Nitrile hydrolysis enzymes

Nitrilases can be used on a commercial scale as demonstrated by the production of acrylic acid, (k)-(—)-mandeHc add (Mitsubishi Rayon-Japan, BASF-Germany) as well as nicotinic add (Lonza-China), thus proving the economic potential of this 

---

Another biocatalytic option is the hydrolysis of p-aminonitriles, compounds that are relatively easily synthesized. There are two hydrolytic routes for the enzymatic conversion of nitriles to the corresponding carboxylic acids. These transformations can be achieved either through a two-step cascade reaction involving a nitrile hydratase followed by an amidase that hydrolyzes the intermediate amide, or through use of a nitrilase, an enzyme able to perform the two sequential transformations (Scheme 14.4). The focus of this chapter is on nitrile hydrolysis enzymes. 

Fischer-Colbrie, G., Matama, T., Heumann, S. et al. (2007) Surface hydrolysis of polyacrylonitrile with nitrile hydrolysing enzymes from Micrococcus luteus BST20. Journal of Biotechnology, 129, 62-68. 

In summary, the formation of optically active compounds through hydrolysis reactions is dominated by biocatalysis mainly due to the availability and ease of use of a wide variety of esterases, lipases and (to a lesser extent) acylases. Epoxide ring-opening (and related reactions) is likely to be dominated by salen-metal catalysts while enzyme-catalysed nitrile hydrolysis seems destined to remain under-exploited until nitrilases or nitrile hydratases become commercially available. 

Apart from nitrile-hydrolyzing enzymes, some esterases and cutinases have been used for surface hydrolysis of PAN [74], These enzyme were shown to specifically hydrolyse vinyl acetate moieties present as co-monomer in many commercial PAN materials, with no changes in crystallinity as determined by X-ray diffraction [74],... 

Most studies of biocatalysis in ionic liquids have been concerned with the use of isolated enzymes. It should not be overlooked, however, that the first report on biocatalysis and ionic liquids involved a whole-cell preparation Rhodococcus R312 in a biphasic [BMIm][PF(s]-water system [7]. It was shown, using a nitrile hydrolysis test reaction, that the microorganism maintained its activity better in ionic liquid than in a biphasic toluene-water system. 

Figure 5.24 Unlike the chemical route, the biocatalytic hydrolysis of acrylonitrile to acrylamide is highly selective, owing to the specific function of the nitrile hydratase enzyme.

Deigner, H., Blencowe, C., and Freyberg, C.E. 1996. Prevalence of steric restrictions in enzymatic nitrile-hydrolysis of apreparation from Rhodococcus sp. 409. Journal of Molecular Catalysis B-Enzyme, 1 61-70. 

Nitrilase, nitrile hydratase Nitrile hydrolysis Chemo- and regio-selectivity Enantioselectivity, no commercial enzyme... 

Hydrolysis and formation of C-N bonds hydrolysis of nitriles, in Enzyme Catalysis in Organic Synthesis, 2nd edn, Vol. 2 (eds K. Drauz and H. Waldmann), Wiley -VCH Verlag GmbH, Weinheim,... 

Glycolic acid can be produced via fermentation process [6] from glycolo-nitrile hydrolysis by mineral acid, such as sulfuric acid [7,8]. Both processes produce a multi-component solution with the acid concentration typically less than 10 wt% for fermentation technology, and less than 40 wt% for glycolo-nitrile hydrolysate. The acid can be produced by the enzymatic conversion (typically the enzyme catalyst used is nitrilase or a combination of a nitrile hydratase and an amidase) of glycolonitrile which results in the production of an aqueous solution of ammonium glycolate [9]. 

On the other hand, nitrilases operate by a completely different mechanism (Scheme 2.101). They possess neither coordinated metal atoms, nor cofactors, but act through an essential nucleophilic sulfhydryl residue of a cysteine [641, 642], which is encoded in the nitrilase-sequence motif Glu-Lys-Cys [643]. The mechanism of nitrilases is similar to general base-catalyzed nitrile hydrolysis Nucleophihc attack by the sulfhydryl residue on the nitrile carbon atom forms an enzyme-bound thioimidate intermediate, which is hydrated to give a tetrahedral intermediate. After the elimination of ammonia, an acyl-enzyme intermediate is formed, which (like in serine hydrolases) is hydrolyzed to yield a carboxyhc acid [644]. 

As a substitute for (expensive) commercial enzyme preparations for nitrile-hydrolysis, whole-cell preparations are recommended Rhodococcus R312 [699] ... 

The similarities between the nitrile hydrolysis and CO2 hydrolysis system relate to the heterolytic H20-assisted splitting of H2O and the importance of the adsorption-induced desorption of the reactant molecule. The primary difference between the enzyme and the zeolite relates to the specific interactions with the imidizole group in the histidine framework, which can simultaniously interact with the reaction center to aid bond cleavage and bond formation reactions. 

This work has demonstrated the utility of high throughput H NMR for the discovery of novel enzymes from genomic libraries. The use of NMR to detect biotransformation products enabled the unexpected observation of multiple product formation for the nitrile hydrolyzing enzyme. This observation resulted in the identification and proposal of a novel mechanism. Additional NMR was used to elucidate the enzymatic activity. Furthermore, it was shown that hydrolysis of the substrate was not due to a combination of nitrile hydratase and amidase. 

Biocatalysis offers two advantages over chemical methodologies for nitrile hydrolysis. First, enzymes operate under mild conditions in the absence of... 

Hydrolysis of nitriles Hydrolysis of [ CJnitrile functions represents a well-established route for the synthesis of [ C]carboxylic acids. For cases in which the harsh reaction conditions of chemical hydrolysis (e.g., 2 N NaOH, reflux) are incompatible with sensitive functionalities, application of enzymes might provide an alternative (Figure 12.17). Enzymatic hydrolysis of nitriles results in amides if catalyzed by nitrile hydratases or in the corresponding carboxylic acids if catalyzed by nitrilases". ... 

Amidases catalyze the second step in die two-enzyme step conversion of nitriles to the corresponding acids, the hydrolysis of amides. Among stereoselective nitrile-converting enzymes, the highest enantioselectivities were found in amidases. Amidases have been mostly characterized as thiol-dependent homodimers (aa) sharing amino acid sequence homologies with other amidases. The functional relationship between nitrile hydratases and amidases is structurally documented by the close proximity of their genes [71,72]. In this chapter, stereospecific amidases alone or with nonselective nitrile hydratases are described. 

---