Whole-cell biocatalysis to oxidize naphthalene to 1-naphthol in liquid-liquid biphasic systems was performed. 40). Toxicity experiments. The cells were cultivated until early log phase in 250-ml shake flasks, and then growing cells (5 ml) were added to 20-ml sterile screw-cap vials. Naphthalene or 1-naphthol dissolved in 50 l dimethyl formamide (DMF) was added to the growing cells to obtain final concentrations of 0.05 g/liter, 0.1 g/liter, 0.5 g/liter, and 1 g/liter. The growth was monitored by determining the Camptothecin distributor optical denseness at 660 nm (OD660). Due to the low solubilities of naphthalene and 1-naphthol in water, the cosolvent DMF was used to suspend the compounds in the aqueous phase. A positive control experiment in which 50 l DMF was added without naphthalene or 1-naphthol was also performed. Cell viability dedication. A Becton Dickinson LSR II circulation cytometer in the University or college of Iowa Circulation Cytometry Facility was used to measure cell viability. For this analysis an L-701 Invitrogen Molecular Probes (Carlsbad CA) LIVE/DEAD TG1/pBS(Kan)TOM-Green cells. Cells were grown to late log phase (OD660, 1.6), when the LB medium appeared to be green due to the production of indigo and isatin (7, 10). HBGF-4 Cells were harvested by centrifugation (10,000 TG1/pBS(Kan)TOM-Green cells (360 ml) Camptothecin distributor were grown to late log phase (OD660, 1.6) and harvested by centrifugation at 10,000 for 10 min. The cells were washed with Tris buffer (pH 7.2). The cells for each experiment were immobilized collectively and later on divided and placed into six independent flasks. A 3% sodium alginate answer was prepared using 120 ml of deionized water. A 1% CaCl2 answer was prepared as the gelation agent using deionized water. Both the sodium alginate and CaCl2 solutions were autoclaved at 121C for 15 min. The sodium alginate answer was allowed to awesome to room heat, and the CaCl2 answer was cooled to 4C. The pelleted cells were resuspended in 25 ml of sterilized deionized water. The cells and sodium alginate answer were combined by stirring them for 5 min on a stir plate. The combination was added dropwise to the stirred gelation agent using a 60-ml syringe with an 18-gauge needle. The combination was stirred for 1 h to harden it. The producing calcium alginate beads were 1 to 2 2 Camptothecin distributor mm in diameter. After hardening, the beads were removed from the perfect solution is and washed twice with sterilized deionized water. The immobilized cells were divided equally among six sterile flasks comprising 19.5 g Camptothecin distributor of beads each. Immobilized cell biotransformation. The immobilized biocatalyst was suspended in 250-ml Erlenmeyer flasks with a working volume of 50 ml. The beads were suspended in 30 ml of Tris-HCl buffer (pH 7.2) supplemented with 20 mM glucose and 100 mg/liter kanamycin. The solvent phase (20 ml) was added to begin the reaction, and the flasks were shaken at 200 rpm and 37C. An HPLC analysis was carried out using samples of the solvent phase. RESULTS Substrate and product toxicities. Whole cells of TG1 expressing TOM-Green were utilized for oxidation of naphthalene to 1-naphthol. The toxicities of both naphthalene and 1-naphthol for the TG1 strain expressing TOM-Green are demonstrated in Fig. ?Fig.1.1. Naphthalene inhibited cell growth actually at a low concentration, 0.05 g/liter. The inhibition of growth improved as the concentration of naphthalene increased to 0.5 g/liter, and no growth was observed with 1 g/liter naphthalene. The inhibitory effect of 1-naphthol was greater than that of naphthalene, and no growth was observed even with 0.5 g/liter 1-naphthol. These results are comparable to the results of similar work carried out previously (38). Consequently, keeping low concentrations Camptothecin distributor of the substrate and the product is critical for keeping the cell viability and the activity for the reaction. Open inside a.