Nitrilases applications

Rey, R, Rossi, J.-C., Taillades, J., Gros, G., and Nore, O. (2004). Hydrolysis of nitriles using an immobilized nitrilase Applications to the synthesis of methionine hydroxy analogue derivatives. Journal of Agricultural and Food Chemistry, 52,8155-8162. 

Nitrilases are quite rare in bacterial genomes and less than 20 were reported prior to the application of metagenomics for their detection in environmental DNA [81]. Two studies targeting environmental genomes report the detection of more than 337 novel nitrilases. This has dramatically increased the amount of information about nitrilases, and the newly discovered diversity can be applied for the enantioselective production of hydroxy carboxylic add derivatives [81]. 

Chapters 5-8 are directed to emerging enzymes, which include oxynitrilases, aldolases, ketoreductases, oxidases, nitrile hydratases, and nitrilases, and their recent applications especially in synthesis of chiral drugs and intermediates. 

Nitrilases convert nitriles to the corresponding carboxylic acids and NH3 through a cysteine residue in the active site [50]. Because of their high enantio- and regio-selectivity, nitrilases are attractive as green catalysts for the synthesis of a variety of carboxylic acids and derivatives (Figure 1.10) [51,52]. Recently, a number of recombinant nitrilases have been cloned and characterized heterologously for synthetic applications [50,53,54]. 

Existing synthetic methods and commercial processes that employ nitrile hydratases (NHases) and nitrilases continue to be improved by directed evolution of existing enzymes, or by the discovery of new enzymes with improved properties, and new applications of these catalysts have recently been described. Numerous reviews have previously been published that describe applications of NHase [ 1-6] and nitrilase [ 1,4—11 ], and in this review we present examples of new applications of these nitrile-utilizing catalysts from journal articles, patent applications, and issued patents that have been published in the past 2-3 years. 

Three cyanide-degrading nitrilases were recently cloned and purified and their kinetic profiles were evaluated in order to better understand their applicability to cyanide bioremediation. CynD from Bacilluspumilus Cl and DyngD from Pseudomonas stutzeri exhibit fairly broad pH profiles with >50% activity retained across pH 5.2 to pH 8.0 while the CHT (NHase) from Gloeocercospora sorghi exhibited a more alkaline pH activity profile with almost all of its activity retained at pH 8.5, slightly lower thermal tolerance, and quite different metal tolerance compared with the two bacterial enzymes [46]. 

Mukherjee, C., Zhu, D., Biehl, E.R. andHua, L. (2006) Exploring the synthetic applicability of a cyanobacterium nitrilase as catalyst for nitrile hydrolysis. European Journal of Organic Chemistry, 5238-5242. 

The ease of the Strecker synthesis from aldehydes makes a-aminonitriles an attractive and important route to a-amino acids. Fortunately, the microbial world offers a number of enzymes for carrying out the necessary conversions, some of them highly stereoselective. Nitrilases catalyze a direct conversion of nitrile into carboxylic acid (Equation (11)), whereas nitrile hydratases catalyze formation of the amide, which can then be hydrolyzed to the carboxylic acid in a second step (Equation (12)). In a recent survey, with a view to bioremediation and synthesis, Brady et al have surveyed the ability of a wide range of bacteria and yeasts to grow on diverse nitriles and amides as sole nitrogen source. This provides a rich source of information on enzymes for future application. 

Recently, another interesting application of nitrilases has been demonstrated for the synthesis of pregabalin-the API of the neurophatic pain drug Lyrica. In this approach, the key step is the resolution of racemic isobutylsuccinonitrile (Scheme 10.8) [18], the process takes place with total regio- and stereoselectivity, and the (S)-acid is obtained and the (R)-substrate can be recycled under basic conditions. To improve the biocatalytic step, directed evolution was applied using the electronic polymerase chain reaction and in the first round of evolution a single C236S mutation led to a mutant with 3-fold increase in activity [19]. 

This screening system has also been applied successfully in the directed evolution of enantioselective EHs acting as catalysts in the kinetic resolution of chiral epoxides 95,96) (Section IV.A.4). Moreover, the firm Diversa has applied the MS-based method in the desymmetrization of a prochiral dinitrile (l,3-dicyano-2-hydroxypropane) catalyzed by mutant nitrilases 46). In this industrial application, one of the nitrile moieties was labeled selectively with as in N-17, which means that the two pseiido-eaaniiovaenc products (S)- N-18 and (J )-18 differ by one mass unit. This is sufficient for the MS system to distinguish between the two products quantitatively 46). 

