Nitrilases enantioselective

Hie bioconversion of a-aminonitriles, although up until now not used on an industrial scale, is of practical interest in the production of optical active a-amino adds. This, however, will only be the case if one can select a nitrilase that enantioselectively hydrolyses die aminonitrile. 

Nitrilases catalyze the synthetically important hydrolysis of nitriles with formation of the corresponding carboxylic acids [4]. Scientists at Diversa expanded the collection of nitrilases by metagenome panning [56]. Nevertheless, in numerous cases the usual limitations of enzyme catalysis become visible, including poor or only moderate enantioselectivity, limited activity (substrate acceptance), and/or product inhibition. Diversa also reported the first example of the directed evolution of an enantioselective nitrilase [20]. An additional limitation had to be overcome, which is sometimes ignored, when enzymes are used as catalysts in synthetic organic chemistry product inhibition and/or decreased enantioselectivity at high substrate concentrations [20]. 

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]. 

Burk and coworkers have used a variety of nitrilases for the DKR of cyanohydrins [48]. Nitrilases catalyze the hydrolytic conversion of cyanohydrins directly to the corresponding carboxylic acids. Racemization was performed under basic conditions (phosphate buffer, pH 8) through reversible loss of HCN. (R)-Mandelic acid was obtained in high yield (86% yield) and high enantioselectivity (98% ee) after 3 hours (Figure 4.23). 

The addition of HCN to aldehydes or ketones produces cyanohydrins (a-hydroxy nitriles). Cyanohydrins racemize under basic conditions through reversible loss of FiCN as illustrated in Figure 6.30. Enantiopure a-hydroxy acids can be obtained via the DKR of racemic cyanohydrins in the presence of an enantioselective nitriletransforming enzyme [86-88]. Many nitrile hydratases are metalloenzymes sensitive to cyanide and a nitrilase is usually used in this biotransformation. The DKR of mandelonitrile has been extended to an industrial process for the manufacture of (R)-mandelic acid [89]. 

Layh N, A Stolz, S Eorster, E Effenberger, H-J Knackmuss (1992) Enantioselective hydrolysis of O-acetyl-mandelonitrile to O-acetylmandelic acid by bacterial nitrilases. Arch Microbiol 158 405-411. 

Robertson DE et al. (2004) Exploring nitrilase sequence space for enantioselective catalysis. Appl Environ Microbiol 70 2429-2436. 

DeSantis, G., Wong, K., Farwell, B. et al. (2003) Creation of a productive, highly enantioselective nitrilase through gene site saturation mutagenesis (GSSM). Journal of the American Chemical Society, 125, 11476-11477. 

Table 8.5 Enantioselective hydrolysis of /3-hydroxynitriles catalyzed by nitrilase bll6402 ...

A novel nitrilase was purified from Aspergillus niger K10 cultivated on 2-cyanopyridine. It was found to be homologous to a putative nitrilase from Aspergillus fumigatus Af293. The nitrilase exhibited maximum activity at 45 °C and pH 8.0 with much less activity observed at slightly acid pH. Its substrate preference was for 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thio-phen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. ( )-2-Phenylpropionitrile was only poorly converted by this enzyme and with minimal enantioselectivity. The enzyme was shown to be multimeric (>650 kDa) and be stabilized in the presence of sorbitol and xylitol [57]. 

An enantioselective nitrilase from Pseudomonas putida isolated from soil cultured with 2 mM phenylacetonitrile was purified and characterized. This enzyme is comprised of 9-10 identical subunits each of 43 kDa. It exhibits a pH optimum at 7.0 and a temperature optimum at 40 °C (Ty2 = 160 min) and requires a reducing environment for activity. This nitrilase was shown to have an unusually high tolerance for acetone as co-solvent, with >50% activity retained in the presence of 30% acetone. The kinetic profile of this nitrilase reveals KM= 13.4mM, cat/ M = 0-9s 1mM 1 for mandelonitrile, ZfM = 3.6mM, kclJKM 5.2 s him-1 for phenylacetonitrile, and KM = 5.3 mM, kC lt/KM = 2.5 s 1 him 1 for indole 3-acetonitrile. Preliminary analysis of this enzyme with 5 mM mandelonitrile revealed formation of (/t)-mandelic acid with 99.9% ee [59]. 

