Rhodococcus nitrilase

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]. 

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). 

The preparation of malonic acid monoesters has been demonstrated using the microbial nitrilase activity of Corynebacterium nitrilophilus ATCC 21 419, Gordona terrae MA-1, or Rhodococcus rhodochrous ATCC 33 025 to hydrolyze methyl cyanoacetate, ethyl cyanoace-tate, M-propyl cyanoacetate, isopropyl cyanoacetate, M-butyl cyanoacetate, tertbutyl cyanoacetate, 2-ethylhexyl cyanoacetate, allyl cyanoacetate, and benzyl cyanoacetate [96]. By maintaining the concentration of nitrile in a reaction mixture at <5 wt%, significant inactivation of the nitrilase activity was avoided for example, a total of 25 g of M-propyl cyanoacetate was added in sequential 5g portions to a lOOmL suspension of Rhodococcus rhodochrous ATCC 33 025 cells (OD630 = 5.6) in 50 mM phosphate buffer (pH 7.0) over 30h at 25 °C to produce mono-M-propyl malonate in 100% yield (Figure 8.17). 

Figure 8.17 Production of mono-n-propyl malonate from M-propyl cyanoacetate using the microbial nitrilase activity of Rhodococcus rhodochrous ATCC 33025...

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... 

Mathew, C.D., Nagasawa, T., Kobayashi, M. and Yamada, H., Nitrilase-catalyzed production of nicotinic acid from 3-cyanopyridine in Rhodococcus rhodochrous Zl.Appl. Environ. Microbiol. 1988, 54, 1030. 

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). 

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). 

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. 

Hoyle, A.J., Bunch, A.W., and Knowles, C.J. 1998. The nitrilases of Rhodococcus rhodochrous NCIMB 11216. Enzyme Microbiology and Technology, 23 475-82. 

Kobayashi, M., Komeda, H., and Yanaka, N. 1992a. Nitrilase from Rhodococcus rhodochrous Jl. Sequencing and overexpression of the gene and identification of an essential cysteine residue. Journal of Biological Chemistry, 267 20746-51. 

Kobayashi, M., Yanaka, N., Nagasawa, T., et al. 1989. Purification and characterization of a novel nitrilase of Rhodococcus rhodococcus K22 that acts on aliphatic nitriles. Journal... 

Nagasawa, T., Wieser, M., Nakamura, T., et al. 2000. Nitrilase of Rhodococcus rhodochrous 11, conversion into the active form by subunit association. European Journal of Biochemistry, 267 138 44. 

Stevenson, D.E., Peng, R., Dumas, E, et al. 1992. Mechanistic and structural studies on Rhodococcus ATCC 39484 nitrilase. Biotechnology and Applied Biochemistry, 15 283-302. 

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]. 

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... 

SP 361 SP 409 Immobilized enzyme mixture from Rhodococcus sp. containing nitrilase, nitril hydratase,esterase, epoxide hydrolase and amidase activity discontinued... 

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