1786 Fig. 4 Western blot analysis of tglA, CBM-tglA, and tglA-CBM fusion proteins from cell wall fractions (lanes 1 to 3), cytosolic fractions (lanes 4 to 6), and supernatant fractions (lanes 7 to 9). Lanes 1, 4, and 7, A. oryzae/tglA-CBM transformant; 2, 5, and 8, A. oryzae/ CBM-tglA transformant; 3, 6 and 9, A. oryzae/tglA transformant fragment encoding the mature region of tglA from A. oryzae that was fused with the gene encoding the FLAG peptide tag at the N terminus was amplified from cDNA prepared from A. oryzae with the following primers, 5′- GGA ATTCCATATGGACTACAAG GATGACGATG ACAAGAACGCTCCCCTGAATGAGTT-3′ and 5′- TCCCATTTAAATTTAGTTCGCAGCCGCAACAG-3′. The amplified fragment was digested with NdeI and SwaI and inserted into pISI-CBM-GFP. The resultant plasmid was designated as pISI-CBM-tglA. All PCR-amplified DNA fragments were confirmed by DNA sequencing (CEQ 8000, Beckman Coulter). All expression plasmids constructed in this study are summarized in Fig. 1. Transformation of A. oryzae was carried out according to the method described by Gomi et al (1987). The resultant transformants were subcultured on CD-NO3 medium plates three times to obtain stable expression transformants. The transformants were named A. oryzae/GFP, A. oryzae/GFP- CBM, A. oryzae/CBM-GFP, A. oryzae/tglA, A. oryzae/ tglA-CBM, and A. oryzae/CBM-tglA. According to our Appl Microbiol Biotechnol (2010) 87:1783–1789 previous study, the copy number of the expression vectors integrated into the genome was assumed to be one (Ishida et al. 2000). Fluorescence microscopy analysis Wild-type A. oryzae and transformants harvested from submerged cultures cultivated for 7 days were washed twice with PBS. GFP fluorescence was detected using a fluorescence microscope (BZ-8000; Keyence Co., Osaka, Japan). Protease treatment Recombinant A. oryzae harvested from submerged cultures cultivated for 10 days were washed with PBS three times, and the surface-displayed protein was cleaved by incubating the suspension with Proteinase K (final concentration, 30 U/L) for 24 h at 37°C. After proteolysis, A. oryzae was washed twice with PBS, followed by gentle centrifugation to remove the protease, and GFP fluorescence was observed using fluorescence microscopy as described above. Western blot analysis Cell wall proteins were extracted from wild-type A. oryzae and transformants. Briefly, the washed frozen cells were ground with a pestle and mortar, and then resuspended in PBS (15 ∼20 μl/mg dry cell). The cytosolic fraction was separated from cell walls and membranes by centrifugation at 15,000 rpm at 4°C for 10 min. Then the resultant precipitate was washed twice with PBS and subjected to the following analysis as a cell wall fraction. Sodium dodecyl sulfate polyacrylamide gel electro- phoresis analysis was performed using a 12.5% gel. The Fig. 5 Time course of lipase activity of the fungus bodies of recombinant A. oryzae displaying tglA-CBM and CBM-tglA fusion proteins (a) and that of culture supernatant (b). Symbols represent wild-type (closed circles and open circles), A. oryzae/tglA trans- formant (closed diamonds and open diamonds), A. oryzae/tglA-CBM transformant (closed squares and open squares), and A. oryzae/CBM- tglA transformant (closed triangles and open triangles)