Nitriles hydratase

Low-spin ferric iron is conunon in heme and in some iron-sulfiir proteins, but nitrile hydratase is presently the lone example among mononuclear, non-heme, non-iron-sulfur iron proteins [31]. The ferric form of the enzyme is obtained by photolysis of the as-isolated nitric oxide d form. The g-values of the photodissoci-ated ferric protein are 2.27, 2.13, and 1.97, and photointermediates of differing g-values are observed. The relatively narrow linewidths allowed the Fe hyperfine to be measured at (40 MHz), establishing that the g- and iron A-tensors are 45° out of alignment. 

RADICAL (5 = 1/2) PROBES OF FERROUS (5 = 2) IRON 3.1. Photosynthetic Reaction Centers 

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Hydrolysis of Nitriles. The chemical hydrolysis of nitriles to acids takes place only under strong acidic or basic conditions and may be accompanied by formation of unwanted and sometimes toxic by-products. Enzymatic hydrolysis of nitriles by nitrile hydratases, nittilases, and amidases is often advantageous since amides or acids can be produced under very mild conditions and in a stereo- or regioselective manner (114,115). 

The hydrolysis of nitriles can be carried out with either isolated enzymes or immobilized cells. Eor example, resting cells of P. chlororaphis can accumulate up to 400 g/L of acrylamide in 8 h, provided acrylonitrile is added gradually to avoid nitrile hydratase inhibition (116). The degree of acrylonitrile conversion to acrylamide is 99% without any formation of acryUc acid. Because of its high efficiency the process has been commercialized and currentiy is used by Nitto Chemical Industry Co. on a multithousand ton scale. 

As illustrated in Figure A8.3 nitrilases catalyse conversions of nitriles directly into the corresponding carboxylic adds (route A), while other nitrile converting enzymes, die nitrile hydratases, catalyse the conversion of nitriles into amides (route B) which, by the action of amidases usually present in the whole cell preparations, are readily transformed into carboxylic adds (route C). 

L-Amino adds could be produced from D,L-aminonitriles with 50% conversion using Pseudomonas putida and Brembacterium sp respectively, the remainder being the corresponding D-amino add amide. However, this does not prove the presence of a stereoselective nitrilase. It is more likely that the nitrile hydratase converts the D,L-nitrile into the D,L-amino add amide, where upon a L-spedfic amidase converts the amide further into 50% L-amino add and 50% D-amino add amide. In this respect the method has no real advantage over the process of using a stereospecific L-aminopeptidase (vide supra). 

Enantioselective transformations of several cyclopropane or oxirane-containing nitriles were studied using nitrile-transforming enzymes [78]. Microbial Rhodococcus sp. whole cells containing a nitrile hydratase/amidase system hydrolyzed a number... 

Both cis- and trans-chrysanthemic nitriles and amides were resolved into highly enantiopure amides and acids by Rhodococcus sp. whole cells [85]. The overall enantioselectivity of reactions of nitriles originated from the combined effects of a higher (lJ )-selective amidase and a (IJ )-selective nitrile hydratase (Figure 6.29). Chrysanthemic acids are related to constituents of pyrethrum flowers and insecticides. 

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]. 

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 discovery and exploitation of enzymes in aldoxime-nitrile pathway nitrile hydratase, amidase, nitrilase, aldoxime dehydratase, etc., are shown along with the use of methodologies, such as organic chemistry, microbial screening by enrichment and acclimation culture techniques, enzyme purification, gene cloning, molecular screening by polymerase chain reaction (PCR). 

We also found that the occurrence of aldoxime dehydratase is as wide as that for nitrile-degrading enzymes such as nitrile hydratase, amidase and/or nitri-lase. All of the nitrile degraders hitherto isolated contained aldoxime dehydratase activities. The author would like to propose that the pathway in which aldoximes are successively degraded via nitrile could be named as the aldoxime-nitrile pathway (Fig. 1). 

The dehydration reaction of aldoxime to form nitriles using the resting cells of Rhodococcus sp. YH3-3 was optimized. We found that the enzyme was induced by aldoxime and catalyzed the stoichiometric synthesis of nitriles from aldoximes at pH 7.0 and 30°C. Phenylacetonitrile once synthesized from phenylacetaldoxime was hydrolyzed to phenylacetic acid, since the strain has nitrile degradation enzymes such as nitrile hydratase and amidase. We have been successful in synthesizing phenylacetonitrile and other nitriles stoichiometrically by a selective inactivation of nitrile hydratase by heating the cells at 40°C for 1 h. Various nitriles were synthesized under optimized conditions from aldoximes in good yields. 

Tauber MM, A Cavaco-Paulo, K-H Robra, GM Gubitz (2000) Nitrile hydratase and amidase from Rhodococ-cus rhodochrous hydrolyze acrylic fibers and granular polyacrylonitrile. Appl Environ Microbiol 66 1634-1638. 

Katayama Y, Y Matsushita, M Kaneko, M Kondo, T Mizuno, H Nyunoya (1998) Cloning of genes coding for the three subunits of thiocyanate hydrolase of Thiobacillus thioparus THI 115 and their evolutionary relationships to nitrile hydratase. J Bacterial 180 2583-2589. 

Another example of a biocatalytic transformation ousting a chemical one, in a rather simple reaction, is provided by the Lonza nitotinamide process (Fig. 2.34) (Heveling, 1996). In the final step a nitrile hydratase, produced by whole cells of Rh. rhodoccrous, catalyses the hydrolysis of 3-cyano-pyridine to give nitotinamide in very high purity. In contrast, the conventional chemical hydrolysis afforded a product contaminated with nicotinic acid. 

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. 

Hydrolases lipase, protease, esterase nitrilase, nitrile hydratase glycosidase, phosphatase hydrolysis reactions in H20... 

Nitrile hydratase (NHase) catalyzes the hydration of nitriles to amides (Figure 1.11) and has been used for production of acrylamide and nicotinamide at large scale. NHases are roughly... 

Kobayashi, M. and Shimizu, S. (1998) Metalloenzyme nitrile hydratase structure, regulation, and application to biotechnology. Nature Biotechnology, 16, 733-736. 

Harrop, T.C. and Mascharak, P.K. (2004) Fe(III) and Co(III) centers with carboxamido nitrogen and modified sulfur coordination lessons learned from nitrile hydratase. Accounts of Chemical Research, 37, 253-260. 

Wu, S., Fallon, R.D. and Payne, M.S. (1997) Over-production of stereoselective nitrile hydratase from Pseudomonas putida 5B in Pichia pastoris activity requires a novel downstream protein. Applied Microbiology and Biotechnology, 48 (6), 704—708. 

Acryl amide is an important bulk chemical used in coagulators, soil conditioners and stock additives. The chemical synthesis has several drawbacks because the rate of acryl amide formation is lower than the formation of the by-product acrylic acid [54]. Further, the double bonds of the reactants and products cause by-product formations as well as formation of polymerization products. As a result of optimization with methods of molecular engineering, a very high activity of the biocatalyst nitrile hydratase at low temperature is yielded, enabling a successful biotransformation that is superior to the chemical route. Here, the synthesis is carried out at a low temperature of about 5°C, showing a conversion of 100%. 

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