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.