 |
INTRODUCTION |
Many microorganisms synthesize poly(3-hydroxybutyrate)
(P(3HB))1 intracellularly and
accumulate it in granular inclusion bodies as a carbon and energy
reserve (1-3). They also synthesize other types of polyhydroxyalkanoic
acids (PHA) in the form of homopolyesters, copolyesters, or polyester
blends. More than 100 different monomer units are known to be
incorporated into the polymer chain (3). Accordingly, various kinds of
copolyesters are expected when a bacterium is grown on the mixtures of
different precursors. However, even if any two copolyesters happen to
have similar overall ratios of the comonomer units, the microstructure
(e.g. local sequence of comonomers) of the two copolyesters
may be quite different, resulting in totally different physicochemical
properties, e.g. thermal transition temperatures,
biodegradability, and mechanical strength (2). Such microstructural
heterogeneity is usually caused by the different assimilation rate of
the monomeric precursors into the bacterium. The width and randomness
of comonomer distribution in the copolymers may depend on the
availability of the comonomers in the form of their CoA thioesters and
the specificity of the PHA synthase.
Most studies of PHA degradation have been performed on extracellular
depolymerases using "denatured" crystalline PHA as substrates. Many
extracellular PHA depolymerases have been isolated and characterized in
terms of their biochemical and molecular biological properties (6). The
rate of enzymatic degradation of microbial PHA is determined by the
specificity of the depolymerases and physicochemical factors such as
the crystallinity and monomer composition of the PHA (2, 6). However,
little is known about the bacterial intracellular PHA depolymerases,
probably because of the difficulty in the isolation and purification of
these enzymes without loss of activity. It is well known that the
"native" PHA inclusions are completely amorphous (7-9). They are
readily crystallized when isolated from cells. Stabilization of the
isolated amorphous granular substrates to prevent them from
crystallizing could be another difficulty in the study of the
intracellular depolymerases because they exhibit little activity
with crystalline PHA (6).
In a previous study, we found that the polyester synthesized by
Hydrogenophaga pseudoflava from glucose plus
-butyrolactone, using a one-step cultivation, was not a homogeneous
random copolymer but a mixture of 3HB-rich and 4HB-rich chains (10).
This microstructural heterogeneity is considered to result from the
different assimilation rate of the two precursors. However, the 3HB-3HV
copolyesters prepared from glucose and -valerolactone were
relatively homogeneous copolyesters. PHA producing microorganisms
usually have intracellular PHA depolymerases. Therefore, any difference
in the microstructural heterogeneity among intracellular polymers,
which even have a similar composition of monomers, is expected to be
strongly reflected in the intracellular degradation reaction by the
depolymerase(s) because of its probable high substrate specificity.
According to our recent study (11), P(4HB) homopolymer was found to be not degraded in H. pseudoflava cells, whereas P(3HB)
homopolymer was almost completely degraded within 24 h in the
presence of ammonium sulfate. This totally different susceptibility of
the two homopolymers to intracellular degradation suggested that
performing an in situ kinetics study of the intracellular
degradation reactions might enable us to obtain information on the
microstructures of the 3HB-4HB copolymer inclusions as well as the
specificity of the intracellular PHA depolymerase.
High resolution NMR spectroscopy has been used as a powerful tool to
determine the microstructural heterogeneity (specifically, heterogeneity in local sequences) of the microbial copolyesters (2, 4,
5). It is possible to assign the 13C resonances present in
local dyad, triad, and tetrad sequences. Therefore, a comparative
analysis of the NMR spectra obtained before and after degradation of a
copolyester in cells should enable us to determine the local sequence
specific degradation of the PHA by the intracellular depolymerase.
