Biomedical Engineering Reference
In-Depth Information
lowing this investigation, Foster
et al.
[59] studied with more details the presence
of depolymerase in P(3HO) granules. The results revealed that (i) the
P. oleovorans
depolymerase remains active in isolated P(3HO) inclusion bodies; (ii) this enzy-
matic activity occurs in association with the organized protein lattice that encom-
passes the stored P(3HO) polymer; and (iii) depolymerase activity of isolated native
P(3HO) granules showed a maximum degradation rate of 1.17 mg h
−
1
at an
optimum pH of 9.
Phasins (PhaP, structural gene
pha
P) are defi ned as a protein class that has a
similar role as oleosins of triacylglycerol inclusions in seeds and pollen of plants
[60]. These proteins have been identifi ed from
A. eutrophus
(nowadays,
C. necator
)
[61] and
Rhodococcus ruber
[62] , and have been shown to infl uence the size of
intracellular PHA granules. Phasins have been suggested to have a role as
amphiphilic proteins (substance readily soluble in polar as well as in nonpolar
solvents) in the interphase between the hydrophilic cytoplasm and the hydro-
phobic PHA molecule, and may also act as an anchor for the binding of
other proteins such as PHA synthase [62]. Inside this group, the GA13 protein,
studied by Schembri and coworkers [63], can be noted. These authors studied
Acinetobacter
RA3123, RA3849, RA3757, RA3762, and
Escherichia coli
DH5
to
identify the 13-kDa PHA (GA13) granule-associated protein as the protein encoded
by a structural gene located within the
Acinetobacter pha locus
. When the P(3HB)
granule samples were examined, a protein of approximately 13 kDa (GA13) was
identifi ed in all four
Acinetobacter
P(3HB)-positive strains, shown to be the
product of the
phaP
AC
(gene encoded PhaP protein by
Acinetobacter
) gene in strain
RA3849 and revealing the presence of two regions containing predominantly
hydrophobic and amphiphilic amino acids. This may be involved in the anchoring
of this protein into the phospholipid monolayer surrounding the PHA granule.
E. coli
showed a small amount of accumulated P(3HB), however, with large-sized
granules. This fact may be related to the poor expression of GA13 protein in this
strain, which is able to reduce PHA synthase (PhaC, structural gene
pha
C)
activity.
Stuart
et al.
[64] reported that different microorganisms (
Ralstonia eutropha
,
Norcadia corallina
,
Azotobacter vinelandii
, and pseudomonads species) showed a
different granule protein boundary in electron microscopy and SDS-PAGE. These
results can be very interesting for biotechnologists since they indicate a natural
“packaging” of polymer during biosynthesis. Maehara
et al.
[65] proposed a struc-
tural PHA granule model determining the distinct target DNA sequences for PhaR
(a repressor protein which regulates PHA synthesis) binding and demonstrated
that PhaR binds not only to DNA but also to PHA. These results confi rm that
PhaR has bifunctional characteristics, namely, binding abilities toward both PHA
and DNA. PhaR is the fi rst protein that interacts directly with PHA polymer. The
recognition requirement for this interaction was relatively nonspecifi c, because
PhaR bound to all forms of P(3HB) - crystalline, amorphous, and 3HB oligomers.
PhaR recognizes and binds directly to the PHA polymer chains being synthesized,
and then the expression of PhaP is initiated at the onset of dissociation of PhaR
from an upstream element for
pha
P. During the elongation of PHA polymer
α
