Cells failed to respire on o-phthalic acid and 3,4-dihydroxybenzoic acid (Table 1). The cell-free extract prepared from phenanthrene-grown cells showed activities of 1-hydroxy-2-naphthoic acid hydroxylase, 1,2-dihydroxynaphthalene dioxygenase, salicylate-1-hydroxylase and catechol-2,3-dioxygenase (Table 2), while salicylic acid-grown cells showed comparatively reduced activities for all enzymes and significantly lower activity of 1-hydroxy-2-naphthoic
acid hydroxylase (Table 2). The cell-free extract prepared from naphthalene-grown cells of P. putida strain CSV86 (this strain does not degrade phenanthrene or 1-H2NA, Mahajan et al., 1994) showed sevenfold less activity of salicylate-1-hydroxylase with 1-H2NA (53 nmol min−1 mg−1) as compared with salicylic acid (362 nmol min−1 mg−1) as substrate. The enzyme preparation from strain PPH failed to show activity of gentisic- and 3,4-dihydroxybenzoic acid dioxygenase selleck chemicals llc (Table 2). Time-dependent spectral changes of catechol dioxygenase reaction showed an increase in A375 nm (Deveryshetty, 2009), indicating the formation of 2-hydroxymuconic semialdehyde due to meta-ring cleavage of catechol by catechol-2,3-dioxygenase (Kojima et al., 1961; Nozaki et al., 1963). Specific activity versus growth profiles showed maximum activity of 1-hydroxy-2-naphthoic acid hydroxylase and 1,2-dihydroxynaphthalene dioxygenase at
18 h, and maximum activity of catechol-2,3-dioxygenase at 21 h (Deveryshetty, 2009). Salicylate-1-hydroxylase activity was detectable, U0126 purchase but at low levels. Cells grown on glucose showed neither O2 uptake nor enzyme activities in the cell-free extract (Deveryshetty, 2009), indicating that the enzymes of the pathway are inducible. 1-Hydroxy-2-naphthoic acid hydroxylase
in the cell-free extract was stabilized by 1-H2NA (0.1 mM), FAD (5 μM), dithiothreitol (2 mM) and glycerol (5%). Interestingly, the enzyme showed stability at 60 °C for 5 min in the presence of 1-H2NA, while the activity of salicylate-1-hydroxylase was lost, suggesting the presence of two distinct enzymes PRKD3 in the strain PPH. Using heat treatment, ammonium sulfate fractionation and DEAE anion-exchange chromatography, 1-hydroxy-2-naphthoic acid hydroxylase was partially purified (81-fold, with a 48% yield and a specific activity of 1518 nmol min−1 mg−1 protein) from phenanthrene-grown cells of Alcaligenes sp. strain PPH (Table 3). Native-PAGE analysis showed a prominent band of lower mobility and two minor contaminating bands with higher mobility (Fig. 1a). SDS-PAGE analysis showed a progressive enrichment of a protein band of ∼34 kDa (Fig. 1b). Additional purification steps such as hydrophobic (Phenyl- and Octyl-Sepharose) or gel filtration chromatography led to the total or a significant (∼70%) loss of activity, respectively, without achieving any further purification.