In this study, we analyzed the microstructure of three different types
of copolymers in H. pseudoflava cells, which included 3HB-3HV, 3HB-4HB, and 3HV-4HB copolymers, by applying first order reaction kinetics to the intracellular degradation. An additional set
of polymers in the form of blend-type polymers (a mixture of two
different homopolymers) 3HB/3HV, 3HB/4HB, and 3HV/4HB were also
analyzed for comparison. Each blend-type polymer had a similar ratio of
two component monomers to that in the corresponding copolymer. All data
obtained from the analyses of intracellular degradation kinetics, NMR,
and thermal transition correlated well with one another. These analyses
showed that the H. pseudoflava intracellular depolymerase is
the most active against 3HB-rich sequences and the least active against
4HB-rich sequences.
| |
EXPERIMENTAL PROCEDURES |
Organism and Culture Media--
Strain ATCC 33668 of H. pseudoflava was purchased from the American Type Culture
Collection. Inocula were grown in 5-ml test tubes containing
nutrient-rich media (1% yeast extract, 1.5% nutrient broth, and 0.2%
ammonium sulfate) for 18 h. The following two media were used in
the cultivation of the bacterium for PHA accumulation: 1) LB medium (10 g of trypton, 5 g of yeast extract, 10 g of NaCl in 1 liter
of distilled water) and 2) modified PHA synthesis mineral medium (11,
12). All growth experiments were performed under aerobic conditions in
a temperature-controlled shaker (Korea Instrument Co., Seoul, Korea) at
35 °C and 190 rpm.
P(3HB) Homopolymer Accumulation--
The precultured (nutrient
broth grown) cells were transferred to a PHA synthesis mineral medium
containing 12 g/liter of glucose and cultivated for 30 h. Cells
were harvested by centrifugation in a Beckman J2-HS (rotor JA-10, 6000 rpm for 10 min).
3HB-4HB Polymer Accumulation--
Two types of 3HB-4HB polymers,
differing in their structural heterogeneity, were accumulated in cells.
The cells containing a 3HB-4HB copolyester were prepared by
transferring the precultured cells to a PHA synthesis mineral medium
containing 10 g/liter of glucose and 2 ml/liter of -butyrolactone
and cultivating them at 35 °C for 72 h. The harvested cells
contained 43 wt % (by dry cell weight) of the copolyester composed of
41 mol % 3HB and 59 mol % 4HB. The 3HB/4HB blend-type polymer, having
a ratio of monomer unit composition similar to that of the 3HB-4HB
copolyester just described, was accumulated in cells by cultivating the
nutrient broth grown preculture in an LB medium containing 4 g/liter of glucose for 22 h and then transferring first step grown cells to a
second step PHA synthesis mineral medium containing 1 ml/liter of
-butyrolactone and cultivating for 18 h. The blend-type polymer in the cells was composed of 45 mol % 3HB and 55 mol % 4HB, and the
cells contained 24 wt % of PHA.
The 3HB-4HB copolymer having more than 90% of 4HB was accumulated by
cultivating the LB grown cells in a PHA synthesis mineral medium
containing 2 ml/liter of -butyrolactone plus 0.6 g/liter of ammonium
sulfate for 48 h. The cells containing 3HB-free P(4HB) homopolymer
were prepared using a three-step cultivation method (11).
3HB-3HV Polymer Accumulation--
To accumulate 3HB-3HV
copolymers in cells, the precultured cells were transferred to an LB
medium and cultivated for 22 h, and then the LB grown cells were
incubated in a PHA synthesis mineral medium containing only ammonium
sulfate (0.6 g/liter) for 12 h to remove the residual P(3HB). The
cells were transferred to a PHA synthesis medium containing
-valerolactone (2 ml/liter) and ammonium sulfate (0.6 g/liter) and
cultivated for 18 h. The harvested cells contained 24.5 wt % of
PHA composed of 21 mol % 3HB and 79 mol % 3HV.
The cells containing a copolymer with a higher level of 3HB[57 mol % 3HB and 43 mol % 3HV] were prepared by cultivating the precultured
cells in a PHA synthesis mineral medium containing glucose (10 g/liter), -valerolactone (2 ml/liter), and ammonium sulfate (0.6 g/liter) for 24 h. The blend-type polymer composed of 3HB- and
3HV-rich chains, having a similar level of comonomer mole ratio
([3HB]:[3HV] =55: 45), was accumulated by cultivating the
precultured cells in an LB medium containing glucose (4 g/liter) for
23 h and then cultivating the LB grown cells in a PHA synthesis mineral medium containing -valerolactone (3 ml/liter) and ammonium sulfate (0.6 g/liter) for 24 h. The cells contained 30.4 wt % of
PHA.
