MOESM4 of Improved secretory expression of lignocellulolytic enzymes in Kluyveromyces marxianus by promoter and signal sequence engineering

Additional file 4: Figure S2. Purification and specific activity of Est1E. Transformants containing pZP28 were grown in 5L fermentor for 56 h. 200 mL culture was centrifuged at 12, 000 rpm for 30 min at 4 degree. 100 mL supernatant was mixed with 400 mL 20mM Bis-Tris buffer (pH 6.6). 200 mL sample was purified by ion exchange chromatography with Q Bestarose FF column (AI0024, Bestchrom, Shanghai, China) and eluted with 20 mM Bis-Trish Buffer with 0~1 M NaCl. Fractions containing enzymatic activity were subjected to gel filtration (Superdex 200 Increase 10/300 GL, GE Healthcare, Illinois, USA) in an AKTA purifier 100 FPLC system (GE Healthcare). Flow rate was controlled at 0.5 mL/min in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10mM Na2HPO4, 1,8 mM KH2PO4, pH 7.4). Peak of Est1E was eluted at 15 mL. The protein concentration of purified Est1E was 90 Îźg/mL, as measured by a BCA Protein Assay Kit (23250, Thermo, Illinois, USA). The enzymatic activity of purified Est1E was 215 U/mL and the specific activity of Est1E was 2400 U/mg. Supernatant of the culture, samples after purification by ion exchange and gel filtration were subjected to SDS-PAGE.