MOESM7 of Improved secretory expression of lignocellulolytic enzymes in Kluyveromyces marxianus by promoter and signal sequence engineering

Additional file 7: Figure S5. Purification and specific activity of MAN330. Transformants containing pZP42 were grown in a 5L fermentor for 56 h. Culture was centrifuged at 12, 000 rpm for 30 min at 4 degree. 100 mL supernatant was mixed with 65 g (NH4)2SO4 and centrifuged at 12,000 rpm for 30 min at 4 degree. Supernatant was discarded. Precipitate was dissolved in 10 mL H2O and subjected to gel filtration (Superdex 75, 10/300, GL, GE Healthcare) in an AKTA purifier 100 FPLC system. Flow rate was controlled at 0.5 mL/min in PBS buffer (pH 7.4). Peak of MAN330 was eluted at 12 mL and fractions containing the peak were pooled. The protein concentration of purified MAN330 was 1mg/mL, as measured by a BCA Protein Assay Kit. The enzymatic activity of purified MAN330 was 1590 U/mL and the specific activity of MAN330 was 1590 U/mg. Precipitate dissolved in H2O and fractions eluted at 12 mL in gel filtration were subjected to SDS-PAGE