3HV-4HB Polymer Accumulation--
For the preparation of the
cells containing a 3HV-4HB copolymer, the LB grown cells were
transferred to a PHA synthesis mineral medium containing only ammonium
sulfate (0.6 g/liter) and cultivated for 15 h, and then the cells
were transferred to a PHA synthesis mineral medium containing
-butyrolactone (1 ml/liter), -valerolactone (1 ml/liter), and
ammonium sulfate (0.6 g/liter) and cultivated for 36 h. The
recovered cells contained 18 wt % of PHA composed of 42 mol % 3HV and
58 mol % 4HB. In the preparation of the cells containing a 3HV/4HB
blend-type polymer having a similar level of the comonomer ratio to
that in the copolymer just described, the LB grown cells were
transferred to a PHA synthesis mineral medium containing
-butyrolactone (2 ml/liter) and ammonium sulfate (0.6 g/liter) and
cultivated for 30 h. Then the cells were transferred to a
third-step PHA synthesis mineral medium containing -valerolactone (2 ml/liter) and ammonium sulfate (0.6 g/liter) and cultivated for 18 h. The cells contained 26 wt % of PHA blend composed of 43 mol % 3HV
and 57 mol % 4HB. Only less than 1-2 mol % of 3HB was detected in
the PHAs.
Monitoring the Intracellular Degradation of Various Polyesters in
Carbon-free Media--
Cells containing various types of polyesters
were recovered by centrifugation and transferred to a carbon-free
mineral medium (a medium containing the same mineral as a PHA synthesis
mineral medium) containing ammonium sulfate (1.0 g/liter) (or not
containing ammonium sulfate for the control experiment). The cells were
incubated under an aerobic shaking condition at 35 °C and 190 rpm. 5 ml of the culture was removed every 5 h to analyze the medium and the cells. The amount of NH4+ remaining
in the medium was measured by the Nessler reagent method (12). The
monomer unit composition of the PHA remaining in cells was determined
by gas chromatography with a Hewlett Packard HP5890A gas chromatograph
equipped with a Carbowax 20M column and a flame ionization detector
(11, 12). Total protein content in cells was determined by the Bradford method.
Polyester Isolation and Characterization--
Polyesters were
extracted from an appropriate amount of cells that had been dried
overnight at 50 °C under a vacuum. Extraction was performed with hot
chloroform in a Pyrex Soxhlet apparatus for 6 h. The concentrated
solvent extract was precipitated in rapidly stirred cold methanol. The
isolated polymers were dried overnight under a vacuum at the ambient
temperature and then weighed. Quantitative determinations of the
monomer units in the polyesters were performed by gas chromatography,
as described above, and by 1H NMR with a Bruker-DRX 500 MHz
spectrometer (10, 12). The 125-MHz 13C NMR spectra were
recorded at 25 °C on a CDCl3 solution of polyester (70 mg/ml) with a 10-µs pulse width, 5-s pulse repetition, 25000 Hz
spectral width, 64,000 data points, and 5,000-10,000 accumulations. Tetramethylsilane was used as an internal chemical shift standard. The
integration of the split spectral signals was performed with standard software.
Thermal transitions of the polyesters were measured under nitrogen
purging by using a TA differential scanning calorimeter (DSC) (DuPont
model 2100, DSC V4.0B) equipped with a data station. The heating rate
was 10 °C/min. The scanning range was between 
100 and
200 °C.
