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 Samples were withdrawn from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 Samples were withdrawn during the incubation of purified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.