Page 83 - Biodegradable Polyesters

3.8 Application Opportunities of Bacterial Polyhydroxyalkanoates 61

3.8.4
Application of PHA Granule Surface Proteins

There are several kinds of proteins found to be located on the surface of in vivo
PHA granules [17]. Among these proteins, PHA synthase has been employed
to covalently immobilize β-galactosidase on the in vivo PHA granule surface by
fusing β-galactosidase to the N-terminus of PHA synthase from Pseudomonas
aeruginosa. Both the substrate binding domain of PHA depolymerase and the
N-terminal domain of PhaF phasin or PhaP (PHA granule-associated protein)
have been used to anchor fusion proteins to PHA microbeads. The auto-regulator
protein PhaR is confirmed to have two separate domains that bind to DNA and
PHB, respectively, and PhaR can be adsorbed to various types of hydrophobic
polymers, such as PHB, poly(L-lactide), polyethylene, and polystyrene, mainly by
nonspecific hydrophobic interactions [64].
It appears that the proteins locating on the in vivo PHA granule surface could
be potential affinity tags for protein purification. Among the nonspecific PHA
granule-binding protein phasin as a hydrophobic affinity tag appears to be the
most attractive because of its richness compared with others.
A novel protein purification method based on phasin, a pH-inducible self-
cleaving intein and PHA nanoparticles has been developed. Genes for the target
proteins to be produced and purified were fused to genes of intein and phasin,
and the genes were jointly overexpressed in vivo,for example, in E. coli.The
fused proteins containing the target protein, intein, and phasing produced by
the recombinant E. coli were released together with all other E. coli proteins
via a bacterial lysis process. They were then adsorbed in vitro to the surfaces
of the hydrophobic polymer nanoparticles incubated with the cell lysates. The
nanoparticles attached to the fused proteins were concentrated via centrifugation.
Following that, the reasonably purified target protein was released by self-cleavage
of the intein and separated with nanoparticles by a simple centrifugation process.
This system was successful to produce and purify the enhanced green fluorescent
protein (EGFP), maltase binding protein (MBP), and β-galactosidase. Using this
technique allows the production and purification of high value added proteins in
a continuous way with low cost [65].

3.8.5
Production of Tailor-Made Biopolyester Nanoparticles and Potential Applications

Extensive studies in the molecular biology and physiology of the bacterial
biopolyester synthesis has been made in the last decade. Previous research
activities were mainly aimed at the biotechnological production of the extracted
and semicrystalline thermoplastic polyester materials. Recently, however, it has
been recognized that the intracellular polyester inclusions or those derived from
in vitro synthesis can be considered as natural bionanoparticles.
These spherical nano- and sub-micro-particles are now being explored with
respect to their commercial potential. Bionanoparticles can be conceived