The change in the molecular weight of P(3HB) in cells during
degradation was monitored by isolating the polymer remaining in cells
and purifying it by reprecipitation in methanol and then by measuring
the intrinsic viscosity of the polymer dissolved in chloroform. The
viscosity of the polymer solutions (1-5 mg/ml) was measured at
30 °C using a capillary viscometer of the Cannon Fanske type
(capillary number 50), which was immersed in a constant temperature
bath. The polymer solution was filtered with a 0.45-µm Gelman
membrane filter before viscosity measurement. The molecular weight of
P(3HB) was calculated using the Mark-Howink equation [
] = kMa, where the constant k is equal to
7.7 × 10
5 (cm3/g) and a is
equal to 0.82 in chloroform (13).
| |
RESULTS |
Intracellular Degradation of P(3HB) Homopolymer in H. pseudoflava--
As shown in Fig. 1,
90% of the P(3HB) accumulated in cells was degraded after the cells
were transferred to and cultivated for 24 h in a carbon-free
mineral medium containing ammonium sulfate, of which the initial
concentration was 1.0 g/liter. The initial dry biomass was 3.69 g/liter, and the content of P(3HB) in cells was 69 wt % by dry cell
weight. The NH4+ in the medium was
consumed exponentially and almost depleted within 24 h during
degradation. The optical density at 660 nm of the medium was
significantly decreased from 9.63 at 0 h to 4.00 at 30 h and
finally reached 3.58 at 60 h. Thus, despite the significant
consumption of NH4+, a substantial
increase in both OD and biomass was not observed. As a control
experiment, the cells containing P(3HB) were also incubated in a medium
free from both carbon and nitrogen. Only 10% of degradation was
observed after 24 h of cultivation, and the extent of degradation
never exceeded 15% after 60 h (data not shown).
View larger version (21K):
[in this window]
[in a new window]
|
Fig. 1.
P(3HB) degradation in H. pseudoflava in the presence of ammonium sulfate (1.0 g/liter). The cells initially contained 69 wt % of P(3HB)
homopolymer.
|
|
As a first order of approximation, the degradation rate of P(3HB) in
cells was analyzed in terms of first order kinetics (Fig. 2). The final value of [3HB
unit]inf at infinite time was approximated by averaging
the last three values in Fig. 1 (hereafter referred to as the
steady-state method). The same approximation was applied to the other
degradation process, in which a steady-state period was reached within
60 h, leading to no further degradation caused by limitations. A
linear regression analysis of the data in Fig. 2 resulted in the
degradation rate constant, k1 of 0.123 h1. The correlation coefficient r2
of the fitted data points in the figure was 0.985 at a 95% confidence interval. The half-life for the degradation of P(3HB) was 5.6 h,
and 95% of the P(3HB) in the cells was degraded after 60 h. A
further increase in the concentration of ammonium sulfate, up to 2.0 g/liter, resulted in the same k1 value of 0.123 h1 (r2 = 0.975) as with the
concentration of 1.0 g/liter, despite slightly less degradation (85%
degradation) at the increased level of nitrogen (data not shown).
View larger version (11K):
[in this window]
[in a new window]
|
Fig. 2.
First order kinetics analysis of P(3HB)
degradation in H. pseudoflava in the presence of
ammonium sulfate (1.0 g/liter). [3HB]0 is the
initial concentration of P(3HB). [3HB]t is the concentration
of P(3HB) at time t. [3HB]inf was approximated
with the averaged value for the last three concentrations in the
steady-state period between 50 and 60 h in Fig. 1 (the
steady-state method, see text for the details).
|
|
Intracellular Degradation of 3HB-4HB Polymers--
The time course
for the degradation in cells containing a blend-type 3HB/4HB polymer
initially composed of 45 mol % 3HB/55 mol % 4HB is shown in Fig.
3. The concentration of the 4HB unit in
the cell suspension was constant throughout the degradation period of
60 h, which indicated that the 4HB units in the blend-type polymer
were not degraded. In contrast, the 3HB units in the cells disappeared
according to the first order degradation process. The apparent first
order degradation rate constant, k1 for P(3HB) in the blend polymer, was determined to be 0.109 h1
(r2 = 0.982) for the initial 45 h
degradation, in which 70% of the total 3HB units was degraded (data
not shown). In this plot, the same final-value approximation as in Fig.
2 was made.
View larger version (18K):
[in this window]
[in a new window]
|
Fig. 3.
