3.1. Use of PhaP as a fusion partner for immobilization of
enzymes
Due to its high affinity for PHB (Zhao et al., 2016) and relatively
small size compared to phasins from other bacterial species (Table S3),
we investigated the use of PhaP from Aeromonas hydrophila as an
affinity tag to immobilize proteins on PHB surfaces. In the initial
experiments, we examined whether fusion with PhaP affected the
expression of target proteins. When PhaP-sfGFP and sfGFP-PhaP were
expressed in E. coli strain BL21(DE3), both fusion proteins were
produced at similar levels to those of the parental sfGFP (Figure 1A,
upper panel). Similarly, expression of LipM7 lipase was not
significantly affected by N- or C-terminal fusion with phasin (Figure
1B, upper panel), indicating that PhaP could be fused upstream or
downstream of target proteins without affecting the efficiency of gene
expression.
The location of the PhaP fusion could significantly affect the
biological activity of target proteins. In the case of sfGFP, the fusion
protein with C-terminal PhaP displayed ~33% higher
fluorescence than the control sfGFP (Figure 1A, lower panel). It has
been reported that the functionality of a fluorescence protein can be
enhanced when fused with a foreign protein sequence (Solovyov et al.,
2011, Huang et al., 2016). However, the enzymatic activity of LipM7
exhibited greater dependence on the location of phasin fusion. Although
LipM7-PhaP was not significantly different from wild-type LipM7 in terms
of its ability to hydrolyze pNPD, PhaP-LipM7 exhibited <25%
the activity of LipM7 (Figure 1B, lower panel). Although the structure
of this lipase has not been determined, this result suggests that the
N-terminus of this enzyme plays an important role in catalytic activity,
in accordance with previous results showing that lipase activity can be
disrupted when fused to N-terminal fusion partners (Gustavsson et al.,
2001; Singh et al., 2018; Malunavicius et al., 2018).