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epsilon-Poly-L-lysine (epsilon-PL) is a homo-poly-amino acid characterized by the peptide bond between the carboxyl and epsilon-amino groups of L-lysine. epsilon-PL shows a wide range of antimicrobial activity and is stable at high temperatures and under both acidic and alkaline conditions. The mechanism of the inhibitory effect of epsilon-PL on microbial growth is the electrostatic adsorption to the cell surface of microorganisms on the basis of its poly-cationic property. Due to this antimicrobial activity, epsilon-PL is now industrially produced in Japan as a food additive by a fermentation process using Streptomyces albulus. In spite of the practical application of epsilon-PL, the biosynthetic mechanisms of epsilon-PL have not been clarified at all. epsilon-PL producers commonly possess membrane-bound epsilon-PL-degrading aminopeptidase, which might play a role in self-protection.

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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,

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Streptomyces sp. GF3587 and 3546 were found to be imine-reducing strains with high R- and S-selectivity by screening using 2-methyl-1-pyrroline (2-MPN). Their whole-cell catalysts produced 91 mM R-2-methylpyrrolidine (R-2-MP) with 99.2%e.e. and 27.5 mM S-2-MP (92.3%e.e.) from 2-MPN at 91-92% conversion in the presence of glucose, respectively.

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A new cobalt-containing nitrile hydratase was purified from extracts of urea-induced cells from Rhodococcus rhodochrous J1 in seven steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. Nitrile hydratase was a 500 -530-kDa protein composed of two different subunits (a subunit 26 kDa, fl subunit 29 kDa).The enzyme contained approximately 11 -12 mol cobalt/mol enzyme. A concentrated solution of highly purified nitrile hydratase exhibited a broad absorption spectrum in the visible range, with an absorption maxima at 410 nm. The enzyme had a wide substrate specificity. Aliphatic saturated or unsaturated nitriles as well as aromatic nitriles, were substrates for the enzyme. The optimum pH of the hydratase was pH 6.5 -6.8. The enzyme was more stable than ferric nitrile hydratases. The amino-terminal sequence of each subunit of R. rhodochrous J1 enzyme was determined and compared with that of ferric nitrile hydratases. Prominent similarities were observed with the subunit. However, the amino acid sequence of the CI subunit from R. rhodochrous J1 was quite different from that of the ferric enzymes. In the present study, we purified and characterized the new cobalt-containing nitrile hydratase of R . rhodochrous J1 and the physicochemical and enzymatic properties were compared with the iron-induced and iron-containing nitrile hydratases from P. chlororaphis B23, Brevibacterium R312 and (') Rhodococcus sp. N-774.The improvement of culture conditions and the mutagenesis of P. chlororaphis B23 greatly increased the production of nitrile hydratase [4-61. As a result, P. chlororuphis B23 nitrile hydratase is used as a catalyst for the industrial production of acrylamide in Japan [7 -91. We purified and crystallized nitrile hydratase from P. MATERIALS AND METHODS Materials