Degradation of 3HB/4HB blend-type polymer in
H. pseudoflava in the presence of ammonium sulfate
(1.0 g/liter). The initial composition of the polymer was 45 mol
% 3HB and 55 mol % 4HB. The cells initially contained 24 wt % of
polymer.
|
|
Contrary to the case of the 3HB/4HB blend-type polymer, the 4HB units
in a copolymer composed of 41 mol % 3HB and 59 mol % 4HB were found
to be degraded under the same incubation condition as in the blend-type
polymer. A first order regressive fitting for the data points over
60 h of cultivation resulted in the apparent k1 values 0.049 h1 and 0.015 h1 for 3HB- and 4HB units, respectively. Their
correlation coefficients, r2, were 0.976 and
0.984, respectively (Fig. 4). In this
plot, the two values at infinite time, [3HB unit]inf and
[4HB unit]inf were assumed to be zero (hereafter referred
to as the initial rate method) because of continuing degradation of the
copolymer even after 60 h. This means that 4HB units can be
degraded if they are copolymerized with 3HB units. It is interesting to
note the remarkable difference in the k1 values
of the two monomer units in the copolymer. With a real random
copolymer, a similar or the same value of k1 is
expected for the adjoining two monomers because the overall reaction is
controlled by the degradation of the monomer unit showing a slower
rate. The dissimilarity in k1 values for the two
monomer units in a copolymer sample thus suggests the existence of two
different types of polymer chains, 3HB-rich and 4HB-rich chains. Such
probable microstructural heterogeneity, as in the 3HB-4HB copolymer,
may be related with the different assimilation rate of the two monomer
precursors, glucose and -butyrolactone, in PHA accumulation by the
bacterium (10).
View larger version (13K):
[in this window]
[in a new window]
|
Fig. 4.
First order kinetics analysis of the 3HB-4HB
(mole ratio, 41:59) copolymer degradation in H. pseudoflava
in the presence of ammonium sulfate (1.0 g/liter).
[3HB]inf or [4HB]inf was assumed to be 0 because of continuing degradation even after 60 h of incubation
(the initial rate method, see text for the details).
|
|
The suggested microstructural heterogeneity in the 3HB-4HB copolymer
was detected in detail at a molecular level by using high resolution
13C NMR spectroscopy, in which each resonance peak is
usually shifted by the presence of a different neighboring monomer
unit, and thus every shifted resonance has information on the local
monomer sequence of the copolyester (2, 4, 5). For the copolymers
composed of 3HB and 4HB monomer units, the sequences of dyad, triad,
and tetrad were assigned assuming a first order Markovian random
copolymerization in cells (4). The expanded spectra of carbonyl carbon
resonance at 169-173 ppm are shown in Fig.
5. The upper two spectra are for the
samples recovered before and after degradation of the 3HB/4HB copolymer
having an initial composition of 41 mol % 3HB and 59 mol % 4HB (the
samples in Fig. 4). The lower two spectra are for the blend-type
polymer having the initial overall composition of 45 mol % 3HB and 55 mol % 4HB (the samples in Fig. 3). As expected from the preparation
procedure of each polyester, resonances ascribable to the sequences 43, 434, and 334 were smaller in the blend-type polymer than that in the
copolymer. This clearly demonstrates that the blend-type polymer is
principally a mixture of P(3HB) and P(4HB), the mixture containing a
minor amount of P(3HB-co-4HB). For both the copolymer and blend-type
polymer, the relative intensities of two absorption signals associated
with 333 and 433 triads were significantly reduced after degradation,
whereas the intensities of the signals for 4HB-rich sequences
(e.g. 444 and 344) were relatively enhanced. Generally, the
sequences having more 4HB units were more slowly degraded, as seen from
the comparison of their signal intensities.
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 5.
