Combination of ammonium sulfate precipitation and ethanol precipitation

The procedure for concentrating PaE from the culture filtrate is shown in Fig 1A. All operations were performed at ambient temperature. Ammonium sulfate (314 mg) was dissolved in 1 mL of P. antarctica L1-S12 culture filtrate to afford 50% saturation. The generated precipitate was collected by centrifugation at 10,000g for 10 min. The precipitate was suspended in 1 mL of 70% (v/v) ethanol and then the supernatant was recovered by centrifugation at 10,000g for 10 min. Ethanol (2 mL) was added to the supernatant to give a final ethanol concentration of 90% (v/v); then, the generated precipitate was collected by centrifugation at 10,000g for 10 min and dissolved in 1 mL of DDW.

(a) Schematic of the concentration steps. The numbers correspond to the lanes in the SDS-PAGE analysis in panel (b). (b) SDS-PAGE analysis. MW, molecular weight marker; 1, culture filtrate; 2, 50% saturated ammonium sulfate precipitate; 3, 70% ethanol insoluble precipitate from 2; 4, 90% ethanol precipitate from supernatant of 70% ethanol suspension of 2; C, purified PaE*. DDW, deionized distilled water. *Preparation of purified PaE is described in S1 File.

The protein pattern of each fraction was checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using a Tris-tricine buffer system (e-PAGEL E-T15S and EzRun T, ATTO, Tokyo, Japan) with Precision Plus Protein Unstained Standards (Bio-Rad) as the molecular weight markers. Protein bands were visualized by silver staining (Silver Staining Kit Protein, GE Healthcare Life Sciences, Buckinghamshire, England).