The nitrilase from cyanobacterium Synechocystis sp. PCC6803 was found to effect the stereoselective hydrolysis of phenyl-substituted /3-hydroxy nitriles to (S)-enriched /3-hydroxy carboxylic acids. The enzyme also effected the conversion of y-hydroxynitrile, albeit with lesser enantioselectivity (Table 8.10). Interestingly, this enzyme was also was found to hydrolyze aliphatic dinitriles, such that for 1,2-dicyanoethane and 1,3-dicyanopropane the... 

Bergeron S, Chaplin D, Edwards JH, Ellis BS, Hill CL, Holt-Tiffin K, Knight JR, Mahoney T, Osborne AP, Ruecroft G (2006) Nitrilase-catalyzed desym-metrization of 3-hydroxyglutaronitrile preparation of a statin side-chain intermediate. Org Proc Res Dev 10 661-665 Burns M, Weaver J, Wong J (2005) Stereoselective enzymic bioconversion of aliphatic dinitriles into cyano carboxylic acids. WO 2005100580 DeSantis G, Zhu Z, Greenberg W, Wong K, Chaplin J, Hanson SR, Farwell B, Nicholson LW, Rand CL, Weiner DP, Robertson D, Burk MJ (2002) An enzyme library approach to biocatalysis development of nitrilases for enantioselective production of carboxylic acid derivatives. J Am Chem Soc 124 9024-9025... 

One of the most attractive biocatalytic options is the nitrilase-catalysed enantioselective hydrolysis of the racemic cyanohydrin. The hydroxyacid is produced directly without need for protection/deprotection steps and cyanohydrins racemize spontaneously at neutral or... 

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). 

In the screening of genomic libraries prepared from environmental samples collected in various parts of the world, more than 200 new nitrilases were discovered that allow mild and selective hydrolysis of nitriles (150). One of them catalyzes the (J )-selective hydrolysis of 35 with an ee value of 94.5% at a substrate concentration of 100 mM (46). However, when experiments were done at a more practical concentration of 2.25 M, activity and enantioselectivity suffered (ee only 87.8%). 

Therefore, directed evolution was applied to solve these problems. To screen for enantioselectivity, the Miilheim MS-based high-throughput ee assay (92,93) (Section III.C) was applied (46). In this case, the necessary isotope labeling focused on the use of in the pseudo-meso compound N-(J )-17 (see Section III.C for a detailed discussion). An (5)-selective nitrilase leads preferentially to N-(5)-18, whereas an 7 -selective variant results in the picw o-enantiomer (J )-18. They differ by one mass unit and can therefore be distinguished by MS, both qualitatively and quantitatively (by integration of the relevant peaks). 

There are two distinct classes of enzymes that hydrolyze nitriles. Nitrilases (EC 3.5.5.7) hydrolyze nitriles direcdy to corresponding acids and ammonia without forming the amide. In fact, amides are not substrates for these enzymes. Nitriles also may be first hydrated by nitrile hydratases to yield amides which are then converted to carboxylic acid with amidases. This is a two-enzyme process, in which enantioselectivity is generally exhibited by the amidase, rather than the hydratase. 

In basic chemicals, nitrile hydratase and nitrilases have been most successful. Acrylamide from acrylonitrile is now a 30 000 tpy process. In a product tree starting from the addition of HCN to butadiene, nicotinamide (from 3-cyanopyridine, for animal feed), 5-cyanovaleramide (from adiponitrile, for herbicide precursor), and 4-cyanopentanoic acid (from 2-methylglutaronitrile, for l,5-dimethyl-2-piperidone solvent) have been developed. Both the enantioselective addition of HCN to aldehydes with oxynitrilase and the dihydroxylation of substituted benzenes with toluene (or naphthalene) dioxygenase, which are far superior to chemical routes, open up pathways to amino and hydroxy acids, amino alcohols, and diamines in the first case and alkaloids, prostaglandins, and carbohydrate derivatives in the second case. 

J. 2002. An enzyme library approach to biocatalysis development of nitrilases for enantioselective production of carboxylic acid derivatives./. Am. Chem. Soc.,124(31), 9024-9025. 

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