Superdex 200 10 300 gl gel filtration

All steps were carried out under anoxic conditions (95% N 2 , 5% H 2 ). Around 3 g of phthalate grown cells (wet mass) were suspended in 9-12 ml buffer A and lysed using a French pressure cell. After ultracentrifugation, the supernatant was filtered through a 0.2-μm sterile filter (Filtropur S 0.2, Sarstedt) and applied to a 12 ml DEAE-Sepharose column (GE Healthcare), equilibrated with buffer A at a flow rate of 0.5 ml min -1 . The column was washed with two bed volumes of buffer A and with buffer A supplemented with 15 mM KCl (PCD) or 20 mM KCl (PCL). Fractions eluting in buffer A at higher KCl concentrations as indicated in the results section were tested for PCL/PCD activities. Activity containing fractions were concentrated and used for SDS-PAGE analysis and enzymatic assays. Other chromatography materials tested for PCL/PCD enrichment were Resource Q-Sepharose anionic exchanger, Superdex 200 10/300 Gl gel filtration (all GE Healthcare), and the affinity dyes Reactive Green 5 Agarose, Reactive Red 120, and Cibacron Blue 3GA (1 ml columns each, all Sigma-Aldrich). All columns were equilibrated with buffer A, and elution was by varying KCl, or in case of the affinity dyes, phthalate concentrations (0.1-1 M).