the coproduction of amide can be avoided. 

Nitrile biodegradation is performed by a variety of microorganisms and proceeds through two different enzymatic pathways direct transformation to carboxylic acids and ammonia, with some exceptions, catalyzed by a nitrilase (EC 3.5.5.1) [1-3] or a two-step reaction, the first catalyzed by nitrile hydratase (EC 4.2.1.84) that produces an amide intermediate, which is further hydrolyzed to the acid and ammonia by an amidase (EC 3.5.1.4) [4, 5],... 

Cyanohydrins are a very useful class of compounds as they can be transformed into a wide variety of compounds while retaining the stereogenic center (32, 35). Hydroxy nitrilases are available from natural sources (13), which can give access to either enantiomer of the product cyanohydrin (Fig. 1) (47). 

However, production of 2,6-difluorobenzamide (Scheme 12.1-18) was effected in 99.5% n-heptane using the nitrile hydratase from Rhodococcus sp. NCIMB 12 21 81841. The enzymatic reaction was found to be activated by light (see 12.1.3.4). More recently, Layh and Willetts have studied nitrile transformations in various organic solvents and biphasic mixtures using a nitrilase from Pseudomonas sp. DSM 11387 and a nitrile hydratase from Rhodococcus sp. DSM 113971 51. The enzymes exhibited good stabilities in biphasic mixtures with hydrophobic solvents when dispersed in... 

Fusarium solani [114], Arthrobacter sp. [115], Escherichia coli, transformed with a KLehsiella ozaenae plasmid DNA [116], Rhodococcus rhodochrous J1 [117,118], and Alcaligenes faecalis JM3 [119, 120]. The enzyme of Rhodococcus rhodochrous J1 was employed for the production of p-aminobenzoic acid from p-aminobenzonitrile [121], and nicotinic acid from 3-cyanopyridine [122]. The conversion of 3 anopyridine to nicotinic add, by a nitrilase of Nocardia rhodochrous IjL100-2, was also reported by Vaughan et al. [123]. These nitrilases were usually inactive on aliphatic nitriles. More recently, a new nitrilase, that acts preferentially on aliphatic nitriles, was purified and characterized in Rhodococcus rhodochrous K22 [124]. 

The metabolism of Bromoxjmil has been studied in some microorganisms, revealing the presence of amide and acid products [3,152-154] (Figure 8). Klebsiella pneumoniae subsp. ozaenae was shown to transform completely the herbicide, with the involvement of a nitrilase enzyme to... 

A variety of nitrilases and even whole cells were used in this transformation, with the aim of obtaining different proportions of starting material and compounds 164 and 166 but not 165 or iy(hydrolised) products. Products 164 and 166 were obtained in up to 99 and 70% ee respectively, both with S absolute configuration. 

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