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Naphthalene oxygenase has been purified 420-fold from naphthalene-adapted Corynehacterium renale. The purified enzyme was obtained in 32% yield and gave an apparent single band on polyacrylainide gel electrophoresis. It has a molecular weight of approximately 99000 and contains two non-identical polypeptide chains of molecular weights 43000 and 56000. The enzyme requires molecular oxygen for its activity and gave a single product, cis-I ,2-dihydroxy-l,2-dihydronaphthalene. The absorption spectrum of the enzyme protein shows a maximum at 278 nm and no absorption in the Soret region, indicating that it is non-heme protcin. Absence of cytochrome P-450 was also confirmed when the protein, treated with CO, showed no absorption at 450 nm. The enzyme followed Michaelis-Menten kinetics with K, values of 2.9 m M and 1.42 mM for naphthalcne and NADH respectively. It exhibited maximal activity at 30 "C and pH 6.5. Studies on the stoichiometry of the reaction showed that one molecule of oxygen is consumed for each molecule of NADH oxidised or product formed.The initial oxidative steps in the metabolism of many aromatic compounds has been shown to be catalysed by an oxygenase which incorporates molecular oxygen into the substrate [I -31. Studies on the metabolism of naphthalene by Pseudomonas sp. has revealed that the bacterium does not use a pathway similar to the one operative in mammals. The latter involves the initial participation of a reduced nicotinamide-dependent monooxygenase system for the formation of arene oxide, which subsequently leads to trans-I ,2-dihydroxy-1 ,2-dihydronaphthalene on hydration [4,5]. In contrast, the bacteria convert naphthalene to cis-1,2-dihydroxy-1,2-dihydronaphthalene catalysed by an NAD(P)H-dependent dioxygenase [6]. Tangnu [7] demonstrated that cultures of Corynebacterium renale convert naphthalene almost quantitatively into salicylic acid. There are limited studies on the mechanism of incorporation of oxygen into the aromatic ring by naphthalene oxygenase owing to the difficulties in obtaining the purified preparation of the enzyme. Attempts to purify this enzyme from Pseudomonas cultures to homogeneity were only partially successful [8]. In this paper we report the purification and characterisation of naphthalene oxygenase from C. reiaale and the formation of cis-1,2-dihydroxy-1,2-dihydronaphthalene from naphthalene by the purified enzyme.

Bacillus amyloliquefaciens alpha-amylase activity is pH-dependent and the plot log (Vmax/Km) versus pH implicated a carboxyl group of aspartic acid/glutamic acid at the active site. Chemical modification of alpha-amylase with EDC confirmed this view. Further, analysis of inactivation kinetics showed that modification of a single carboxyl group led to complete loss of the enzymic activity.

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Clostridium pasteurianum possesses a high level of glutamate synthase (EC 1.4.1.14) activity and cell yield when grown on 4 mM ammonium chloride and molasses as the sole nitrogen and carbon sources, respectively. The enzyme activity is stabilized by addition of alpha-ketoglutarate, EDTA, and 2-mercaptoethanol. Ammonium sulfate precipitation and single-step combined gel and ion-exchange chromatography followed by fractional dialysis yield a homogeneous protein with 40% recovery of the glutamate synthase activity. The native enzyme (Mr congruent to 590,000) gives five different subunits (as dimers) upon SDS gel electrophoresis. The enzyme has been characterized for pH and temperature optimum, substrate specificity, Kmapp values, energy of activation, half-life, and thermal stabilization. Metal ions and citric acid cycle metabolites do not affect the enzyme activity. Glutamate synthase shows fluorescence maximum at 370 nm when excited at 280 nm. The fluorescence is quenched upon the addition of NADH. Spectroscopic examination of the enzyme gave absorption maximum at 280 and none at 380 and 440 nm, indicating the absence of iron and flavin. The absence of iron and flavin was also confirmed by atomic absorption, chemical analysis, and fluoroscopy, respectively. The C. pasteurianum enzyme differs from that of other aerobic bacterial sources.