125 MHz 13C NMR absorption
signals in the carbonyl region for the polymers isolated before and
after degradation in H. pseudoflava cells. The
spectra in a are for the 3HB-4HB (initial mole ratio, 41:59)
copolymer, and the spectra in b are for the 3HB/4HB (initial
mole ratio, 45:55) blend-type polymer. The 3HB unit was designated 3 and the 4HB unit was designated 4. The carbonyl-region signals exhibit
dyad (33, 34, 43, and 44) and triad (33*3, 43*3, 44*4, etc.) sequence
distribution of the polymers.
|
|
In a similar manner, sequence-dependent changes in the
absorption for the other six 13C resonance-peaks in the
region of 15-70 ppm occurred before and after degradation of the two
types of 3HB/4HB polymers. In the methylene carbon CH2 (2)
associated with the 3HB unit (Fig. 6),
the relative intensity of the 33*33 sequence peak decreased, whereas
the intensities for the other three tetrad sequences (43*34, 43*33, and
33*34) and 4HB-inserted triad sequences (43*4 and 33*4) were strongly
enhanced after 60 h degradation. As in the case of the split
carbonyl absorption described, the more 4HB units in a sequence, the
higher the relative intensity of the sequence after degradation. Thus,
the degradation of tetrad sequences occurred in the decreasing rates of
33*33 > 43*33 and 33*34 > 43*34. In the blend-type polymer,
the signals associated with the 4HB-containing tetrad sequences were
only barely detectable, as expected. However, the intensity of the
signal associated with the triad 43*4 sequence was more strongly
enhanced than that of 33*4 sequence after degradation in a similar
fashion to the case of the copolymer. The methyl carbon
CH3(4) in 3HB was assigned to the resonances around 20 ppm.
Four peaks associated with triad sequences 43*4, 43*3, 33*4, and 33*3
were identified (spectra not shown). In the blend polymer the two triad
sequences 43*3 and 33*4 showed little absorption, which agreed with the
observation of the carbonyl and methylene carbon described above. For
both types of polymers, the intensity of the signal associated with the
33*3 sequence decreased, whereas that with the 43*4 sequence increased
after degradation, as expected. The other signals associated with
carbons such as CH(3), CH2(6), CH2(7), and
CH2(8) showed similar sequence-dependent
intensity changes to that observed for the four carbons described (data not shown), except for the fact that the carbons CH2(6),
CH2(7), and CH2(8) in the 4HB unit of the
blend-type polymer showed little change in the ratios of the related
local sequence-associated 13C NMR signals. This must be due
to the preferential degradation of P(3HB) in the blend-type polymer.
However, in the 3HB-4HB copolymer, a significant change in the ratios
of the signal intensities of the sequences related to the three 4HB
carbons was noticed after degradation, which additionally demonstrates
that the initial copolymer was a mixture of 3HB-rich and 4HB-rich
chains, not a homogeneous random copolymer.
View larger version (15K):
[in this window]
[in a new window]
|
Fig. 6.
125 MHz 13C NMR absorption
signals for CH2(2) associated with 3HB unit in the polymers
isolated before and after degradation in H. pseudoflava
cells. The CH2(2) group is marked by
italicizing the CH2(2) in the chemical formula
in the figure. The spectra in a are for the 3HB-4HB (initial
mole ratio, 41:59) copolymer, and the spectra in b are for
the 3HB/4HB (initial mole ratio, 45:55) blend-type polymer. The
absorption signals exhibit triad (33*4 and 43*4) and tetrad (33*33,
33*34, 43*33, and 43*34) sequence distribution of the polymers.
|
|
The structural heterogeneity of the 3HB/4HB blend-type polymer was also
confirmed by analyzing the two samples obtained before and after
degradation using differential scanning calorimetry. The blend-type
polymer exhibited two melt transitions at 45 and 160 °C,
respectively (Fig. 7). Two different
polymers are usually immiscible because of unfavorable interactions
between them (14). Therefore, after melting a mixture of two
crystallizable polymers, the component polymers crystallize in separate
domains. Melting of the resulting immiscible polymer blend thus reveals
two melting endotherms. P(3HB) homopolymer melts at 175 °C, and
P(4HB) homopolymer melts at 55 °C (11). The exhibition of the two
melting transitions (Fig. 7) indicates that the polymer sample was a
blend, not a copolymer. The significant decrease in the relative area
of the endothermic peak at 160 °C, observed after degradation,
additionally supports that 3HB-rich polymer preferentially degraded in
the 3HB/4HB blend-type polymer. However, the 3HB-4HB copolymer showed a
single endotherm at 52 °C both before and after degradation, indicating the absence of the high degree of structural heterogeneity as in the blend-type polymer.
