Short nanofibers-based hierarchical structures for lipase immobilisation

Short nanofibers are fibers with diameters in the sub-micron scale and with finite length (up to 1mm). A major advantage of short – compared to electrospun (long) – nanofibers is their suspendability in liquid media. Such advantage enables a user to use the fibers in suspension or to prepare constructs of pre-determined density, such as paper-like membranes or thin coatings. It is here proposed that short nanofibers may be useful as reusable substrates for enzyme immobilisation.

This investigation is focused on lipase immobilisation onto the surface of short chitosan nanofibers (SNFs) native or modified with other functional groups. The objective of the study was to understand how the
functionality of surface modified SNFs affected lipase immobilisation and to identify optimal reaction conditions for immobilisation. SNFs were functionalized via established chemistry approaches by using two
pathways, glutaraldehyde and EDC-NHS. The so-functionalized SNFs were then characterized by testing different fiber diameters or preparing constructs with different fiber-per-unit-volume density (paper, loose fibers in suspension), as the shear-dispersion approach offers a wide choice in the assembly of SNFs. The immobilisation of the enzyme on nanofibers was demonstrated, with high-efficiency protein binding and
reusability. Lipase immobilised on loose fibers retained over 90% of its specific activity when reusing it up to 10 times, in comparison to free lipase (which cannot be reused), whereas lipase bound on paper samples, retained around 60% of its original specific activity (Figure 1). Furthermore, lipase immobilised on short chitosan nanofibers (in both forms) displayed significant thermal stability at high temperatures. Aside from thermal stability, the main advantage identified for lipase immobilised on SNFs is its easier separation, recovery and re-use.

The nature of short nanofibers is such that they can be treated as if they were particles, and fibers of different type, geometry, functionality can be mixed in suspension with great ease. This unique set of properties will allow to build multi-functional coatings or constructs, otherwise not accessible. This work also demonstrates that short chitosan nanofibers are a viable support for the immobilisation of enzymes (lipase acting as a model molecule), opening the way to novel constructs with high-application and tuning potential.