Assay for 1-hydroxy-2-naphthoate hydroxylase was performed using a modification of the method of Kamin et al. (1978). The assay system contained, in a combined volume of 3 mL, 50 μmol 1-hydroxy-2-naphthoic Pirfenidone ic50 acid, 65 μmol NADH, 1 mmol EDTA and a suitable amount of cell-free extract in phosphate buffer (20 mmol, pH7.6). The reaction was
initiated by the addition of the substrate. Enzyme activity (Shamsuzzaman & Barnsley, 1974) was measured spectrophotometrically by monitoring the decrease in the absorbance at 340 nm due to the oxidation of NADH (ɛ=6221). Salicylaldehyde dehydrogenase activity was determined from the rate of increase in the absorbance at 340 nm (ɛ=3840) due to the formation of NADH. The reaction mixture contained 2.75 mL of 20 mM tetrasodium pyrophosphate HCl (pH 8.5), 0.1 mL salicylaldehyde (3 mM aqueous solution of freshly redistilled Palbociclib datasheet aldehyde) and 0.1 mL NAD+ (150 mM). Catechol-1,2-dioxygenase (Hegeman, 1966) activity was measured spectrophotometrically by an increase in absorbance at 260 nm due to formation of cis,cis-muconic acid (ɛ=1690). Catechol-2,3-dioxygenase
activity was measured by determining the rate of accumulation of 2-hydroxymuconic semialdehyde (ɛ=3600) at 375 nm (Feist & Hegeman, 1969). The reaction mixture contained 100 μmol Tris-hydrochloride buffer (pH 7.6) and 0.2 μmol catechol. The reaction was initiated by the addition of 0.1 mL of crude enzyme. Gentisate-1,2-dioxygenase activity (Crawford et al., 1975) was measured spectrophotometrically by an increase in absorbance at 334 nm due to formation of maleylpyruvate (ɛ=1080). The assay mixture contained 0.15 μmol gentisic acid in 3 mL of 0.1 M Na–K phosphate buffer (pH 7.4) and the reaction was started by the addition
of enzyme. The protein concentration of the enzyme solution was Bay 11-7085 determined using bovine serum albumin as standard (Lowry et al., 1951). Specific activity of crude enzyme was expressed as μmol of substrate degraded/product formed per minute per mg of protein under assay conditions. Strain PNK-04 was tested for the utilization of various aromatic compounds, such as naphthalene, 1-methylnaphthalene, 2-methylnaphthalene, phenanthrene, 1-naphthol, 1-naphthoic acid, phthalic acid, 4-hydroxybenzoic acid, gentisic acid, protocatechuic acid, ortho and para cresols, salicylic acid and catechol. In all the cases this bacterium was grown on PMS medium (pH 7), with appropriate carbon source added to the shake flask (1 g L−1). The culture was incubated on a rotary shaker (180 r.p.m., 37 °C). Growth at the expense of the respective aromatic compounds was verified by demonstrating an increase in bacterial protein. Pseudoxanthomonas sp. PNK-04 was able to grow on chrysene as the sole source of carbon and energy. The typical growth pattern of this bacterium on chrysene (Fig.