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 7.
Differential scanning calorimetric traces of
the polymers isolated before and after degradation in H. pseudoflava cells. The traces in a are for
the 3HB-4HB (initial mole ratio, 41:59) copolymer, and the traces in
b are for the 3HB/4HB (initial mole ratio, 45:55) blend-type
polymer.
|
|
Intracellular Degradation of 3HB-3HV Polymers--
Degradation
experiments were carried out for two types of cells, one containing a
copolymer composed of 57 mol % 3HB/43 mol % 3HV and the other a
blend-type polymer composed of 55 mol % 3HB/45 mol % 3HV. Both type
of cells almost completely consumed the added
NH4+ (1.0 g/liter) at 60 h of
incubation. 24% of PHA remained in the copolymer cells, and 18% of
PHA remained in the blend-type polymer cells. In addition, the amounts
of the remaining P(3HB) and P(3HV) were 5.5 and 30.6%, respectively,
for the blend-type polymer. Contrary to the case of the 3HB/4HB
blend-type polymer, degradation of the second monomer 3HV unit in the
3HB/3HV blend-type polymer cells was observed. First order rate plots
for the degradation of the two types of 3HB-3HV polymers also showed
good linear correlation over the entire degradation period (data not
shown). A linear regression analysis for the degradation of the
copolymer resulted in the k1 value of 0.026 h1 for the 3HB unit (r2 = 0.949)
and that of 0.022 h1 for the 3HV unit
(r2 = 0.975) at a 95% confidence interval. In
this calculation, the initial rate method was employed because of
continuing degradation of the copolymer after 60 h. The
k1 values for the 3HB and 3HV units in the
blend-type polymer were determined to be 0.077 h1 with
r2 = 0.998 and 0.055 h1 with
r2 = 0.991, respectively, by the steady-state
method. Thus, the degradation of the 3HV unit was slower than that of
the 3HB unit. The similar degradation rate constants for both monomer
units in the 3HB-3HV copolymer indicate the almost complete random
nature of the copolymer, containing no long blocked chains as well as being less heterogeneous than in the 3HB-4HB copolymer. In addition, the higher degradation rate of the 3HB-rich chains in the blend-type polymer may imply that the H. pseudoflava intracellular
depolymerase is more specific to 3HB units than to 3HV units.
First order degradation kinetics analysis was also applied to the
3HB-3HV copolymer containing a high level of 3HV, poly (21 mol % 3HB-co-79 mol % 3HV). Fifty-one % of the
NH4+ added at the concentration of 1.0 g/liter remained unconsumed and 37 wt % of PHA remained not degraded
after 60 h of incubation (data not shown). The
k1 values for 3HB and 3HV units were calculated to be 0.099 h1 (r2 = 0.992) and
0.083 h1 (r2 = 0.992),
respectively, by the steady-state method
Local sequence-specific degradation of 3HB-3HV polymers was also
investigated by using 125 MHz 13C NMR spectroscopy. The
sequence assignment was made according to the method of Doi and
co-workers (5). The carbonyl absorption region contained the
information on the distribution of dyad sequences, VV, BV, and BB (Fig.
