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,
Summary. In this paper an explanation is given of how Pseudomonas (P.) chlororaphis B23 can accumulate so much acrylamide of such high purity. One reason is that P. ehlororaphis' B 23 exhibits much greater nitrile hydratase activity than amidase activity; the rate of formation of acrylamide through the nitrile hydratase reaction is at least 4000 times higher than its breakdown catalyzed by amidase. Furthermore, acrylonitrile, a powerful nucleophilic reagent, inactivates the active thiol residue of the amidase, whereas nitrile hydratase is not so susceptible to acrylonitrile. Thus acrylamide is produced but not transformed further. In addition, the nitrile hydratase purified from P. chlororaphis B 23 exhibits high resistance to a high concentration of acrylamide. Some other explanations, and the results of evaluation of the P. chlororaphis B 23 enzyme as a catalyst for the production of acrylamide are discussed.
Advantageous mutants of Pseudomons chlororaphis B23 for the enzymatic production of acrylamide were isolated. A mucilage polysaccharide non-producing mutant, Am-3, was precipitated completely by brief centrifugation, in contrast with the parent strain. A mutant, AM-324, derived from Am-3 exhibited about 3.8-fold higher nitrile hydratase activity than that of the parent strain. These mutants are promising for acrylamide production on an industrial scale. Nitrile hydratase catalyzes the hydration of nitriles to amides.1~3) We have proposed a new industrial scale enzymatic production process for acrylamide involving nitrile hydratase as a catalyst (equation I).4) CH2 =CHCN+H2O *CH2 =CHCONH2
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