Appl Microbiol Biotechnol (2010) 87:1783–1789 separated proteins were transferred from the gel to a polyvinylidene difluoride (PVDF) membrane (Atto, Tokyo, Japan) using a HorizBlot AE-6677 (Atto), according to the supplier's instruction. The PVDF mem- brane was blocked with 5% non-fat milk and incubated with primary mouse anti-GFP IgG (Sigma, St. Louis, MO) or mouse anti-FLAG IgG (Sigma) for 1 h at room temperature. The PVDF membrane was subsequently incubated with a secondary alkaline phosphatase- conjugated mouse anti-mouse IgG antibody (Promega, Madison, WI) and washed twice with TBS-T buffer. For detecting GFP, the PVDF membrane was incubated with a mixture of nitroblue tetrazolium chloride and 5-bromo-4- chloro-3-indolyl phosphate toluidine salt (Promega), according to the manufacturer’s procedure. For detecting tglA, the PVDF membrane was incubated with CDP-star (Applied Biosystems), according to the manufacturer's procedure. Measurement of lipase activity Wild-type A. oryzae and each of the transformants were cultivated in GPY medium and harvested by filtration with Miracloth (Calbiochem, Darmstadt, Germany). Cells were washed with distilled water and lyophilized. Lyophilized cells were crushed with an SK-Mill (Funakoshi, Tokyo, Japan). Culture supernatants and crushed cells were used for the assay of lipase activity using Lipase Kit S (DS Pharma Biomedical, Osaka, Japan) according to the supplier's protocol, and the resulting values were expressed in international units (IU). One unit of lipase activity was defined as the amount of enzyme catalyzing the formation of 1 mmol of 2,3-dimercaptopropan-1-ol from 2,3-dimercaptopropan-1-ol tributyl ester per min. Triplicate experiments were performed. Results Localization of GFP-CBM and CBM-GFP fusion proteins evaluated by western blotting We constructed GFP-CBM and CBM-GFP fusion protein expression vectors (pISI-GFP-CBM and pISI-CBM-GFP, respectively) as shown in Fig. 1. The GFP secreting vector (pISI-GFP) was used as a control (Adachi et al. 2008). These vectors were transformed into the A. oryzae IF4 niaD− strain to obtain each transformant. To investigate the expression and localization of GFP-CBM and CBM-GFP proteins, the cell wall fractions of recombinant A. oryzae were subjected to western blotting with an anti-GFP monoclonal antibody. The bands corresponding to the GFP-CBM and CBM-GFP fusion proteins were observed 1787 in the cell wall fractions of A. oryzae/GFP-CBM and A. oryzae/CBM-GFP transformants (Fig. 2, lanes 1 and 2). Although weak bands of GFP-CBM were observed in the cytosolic and supernatant fractions (Fig. 2, lanes 4 and 7), GFP-CBM and CBM-GFP were mainly expressed in the cell wall fraction. For the A. oryzae/GFP transformant, GFP was secreted in the supernatant (Fig. 2, lane 9) and did not accumulate in the cell wall fraction (Fig. 2, lane 3). For GFP-CBM, the band corresponding to GFP was also observed in the supernatant fraction (Fig. 2, lane 7), which might have been caused by non-specific proteolysis. Localization of GFP-CBM and CBM-GFP fusion proteins visualized by fluorescence microscopy To visualize the localization of GFP-CBM or CBM-GFP, each transformant was directly observed using fluorescence microscopy. Bright green fluorescence was clearly observed on the cell surface of A. oryzae/GFP-CBM (Fig. 3a and b) and A. oryzae/CBM-GFP (Fig. 3d, e) transformants. In contrast, no green fluorescence signal was detected on the cell surface of the A. oryzae/GFP transformant (Fig. 3g, h). These results clearly show that CBM is a useful anchor protein on the A. oryzae cell surface allowing function of the target protein to be retained. To evaluate the accessibility of cell surface displaying GFP, protease treatment was carried out. After proteinase K treatment, green fluorescence on the cell surface of A. oryzae/GFP-CBM and A. oryzae/CBM-GFP transformants was diminished (Fig. 3c, f), whereas the A. oryzae/GFP transformant was scarcely different before and after treatment (Fig. 3h, i). We assumed that we could visualize the produced GFP fused with anchor proteins that were successfully localized on the cell surface. Evaluation of triacylglycerol lipase-displaying A. oryzae To construct A. oryzae transformants displaying triacylgly- cerol lipase, the pISI-tglA-CBM and pISI-CBM-tglA (Fig. 1) vectors were transformed into the A. oryzae IF4 niaD− strain. As a control, an A. oryzae transformant secreting triacylglycerol lipase was constructed using the pISI-tglA vector. First, the localization of the tglA-CBM and CBM-tglA fusion proteins was investigated by western blot analysis using an anti-FLAG monoclonal antibody (Fig. 4). The bands corresponding to the tglA-CBM and CBM-tglA fusion proteins were observed only in the cell wall fractions from A. oryzae/tglA-CBM and A. oryzae/ CBM-tglA transformants (Fig. 4, lanes 1 and 2). With respect to the cytosolic and supernatant fractions of the A. oryzae/CBM-tglA transformant, bands corresponding to tglA were detected (Fig. 4, lanes 5 and 8), which might have been caused by non-specific proteolysis. In the A.