However, no protein accumulation occurred in the PMS controls.
After 10 days of incubation the Bafilomycin A1 culture entered the stationary phase. During this period the concentration of chrysene in the medium decreased from 400 to 140 mg L−1, i.e. 60% of the chrysene was degraded during the 12 days of incubation. TLC of the ethyl acetate extract of the supernatants from the washed-cell incubations with chrysene showed the presence of polar metabolites. Metabolic intermediates were tentatively identified by comparing their Rf values with those of the respective standard reference compounds. Chrysene moved along with the solvent front. 1-Hydrox-2-naphthoic acid (Rf 0.43) and salicylic acid (Rf 0.15) were identified as the probable intermediates. A spot with Rf value of 0.86 did not match with any standards tested. The extracts were then analysed by HPLC and the individual spots on TLC were further characterized by LC-ESI-MS. Retention times from HPLC analysis (Fig. 2) and LC-ESI-MS
characteristics of the metabolites are given in Table 1. HPLC retention times of identified metabolites were identical to those of respective standard reference compounds. LC-ESI-MS of metabolite C1 gave a molecular ion (M+) at m/z 138 and Selleckchem Dasatinib subsequently at 121 (M+– 17, probably due to loss of OH), 110, 93 (M+– 45, loss of COOH), 80, 77 and 63 (Table 1, C1). The fragmentation pattern is identical to that of standard salicylic acid. The mass spectrum of metabolite C2 showed a base peak at 187 (M+– 1), and subsequent ion fragments at m/z 170 (M+– 17, loss of OH), 154, 143 (M+– 45, loss of COOH), 126 (M+– 17 – 45, losses of OH and COOH), 115 and 79 (Table 1, C2). The fragmentation pattern of this metabolite matched well with that of standard 1-hydroxy-2-naphthoic acid. The LC-MS spectrum of metabolite C3 showed an ion fragment at m/z 239 (M+– 1), a base peak m/z 222 (M++1−OH), and subsequent fragments at 204, 193 (M+– COOH) and 176 (phenanthrene ion). This fragmentation pattern is characteristic of hydroxyphenanthroic
acid (Baboshin et al., 2008). The mass spectra of standards and metabolites are Ribose-5-phosphate isomerase provided as Supporting Information, Figs S1–S3. The enzyme extract prepared from cells grown on different carbon sources showed high activity of 1,2-dihydroxynaphthalene dioxygenase, moderate activity of 1-hydroxy-2-naphthoate hydroxylase and catechol-1,2-dioxygenase, and low activity of salicylaldehyde dehydrogenase; catechol-2,3-dioxygenase and gentisate-1,2-dioxygenase activity was not detected (Table 2). As expected, the crude extract prepared from glucose-grown cells did not show any activity of the above enzymes, thus suggesting the inducible nature of the enzymes involved in the degradation of chrysene. To elucidate the chrysene degradation pathway operating in PNK-04, the expected intermediates of the pathway were supplied as sole source of carbon.