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).