Chemical reactions enhanced by catalysts or enzymes are an integral part of the manufacturing processes for the majority of chemical products. The total market for catalysts and enzymes amounts to 11.5 billion (2005), of which catalysts account for about 80%. It consists of four main applications environment (e.g., automotive catalysts), 31% polymers (e.g., polyethylene and polypropylene), 24% petroleum processing (e.g., cracking and reforming), 23% and chemicals, 22%. Within the latter, particularly the catalysts and enzymes for chiral synthesis are noteworthy. Within catalysts, BINAPs [i.e., derivatives of 2,2 -bis(diphenylphosphino) -1, l -bis-l,l -binaphthyl) have made a great foray into chiral synthesis. Within enzymes, apart from bread-and-butter products, like lipases, nitrilases, acylases, lactamases, and esterases, there are products tailored for specific processes. These specialty enzymes improve the volumetric productivity 100-fold and more. Fine-chemical companies, which have an important captive use of enzymes, are offering them to third parties. Two examples are described here ... 

Environmental application for bioremediation purposes must also get the benefit of the technology. Since haloalkane dehalogenase activity was tested successfully, as shown in Example 2 above for the dehalogenation of toxic VOCs, many others enzymatic activities should be of great interest, such as oxygenases, nitrilases, and organophosphorus hydrolases, for example. 

DuPont has developed another commercial process, based on catalysis by a nitrilase (E.C. 3.5.5.1), to the solvent l,5-dimethyl-2-piperidone (1,5-DMPD) (Xolvone ) with applications in electronics and coatings (Thomas, 2002). The raw material is 2-methylglutaronitrile (MGN), a by-product during the manufacture of adipodinitrile (ADN) for nylon 6,6 discussed in the previous section. Such a raw material situation leads to coupling of nylon-6,6, 5-cyanovaleramide, and l,5-dimethyl-2-piperidone production, a situation that most likely is specific to DuPont and thus not prone to much competition. 

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]. 

Nitrilase from Rhodococcus R312 was found to be (/ )-enantiospecific toward 2-cyano 1,4-benzodioxane, in contrast to other screened nitrilases that showed (5)-preferences however, the anthors do not report on the absolute configuration of the enantiopure synthesized l,4-benzodioxane-6-formyl-2-carboxylic acid. An organic cosolvent was added to the reaction mixtnre to increase solubihty of substrates (Table 17.7). A possible hypothesized application of enantiopure l,4-benzodioxane-2-carboxylic acid is the synthesis of doxazosine methylate, member of the quinazoline family of drugs, and indicated for the treatment of hypertension. 

Winkler, M., Kaplan, O., Vejvoda, V. et al. 2009. Biocatalytic application of nitrilases from Fusarium solani 01 and Aspergillus niger KIO Journal of Molecular Catalysis B-Enzyme, 59 243-7. 

In order to assess the potential applications of these new nitrilases in biocatalytic processes, data on their operational stabiUty were required. To this end, we investigated the kinetic behavior of enzymes from F. solani and A. niger either immo-biUzed on solid supports or retained in stirred ultrafiltration membrane reactors in continuous experiments. 

Fungal nitrilases immobilized on these columns were applicable to continuous biotransformation of heteroaromatic nitriles like 3- and 4-cyanopyridine, the products of which, nicotinic and isonicotinic acid, respectively, are of commercial interest. The enzyme from F. solani exhibited a higher stability than that from A. niger at 35 °C. The conversion of 3-cyanopyridine by the former enzyme was nearly quantitative within 24h [50], while it decreased by 30% within 15h in the case of the latter [49]. Similar differences in operational stabiUties were observed during conversion of 4-cyanopyridine. The stabiUty of the enzymes depended on the substrate used, both nitrilases being more stable during the conversion of 4-cyanopyridine in comparison with 3-cyanopyridine. 

A method of efficient induction of fungal nitrilases in wild strains was established and seems to be of wide application throughout this enzyme group. However, heterologous expression of fungal nitrilases remains to be solved in order to facilitate the use of these enzymes in industrial biotechnology. To what extent the desirable properties (broad substrate specificity, thermostability, chemoselectivity and enantioselectivity) can be improved by mutagenesis of these enzymes should also be examined. 

An enantioselective nitrilase has also been shown to be applicable in the dynamic kinetic resolution of mandelonitrile. Using the nitrilase produced by Alcaligenes faecalis ATCC 8750 Yamamoto et al. showed that they could derive (Rj-(-)-mandelic acid from mandelonitrile in 91% yield with an ee of 100%. Under the reaction conditions used non-reacting (S) -mandelonitrile undergoes spontaneous racemiza-tion leading to the high yield (see Scheme 12.1-8)[48]. Currently (R)-mandelic acid and (R)-chloromandelic acid are produced using nitrilases on an industrial scale by the Mitsubishi Rayon Corp. 

---