1788 oryzae/tglA transformant, although bands corresponding to tglA were detected in all of the fractions, it was mainly observed in the supernatant fraction (Fig. 4, lanes 3, 6, and 9). We then evaluated the time course of lipase activity of the fungus body and culture supernatant (Fig. 5a, b). About 800 mg of dry cells were obtained from the 100 ml of culture medium after 8 days cultivation and there was no significant difference of the dry-cell weight between each transformants (data not shown). There was no activity in the fungus body or culture supernatant of A. oryzae, which served as a negative control. As for the culture supernatant, lipase activity (maximal activity was 190 IU/L) was detected only in the A. oryzae/tglA transformant (Fig. 5b). In terms of the fungus body, the lipase activity of the A. oryzae/CBM-tglA transformant (maximal activity was 3.8 IU/mg dry cells) was about 4-fold higher than that of A. oryzae/tglA (maximal activity was 1.1 IU/mg dry cells) and A. oryzae/tglA-CBM (maximal activity was 1.3 IU/mg dry cells) transformants (Fig. 5a). These results indicate that tglA was successfully displayed on the cell surface by the A. oryzae/CBM-tglA transformant while retaining its enzymatic activity. Discussion In this study, to expand the utility of the cell surface display system of A. oryzae, we successfully developed a novel cell surface display system using CBM as the anchor protein. CBM has two putative advantages. One is its small size (about 9 kDa) compared to the previously reported anchor protein MP1 (about 25 kDa; Adachi et al. 2008) which may reduce the steric hindrance between anchor protein and target protein, and the other is that a target protein can be fused to either its N or C terminus. Western blot analysis revealed that both GFP-CBM and CBM-GFP fusion proteins were located in the cell wall fraction (Fig. 2). GFP fluorescence was also clearly detected ubiquitously on the cell surface of A. oryzae (Fig. 3a, b, d, e). To our knowledge, CBM is the first anchor protein whose N or C terminus can be fused with a target protein that can be expressed on the cell surface of A. oryzae while retaining its activity. The expression level of GFP-CBM is higher compared with that of GFP-MP1 (Adachi et al. 2008), showing one of the advantages of CBM as an anchor protein. The expression level of GFP- CBM is higher compared to that of CBM-GFP, suggesting the importance of the fusion site for the production levels of the fusion protein. In all transformants, the GFP fluorescence localization at the both hyphal tips and septa was caused by the signal sequence, which is consistent with a previous report (Maruyama and Kitamoto 2007). Additionally, the decrease Appl Microbiol Biotechnol (2010) 87:1783–1789 in cell surface fluorescence after proteinase K treatment clearly shows that GFP is accessible enough for the protease (Fig. 3c, f), which suggests that both GFP-CBM and CBM-GFP were oriented outside the cell wall. Using CBM as an anchor protein, the lipase tglA was displayed on the cell surface of A. oryzae. Both tglA-CBM and CBM-tglA fusion proteins were also successfully located on the cell wall (Fig. 4). Because tglA activity was detected only in the fungus body, this demonstrates that CBM can immobilize a fused protein tightly on the cell surface in the same manner as the glycosylphosphatidyl inositol anchor protein MP1 (Adachi et al. 2008). However, the lipase activity on the fungus body expressing tglA- CBM was lower compared to that expressing CBM-tglA. One possible explanation is that the activity of tglA-CBM may be inhibited because the location of the catalytic triad of tglA is on the C-terminal side (Toida et al. 2000). Although introduction of some linker region between target protein and anchor protein may increase the activity of target protein, it is time-consumable and troublesome. These results also suggest that the site of the displayed protein and anchor protein fusion is important for protein display without the loss of function, and that CBM is an appropriate anchor protein due to the availability of both its N and C termini. In conclusion, we developed a novel cell surface display system for A. oryzae using CBM as an anchoring motif. This system is convenient for genetic manipulation because CBM has a small molecular weight and target proteins can be fused to either its N or C terminus. Furthermore, it should be possible to catalyze sequential reactions by combining this CBM-based system with cell surface display using MP1 or other anchor proteins. Acknowledgements This work was supported by the 2005 Regional Innovative Consortium Project of the Ministry of Economy, Trade and Industry, Japan, and Bio-oriented Technology Research Advancement Institution, Japan, and partially supported by Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction, Kobe), MEXT, Japan. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. References Adachi T, Ito J, Kawata K, Kaya M, Ishida H, Sahara H, Hata Y, Ogino C, Fukuda H, Kondo A (2008) Construction of an Aspergillus oryzae cell-surface display system using a putative GPI-anchored protein. Appl Microbiol Biotechnol 81:711–719 Akao T, Sano M, Yamada O, Akeno T, Fujii K, Goto K, Ohashi-Kunihiro S, Takase K, Yasukawa-Watanabe M, Yamaguchi K, Kurihara Y,