Modification of Polyhydroxyalkanoates (PHAs) - Polyhydroxyalkanoate (PHA) Based Blends, Composites and Nanocomposites - page 170

substrate for PHA biosynthesis. On the other hand, during in vitro PHA
synthesis, PHA synthase and the hydrophobic chain of the polymer were
covalently bound resulting in the formation of an amphiphilic block co-
polymer in which PHA synthase can further be modified through protein
engineering to improve the protein-PHA copolymer. 165,166 It has been re-
ported that the first 100 N-terminal amino acid residues of PHA synthase can
be removed without affecting the enzyme's activity. 167 Thus, additional lig-
and fusion can still be carried out without affecting the protein's native
catalytic activity. Incorporating a tumor specific ligand, RGD4C, with PHA
synthase helps PHA nanoparticles adhere more effectively on MDA-MB 231
breast cancer cells. 168 Modifications of the PHA nanoparticles were de-
scribed by Kim et al. 165 where the use of PHA synthase from Ralstonia
eutropha H16 for the formation of protein-PHB copolymer micelles
(involving polymerization and self-assembly of 3-hydroxybutyryl-CoA (3HB-
CoA)) in aqueous solution at room temperature resulted in a hydrated shell.
Concomitantly, hydrophobic drug molecules are integrated into the core of
the shell. On the other hand, Lee et al. 168 proposed a coupling between PHB
emulsion and enzymatic protein functionalization. Then growing PHB tail
chain arising from the action of PHA synthase that was fused earlier with
RGD4C and this arrangement was able to form hydrophobic interactions
with the surface of the PHB nanoparticle emulsion containing drug mol-
ecules. As the tail chains grew continuously, the surface of the nanoparticles
was covered with RGD4C peptide-PHB copolymer that targets breast cancer
cells. Paik et al. 166 showed that PHB nanoparticles can also bind to a solid
surface. In their study, PHA synthase was fused to a His-tag (10x-histidine)
expressed in a recombinant E. coli synthesizing PHB to produce a protein-
polymer hybrid with His-tag end-functionality. The His-tag bound tightly
with a Ni 21 -nitrilotriacetic acid (Ni-NTA) derivatized solid surface (silicone
or agarose). All these modification methods are excellent demonstrations of
synthesizing a wide variety of protein functionalized PHAs with novel
properties.
PHA surface erosion is normally initiated at micro-holes on the PHA's
surface that allow enzymes and water molecules to adhere to the film sur-
face, commencing the hydrolytic process. 169,170 At the beginning, water
molecules enter the amorphous regions within the film, which triggers the
enzyme-catalyzed hydrolysis of the ester bond. 171 During the hydrolysis
process, the roughness of the PHA film increases over time. 172 While the
enzyme is thought to attack mainly the amorphous region, wide angle X-ray
diffractography revealed a decline in the crystalline peak after 22 hours of
reaction, which indicated that the crystalline region was also hydrolyzed by
lipase after the amorphous region was eroded. 172 Selective surface erosion
using PHB depolymerase helps to liberate the 3HB monomers from the
surface of P(3HB-co-4HB) and leave the undegradable 4HB monomers on the
polymer surface. 173 Different polymer constitutions on the polymer surface
may exhibit different degradation properties. Among the parameters that
contribute to the enzymatic degradation of PHA films are the molecular

d n 2 r 4 n g | 1

.