Nitrile hydratase of Pseudomonas chlororaphis B23

Abstract

Nitrile hydratase of Pseudomonas chlororaphis B23 was completely stabilized by the addition of 22 mM n-butyric acid. The enzyme was purified from extracts of methacrylamide-induced cells of P. chlororaphis B23 in eight steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. The crystallized enzyme appeared to be homogeneous from analysis by polyacrylamide gel electrophoresis, analytical ultracentrifuge, and double diffusion in agarose. The enzyme has a molecular mass of about 100 kDa and consists of four subunits identical in molecular mass (approximately 25 kDa). The enzyme contained approximately 4 mol iron/mol enzyme. The concentrated solution of highly purified nitrile hydratase had a pronounced greyish green color and exhibited a broad absorption in visible range with a absorption maxima at 720 nm. A loss of enzyme activity occurred in parallel with the disappearance of the absorption in the visible range under a variety of conditions. The enzyme catalyzed stoichiometrically the hydration of nitrile to amide, and no formation of acid and ammonia were detected. The enzyme was active toward various aliphatic nitriles, particularly, nitriles with 3 -6 carbon atoms, e.g. propionitrile, n-butyronitrile, acrylonitrile and cyclopropyl cyanide, served as the most suitable substrates.Relatively little is known about the ability of microorganisms to utilize nitriles as carbon and/or nitrogen sources [I, 21. Benzonitrile and related aromatic nitriles [3 -61 and heterocyclic nitriles [7, 81 have been shown to be converted directly to the corresponding acids and ammonia with little release of the amide as an intermediate (Eqn 1). These nitrilases were purified, and there was no separate amidase required for formation of the acid product. Aliphatic nitriles are catabolized in two stages, via conversion to the corresponding amide and then to the acid plus ammonia [9 -141. Recently, we termed the enzyme that catalyzes the hydration of nitrile to amide as 'nitrile hydratase' [14] (Eqn 2). This enzyme is clearly distinguishable from the nitrilase based on the mode of degradation of nitrile.(2) (R, can be phenyl and a,/l-alkenyl; R2 can be alkyl).Recently, we proposed a new enzymatic production process of acrylamide on an industrial scale involving nitrile hydratase as a catalyst. Pseudomonas chlororaphis B23 was selected as a favorable strain produced more than 400g acrylamide/l reaction mixture from acrylonitrile under suitable conditions 11 51. Thus, nitrile hydratase is promising as Carrespondence to T. Nagasawa,

Enantioselective hydrolysis of racemic naproxen nitrile and naproxen amide to S-naproxen by new bacterial isolates

“…The alternative pathway involves nitrile hydratases which mediate the conversion of nitriles to the corresponding amides. In a subsequent step amidases convert the amides to the corresponding carboxylic acids and ammonia (Harper, 1977a,b;Nagasawa et al, 1987Nagasawa et al, , 1988aNagasawa et al, ,b, 1990Watanabe et al, 1987). The enzymatic hydrolysis of nitriles represents a very convenient synthetic method for amides and/or carboxylic acids due to the mild reaction conditions.…”

Nitrile Pathway Involving Acyl-CoA Synthetase

“…In the case of the aldoxime dehydratase (OxdA) of this strain (33), the k cat value for butyraldoxime (5.4 min Ϫ1 ) is much higher than that for pyridine-4-aldoxime (0.090 min Ϫ1 ), and OxdA prefers aliphatic aldoximes to aromatic ones as substrates among the tested aldoximes. For the NHase of this strain (29), butyronitrile is a better substrate and exhibits the highest affinity (K m ϭ 1.03 mM) among all of the tested nitriles. Other aliphatic nitriles such as propionitrile and acrylonitrile are remarkably active as substrates for the enzyme, although aromatic nitriles are barely hydrated by the NHase (29).…”

“…Aldoxime dehydratase activity was measured anaerobically under reduced conditions (standard assay B) as described previously (33). NHase activity was assayed by the method reported previously (29). Amidase activity was measured by the same method as reported previously (21).…”

“…One of the fruits of our application-oriented nitrile studies is the current industrial production of acrylamide and nicotinamide using NHase of an actinomycete, Rhodococcus rhodochrous J1 (1,10). On the other hand, NHase of Pseudomonas chlororaphis B23 (29), which was previously used as a catalyst for acrylamide manufacture (10,22), is now used for the production of 5-cyanovaleramide, a herbicide intermediate, at the industrial level (30). Because the amount of NHase produced comprises Ͼ50% of the total soluble proteins in P. chlororaphis B23 when this strain is cultured in the presence of methacrylamide (31), there must be a very interesting regulation mechanism for the enzyme expression (9).…”

Incorporation of thiolate donation using 2,2′-dithiodibenzaldehyde: Complexes of a pentadentate N2S3 ligand with relevance to the active site of Co nitrile hydratase

“…Examples include nitrile hydratase [1][2][3][4][5][6][7][8][9][10] and the A-cluster of carbon monoxide dehydrogenase/acetyl CoA synthase [11][12][13][14][15][16][17][18][19][20][21][22][23]. Understanding the catalytic mechanisms of these enzymes can be greatly aided by the study of small-molecule analogs, or model complexes, which reproduce the active-site structure and/or the function of the enzyme.…”