Figure Legends
Figure 1. Expression of PhaP-fused proteins in E. colicells. (A) sfGFP, sfGFP-PhaP, and PhaP-sfGFP were overexpressed in the transformed E. coli strains. After lysis of the E. colicells, expressed proteins were analyzed by SDS-PAGE (upper panel), or by the measurement of sfGFP fluorescence (lower panel). (B) LipM7, LipM7-PhaP, and PhaP-LipM7 overexpressed in the E. coli cells were analyzed by SDS-PAGE (upper panel), or by the measurement of lipase activity (lower panel). Lanes: M, molecular weight markers; C, lysates of E. coli cells without IPTG induction; T, lysates of IPTG-induced E. coli cells; S, soluble lysate fractions of the IPTG-induced E. coli cells.
Figure 2. Comparison of sfGFP immobilized on PHB nanofibers via N-terminal or C-terminal fusion of PhaP. sfGFP-PhaP, and PhaP-sfGFP were incubated in a buffer solution containing the PHB-nanofibers. After the incubation, PHB-nanofibers were recovered by centrifugation and washed with 50 mM Tris-HCl buffer (pH 8.0). sfGFP fluorescence on the surface of the nanofibers were observed under a fluorescence microscope (Eclipse 80i microscope, Nikon, Tokyo, Japan).
Figure 3. Immobilization of PhaP-fused LipM7 on PHB nanofibers.(A) The amounts of immobilized proteins on PHB-nanofibers. (B) Stability of immobilized enzymes on PHB nanofibers. Samples were withdrawn from the storage buffer at the indicated time points and protein concentrations were measured to determine the amounts of LipM7 remained on the PHB nanofibers. Open circles, LipM7; filled circles, LipM7-PhaP.
Figure 4. Characterization of LipM7-PhaP immobilization on the PHB nanofibers. (A) Kinetics of LipM7-PhaP immobilization on PHB-nanofibers. Samples were withdrawn during the incubation of purified LipM7-PhaP with PHB nanofibers, and measured for protein concentration to monitor the binding of LipM7-PhaP over time. (B) Selective binding of LipM7-PhaP on the PHB nanofibers. After overexpression of LipM7 or LipM7-PhaP, crude cell lysate was incubated with PHB nanofibers as described in Materials and Methods. After washing (lanes marked W), immobilized proteins were eluted with SDS solution (lanes marked E). M, molecular weight markers.
Figure 5. Comparison of LipM7-PhaP immobilized on conventional supports and PHB nanofibers. (A) Amounts of LipM7-PhaP immobilized on various supports of same weight. (B) Specific activity of LipM7-PhaP immobilized on various supports.
Figure 6. Repeated use of the PHB nanofiber-immobilized enzyme.Enzymatic activity of the immobilized LipM7 or LipM7-PhaP was measured over fifty reaction cycles. Open circles, LipM7; filled circles, LipM7-PhaP.