2.12 of National Center for Biotechnology Information

(Altschul et al., 1990). Cells were grown at 28 °C on a rotary shaker (180 r.p.m.) in 100-mL Erlenmeyer flasks containing 25 mL mineral salt medium (MSM, pH 7.2) and 1 g L−1 of either phenanthrene or succinate as the sole carbon source as described earlier (Mallick et al., 2007). To determine the optimal conditions for phenanthrene degradation by the test organism, CDK activity different pH values in the range of 5.0–8.0 of the medium, different cultivation temperatures in the range of 15–40 °C and different phenanthrene concentrations in the range of 0.1–2.0 g L−1 were tested individually for growth in MSM. For resting cell transformations, cells were harvested in the selleck late exponential phase by centrifugation (8000 g, 10 min), washed twice with an equal volume of potassium phosphate buffer (50 mM, pH 7.2) and finally resuspended in the same buffer to yield an OD660 nm of 1.0. Phenanthrene and pathway intermediates, viz, 2-hydroxy-1-naphthoic acid, 1-hydroxy-naphtoic acid, 1-naphthol, 2-naphthol, naphthalene-1,2-diol, salicylic acid, o-phthalic acid, protocatechuic acid and catechol in the range of 0.1–1 g L−1 were added individually

to washed cell suspensions, and incubated at 28 °C for different periods of time up to 48 h. Unless stated otherwise, each experimental set was performed in triplicate. To isolate phenanthrene-degraded metabolites and unutilized phenanthrene, the spent broth and resting cell culture were centrifuged (8000 g, 10 min) SB-3CT and the supernatants were acidified to pH 1.5–2.0 by 6 N hydrochloric acid and extracted three times with equal volumes of ethyl acetate. The combined organic layer was re-extracted with aqueous sodium hydroxide (10 mM). The organic phase was evaporated under reduced pressure (neutral fraction). The aqueous NaOH extracts were acidified as above and then extracted with ethyl acetate (acidic fraction). The combined extracts were dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residues

were methylated with a boron trifluoride/methanol solution (Merck) as needed before analysis. Measurements were performed at 25 °C using a YSI model 5300A biological oxygen monitor (Yellow Springs Instrument Co., Yellow Springs, OH) equipped with a Clark-type polarographic oxygen electrodes (YSI model 5331A oxygen probes) and a sample chamber fitted within a YSI model 5301B standard bath. The sample size was 2.0 mL, and the reaction mixture contained 0.5 mL cell suspension (25 mg cells, wet weight), substrate (0.5 mL) and 1 mL phosphate buffer (50 mM, pH 7.0). The reaction was initiated by injecting a suitable amount of the assay substrate and oxygen uptake was monitored for 5 min. Phenanthrene (0.5 mL) was added as a saturated solution (∼1.