Rhodococcus nitrile hydrolysis

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. 

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. 

Langdahl, B.R., Bisp, P., and Ingvorsen, K. 1996. Nitrile hydrolysis hy Rhodococcus erythro-polis BLl, an acetonitrile-tolerant strain isolated from amarine sediment. Microbiology, 142 145-54. 

For instance, 1,3- and 1,4-dicyanobenzenes were selectively hydrolyzed by Rhodococcus rhodochrous to give the corresponding monoacids [672, 673]. In the aliphatic series, tranexamic acid (iratw-4-aminomethyl-cyclohexane-l-carboxylic acid), which is a hemostatic agent, is synthesized fi om trans-, 4-dicyanocyclohexane. Complete regioselective hydrolysis was achieved by using an Acremonium sp. [674]. The outcome of regioselective nitrile hydrolysis is believed to depend on the distance of the nitrile moieties and the presence of other polar groups within the substrate [675, 676]. 

For organisms which express both pathways for nitrile hydrolysis, the stereochemical pathways can be very complex. The latter is illustrated by the microbial resolution of cx-aryl-substituted propionitriles using a Rhodococcus butanica strain (Scheme 2.109) [697]. Formation of the natural L-acid and the o-amide indicates the presence of an L-specific amidase and a nonspecific nitrile hydratase. However, the occurrence of the (5)-nitrile in case of Ibuprofen (R = i-Bu, e.e. 73%) proves the enantioselectivity of the nitrile hydratase [694]. In a related approach, Brevibacter-ium imperiale was used for the resolution of structurally related a-aryloxypropionic nitriles [698]. 

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

The biocatalytic differentiation of enantiotopic nitrile groups in prochiral or meso substrates has been studied by several research groups. For instance, the nitrilase-catalyzed desymmetrization of 3-hydroxyglutaronitrile [92,93] followed by an esterification provided ethyl-(Jl)-4-cyano-3-hydroxybutyrate, a useful intermediate in the synthesis of cholesterol-lowering dmg statins (Figure 6.32) [94,95]. The hydrolysis of prochiral a,a-disubstituted malononitriles by a Rhodococcus strain expressing nitrile hydratase/amidase activity resulted in the formation of (R)-a,a-disubstituted malo-namic acids (Figure 6.33) [96]. 

A variety of commercial kits and automated systems are available to test the abilities of bacteria to assimilate, ferment, decarboxylate, or cleave selected organic compounds.46 Their reliability for species identification is usually greater with cultures from clinical samples, where a limited number of bacteria are commonly encountered, and less with environmental soil and water samples, where a great many uncommon or previously unidentified species not in the database are likely to be present.29,45 Additional tests beyond those found in the commercial kits may be necessary for example, the hydrolysis of various nitriles and amides is useful for identifying Rhodococcus spp.47 Some commercial kits for clinical use feature antimicrobial susceptibility testing.21... 

Reisinger, C., Osprian, I., Glieder, A. et al. (2004) Enzymatic hydrolysis of cyanohydrins with recombinant nitrile hydratase and amidase from Rhodococcus erythropolis. Biotechnology Letters, 26, 1675-1680. 

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). 

One of the best examples for discussing biotransformations in neat solvents is the enzymatic hydrolysis of acrylonitrile, a solvent, to acrylamide, covered in Chapter 7, Section 7.1.1.1. For several applications of acrylamide, such as polymerization to polyacrylamide, very pure monomer is required, essentially free from anions and metals, which is difficult to obtain through conventional routes. In Hideaki Yamada s group (Kyoto University, Kyoto, Japan), an enzymatic process based on a nitrile hydratase was developed which is currently run on a commercial scale at around 30 000-40 000 tpy with resting cells of third-generation biocatalyst from Rhodococcus rhodochrous J1 (Chapter 7, Figure 7.1). 

Matoishi, K. Sano, A. Imai, N. Yamazaki, T. Yokoyama, M. Sugai, T. Ohta, H. Rhodococcus rhodochrous IFO 15564-mediated hydrolysis of alicyclic nitriles and amides stereoselectivity and use for kinetic resolution and asymmetri-zation. Tetrahedron Asymmetry 1998, 9, 1097-1102. 

Rhodococcus rhodochrous was used for the hydrolysis of both granular PAN and acrylic fibers by nitrile hydratase and amidase (Tauber et al., 2000). Similarly, Agrobacterium tumefaciens (BST05) was found to convert polyacylonitrile into polyacrylic acid by nitrile hydratase and amidase (Fischer-Colbrie et al., 2006). Nitrilase was also used for the surface hydrolysis of PAN from Micrococcus luteus BST20 (Fischer-Colbrie et al., 2007). However, polyamidase from Nocardia farci-nica leads to an increase of polar groups on the surface of PA, which was measured by tensiometry (Heumann et al., 2009). 

Effenberger, F. and Graef, B.W. 1998. Chemo- and enantioselective hydrolysis of nitriles and acid amides, respectively, with resting cells of Rhodococcus sp. C3II and Rhodococcus erythropolis MP50. Journal of Biotechnology, 60 165-74. 

Kinfe, H.H., Chhiba, V., Erederick, J., et al. 2009. Enantioselective hydrolysis of 3-hydroxy nitriles using the whole cells biocatalyst Rhodococcus rhodochrous ATCC BAA-870. Journal of Molecular Catalysis B Enzyme, 59 231-6. 

Klempier, N., Harter, G., de Raadt, A. et al. 1996. Chemoselective hydrolysis of nitriles by Rhodococcus rhodochrous NCIMB 11216. Food Technology and Biotechnology, 34 67-70. 

The degradation of nitriles by nitrilases (EC 3.5.5.1) has been the subject of intense study, especially as it relates to the preparation of the commodity chemical acrylamide. Nitrilases catalyze the hydrolysis of nitriles to the corresponding acid plus ammonia (Figure 1 reaction 5), whereas nitrile hydratases (EC 4.2.1.84) add water to form the amide. Strains such as Rhodococcus rhodo-chrous Jl, Brevibacterium sp., and Pseudomonas chlororaphis have been used to prepare acrylamide from acrylonitrile, which contain the hydratase and not nitrilase activity [12]. A comparison of these strains has been discussed elsewhere [98]. Other uses of nitrilases, however, have primarily been directed at resolution processes to stereoselectively hydrolyze one enantiomer over another or regiose-lectively hydrolyze dinitriles [99-101]. 

Amides can be made by the enzymatic hydrolysis of nitriles. Nitto Chemical Industry of Japan uses Rhodococcus rhodocrous to prepare acrylamide from acrylonitrile... 

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