1. Introduction
Enzymes have many advantages over conventional chemical catalysts, including outstanding substrate specificity and more rapid catalytic turnover (Jaeger and Eggert, 2004). Since they operate under milder conditions and consume less energy, enzyme-based processes also provide an eco-friendly route for the production of chemicals (Houde et al., 2004). Enzyme immobilization is an attractive option to reduce production cost and develop novel biotransformation processes. While stable immobilization of enzymes can be achieved by covalent linkage of functional groups between enzymes and support materials, it often perturbs the native conformation of enzymes, leading to diminished enzyme activity (Homaei et al, 2013; Tischer and Wedekind, 1999; Zhou et al., 2015). By contrast, immobilization methods based on non-covalent sorption can be performed under milder conditions, allowing immobilized enzymes to better retain their activity. However, in general, non-covalently attached enzymes are easily displaced from supports, limiting their reusability. Furthermore, most current immobilization methods, including both covalent and non-covalent types, result in a random orientation of enzymes on the support surface, and only a fraction of immobilized enzymes are optimally oriented toward their substrates. This can also contribute to a decrease in enzyme activity after immobilization.
Polyhydroxyalkanoates (PHA) are a class of biodegradable polyesters produced by a number of bacterial species (Chodak, 2008). In the cytoplasm of bacterial cells, a few surface-associated proteins are present on the surface of PHA granules, including PHA synthases and phasins. Phasins are amphiphilic proteins located at the PHA-cytoplasm interface that form stable monolayers with a nearly ordered orientation on PHA granules (Dong et al. 2010; You et al. 2011; Prieto et al. 2016; Tarazona et al. 2019). From a biotechnological perspective, these proteins have a high affinity toward the polymeric surface, and they can be usefully employed for immobilization of target proteins by engineering genetic fusions. Indeed, a number of groups have demonstrated successful immobilization of proteins onto PHA supports (Moldes et al., 2004; Peters et al., 2006; Seo et al., 2016; Wong et al., 2018; Yang et al., 2015) through fusion with PHA-binding proteins, including phasins. However, most of these studies focused on in vivo immobilization of enzymes onto PHA granules accumulated inside cells, with drawbacks including uncontrolled size distribution and limited surface area.
In the present study, we investigated the use of electrospun PHA nanofibers as an alternative support to immobilize phasin-fused recombinant enzymes. A lipase discovered via metagenomics analysis was immobilized onto electrospun polyhydroxybutyrate (PHB) nanofibers using phasin from Aeromonas hydrophila (PhaP) as an affinity fusion tag. Due to a high surface-area-to-volume ratio, the amount of enzyme that could be loaded onto PHB nanofibers was >100-fold greater than that which could be loaded onto PHB granules. Additionally, the immobilized lipase exhibited markedly higher stability and activity, allowing for repeated use without significant loss of activity. Our results demonstrate that PHB nanofibers can be used as a highly efficient and versatile support to immobilize enzymes when used in combination with a phasin tag.