For expression of proteins, the transformed yeasts were grown at 30 °C with shaking at 200 r.p.m. in 300 mL of YPD medium in 500-mL baffled shake flasks. For fermentation, recombinant strains were allowed to grow aerobically at 30 °C with shaking at 200 r.p.m. in 800 mL of SD medium in 1-L Erlenmeyer flasks to an OD600 nm value of 1.5–2.0. The inoculum culture was harvested by centrifugation, washed twice with sterile distilled water, and then inoculated

into 20 mL of CMC medium in a 50-mL closed bottle to an OD600 nm value of 20. These cultures were cultivated at 30 °C with shaking at 100 r.p.m. Yeast transformants containing the chimeric endoglucanase CelE with the altered dockerin domain were screened for CMC-degrading ability by patching on YPD plates containing 1 g L−1 CMC. After 48 h of growth, colonies on the plate were washed, and the remaining CMC was stained with 1 g L−1 Congo red and destained with 1 g L−1 Protein Tyrosine Kinase inhibitor NaCl (Den Haan et al., 2007). To confirm the secretion of endoglucanase, halos were detected on YPD–CMC plates that adsorbed 5 μL of culture supernatant. β-Glucosidase activity was detected by screening on YPD plates containing 5 mM p-nitrophenyl-β-d-glucopyranoside, as described previously (Jeon et al., 2009). For the production and secretion of proteins, recombinant selleck chemical yeasts were grown at 30 °C

for 48 h in YPD medium. Medium supernatant was then obtained by centrifugation. The supernatant was concentrated by ultrafiltration using an Ultrafree Biomax centrifugal filter unit (Millipore Co.) with a 10-kDa cut-off membrane. The concentration of secreted proteins was measured using the Bradford method (Bradford, 1976) with a Quick Start™ protein assay kit (Bio-Rad Laboratories Inc.) using bovine serum albumin as the standard. Purification was performed using cellulose (Sigmacell Type 50) at a concentration 2-hydroxyphytanoyl-CoA lyase of 10 mg protein per 1 mg cellulose and binding was performed at room temperature for 1 h with continuous shaking (Shpigel et al., 1999). The CBD-fusion protein

bound to the cellulose was centrifuged at 1600 g. Nonspecific proteins bound to cellulose were removed by washing cellulose samples three times, once with 1 M NaCl in 20 mM Tris, pH 8.0, and twice with 20 mM Tris, pH 7.5. Subsequently, bound proteins were eluted with 50 mM Tris, pH 12.5. Proteins in the cellulose-bound fraction were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The assembly of minicellulosomes was confirmed by native PAGE and zymogram analysis as described previously (Murashima et al., 2002). A zymogram with CMC was obtained by incorporating 0.2% of the substrate into the polyacrylamide gels used for native PAGE (Zhou et al., 2008). After electrophoresis, the gel was washed at room temperature in solution A (50 mM sodium acetate buffer, pH 5.0, containing 30% isopropanol) for 1 h and then solution B (50 mM sodium acetate buffer, pH 5.0) for 1 h.