3.3. Characterization of LipM7-PhaP binding to PHB nanofibers
A 0.2 mg sample of LipM7-PhaP was added to 1 mL of immobilization buffer
(50 mM TRIS-HCl, pH 8.0) containing 5 mg of PHB nanofibers, and the
amount of protein in buffer solution was measured periodically to
estimate the quantity of fiber-bound proteins. As shown in Figure 4A,
LipM7-PhaP rapidly partitioned onto the PHB nanofibers, and 50%
saturation was reached in 15 min. The binding of LipM7-PhaP reached a
plateau after 70 min.
To test the selectivity of LipM7-PhaP binding to PHB nanofibers, PHB
nanofibers were incubated with a clarified lysate of E. colicells expressing LipM7-PhaP. After extensive washing, proteins bound to
PHB nanofibers were eluted with 10% SDS solution. The results in Figure
4B demonstrate that PHB nanofibers specifically interact with
LipM7-PhaP. While the relative abundance of LipM7 in SDS-eluted proteins
was comparable to that in the cell lysate (lanes A and B of the LipM7
group), LipM7-PhaP was eluted in high purity. This indicates that most
of the E. coli proteins were outcompeted by LipM7-PhaP, and were
removed during the washing step. The maximum loading capacity
(q max) and the dissociation constant
(k d) determined by Langmuir isotherm model
analysis were 12.5 mg per gram of PHB nanofibers and 0.92 µM,
respectively (Table 1 and Figure S1). Although thek d value for electrospun nanofibers was 5-fold
higher than for the granular supports, the enlarged surface area of the
nanofibers allowed them to anchor 120-fold more enzyme.
3.4. Enzymatic activity of lipases immobilized on PHB
nanofibers
The advantages of phasin-mediated enzyme immobilization on PHB
nanofibers was highlighted by comparing with conventional immobilization
methods. Compared with Duolite A568 and Sipernat D17, the same weight of
PHB nanofibers could retain 2- to 3-fold more enzyme (Figure 5A).
Interestingly, differences in enzymatic activity between different
immobilization supports were pronounced for immobilized LipM7-PhaP. PHB
nanofibers loaded with LipM7-PhaP exhibited 3- to 10-fold higher lipase
activity than Duolite A568 and Sipernat D17 (Figure 5B), indicating that
the enzyme immobilized on PHB nanofibers possessed higher specific
enzymatic activity than enzyme immobilized on conventional supports.
This was attributed to the oriented immobilization of enzymes onto the
PHB surface.
Furthermore, nanofiber-immobilized enzyme could be used without
substantial loss of activity over repeated reaction cycles. As shown in
Figure 6, LipM7-PhaP immobilized on PHB nanofibers retained
>74% of initial activity after 50 reaction cycles. By
contrast, LipM7 adsorbed onto PHB nanofibers lost more than half of
initial activity after only three reaction cycles.
Additionally, nanofiber-immobilized LipM7-PhaP was successfully used for
a synthetic reaction. When employed for the transesterification of
octanoic acid to produce methyl octanoate, ~70% of
octanoic acid was converted into methyl octanoate after 60 h (Figure
S2).