8). For the sample before degradation,
the absorption intensity of the doublet signal at 169.3 ppm associated with VB and BV was very low in the blend-type polymer (55 mol % 3HB/45
mol % 3HV). However, in the copolymer having a similar composition (57 mol % 3HB/43 mol % 3HV), it was almost comparable with the sum of the
VV and BB signal intensities indicative of a rather homogeneous random
copolymer, considering the similar 3HB/3HV monomer ratio in the sample
before degradation. For both polymers, the relative intensity of the
signal associated with BB was significantly reduced after 60 h of
degradation. This again shows the higher specificity of the
intracellular depolymerase to 3HB units than to 3HV units. The other
dyad absorption signals associated with the carbons CH(3), CH(7),
CH2(2), CH3(4), and CH3(9) showed a
similar trend (data not shown). An increase in the relative signal
intensity of the BV sequence and a decrease in that of the BB sequence
were generally observed for the carbons CH(3), CH2(2), and
CH3(4) during degradation. For the carbons CH(7) and
CH3(9) exhibiting VB and VV signals, the signal ratio of VB
to VV decreased, as expected. A spectral analysis for the triad
sequences associated with CH2(6) and CH2(8) in
the 3HV unit revealed that an increase in the number of 3HV units in a
sequence caused the relative intensity of the corresponding signals to increase during degradation (Fig. 9). The
change is significant enough to distinguish between BVB and VVB or BVV
sequences of the copolymer (Fig. 9). However, in the blend-type
polymer, the change in resonance absorption of the carbons CH(7),
CH2(6), and CH2(8) in the 3HV unit was
negligible, probably because of the rather slower degradation of the
3HV-rich polymer chains giving rise to the absorption signals.
Actually, 95% of the 3HB unit in the blend-type polymer was degraded,
but 31% of the 3HV unit remained not degraded after 60 h of
incubation.
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 8.
125 MHz 13C NMR absorption
signals in the carbonyl region of the polymers isolated before and
after degradation in H. pseudoflava cells. The
spectra in a are for the 3HB-3HV (the initial mole ratio,
57:43) copolymer, and the spectra in b are for the 3HB/3HV
(the initial mole ratio, 55:45) blend-type polymer. The 3HB unit was
designated B and the 3HV unit was designated V. The carbonyl-region
signals exhibit dyad (BB, BV, VB, and VV) sequence distribution of the
polymers.
|
|
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 9.
125 MHz 13C NMR absorption
signals for CH2(6) (a) and
CH2(8) (b) in the 3HV unit of the polymers
isolated before and after degradation in H. pseudoflava
cells. The spectra are for the 3HB-3HV (the initial mole
ratio, 57:43) copolymer. The positions of the two carbons
CH2(6) and CH2(8) are marked by
italicizing them in the chemical formula in the
figure.
|
|
The DSC trace of the 3HB-3HV (the initial molar ratio, 57:43) copolymer
before degradation was very similar to that of the polymer remaining
after degradation (data not shown). However, the DSC trace of the
3HB/3HV blend-type polymer exhibited two strong melting endothermic
peaks of a similar area at 110 and 165 °C, ascribable to the melting
of P(3HV) (15) and P(3HB), respectively. Similar to the case of the
3HB/4HB blend-type polymer, the presence of the two endothermic peaks
indicates the existence of two separate crystalline domains associated
with 3HV-rich P(3HB-co-3HV) and P(3HB). The relative area of the
endothermic peak for P(3HB) was significantly reduced after degradation
with the area for the 3HV-rich polymer being remarkably increased.
Intracellular Degradation of 3HV-4HB Polymers--
For direct
comparison of the relative degradation rate between 3HV units and 4HB
units, we prepared two types of 3HV-4HB polymers, a blend-type polymer
(P(3HV):P(4HB) = 43:57, in terms of monomer mole ratio) and a poly
(42 mol % 3HV-co-58 mol % 4HB) copolymer. Both polymers contained
less than 1-2 mol % of 3HB. Only 30% of the
NH4+(1.0 g/liter) was consumed for
60 h of incubation. The amount of the remaining 3HV unit was 85%
for the copolymer and 47% for the blend-type polymer. This indicates
that the introduction of the 4HB unit into the 3HV-containing polymer
retarded the degradation of the polymer. No degradation of 4HB unit was
observed for the two polymers. The k1 values for
the 3HV unit were determined to be 0.076 h1
(r2 = 0.987) and 0.079 h1
(r2 = 0.979) for the blend-type and copolymer
sample, respectively, by the steady-state method. This result clearly
shows again that the intracellular depolymerase has no activity
against the 4HB unit in P(4HB).
| |
DISCUSSION |
NMR Parameter D as an Index of the Microstructural Heterogeneity of
PHA--
Table I shows that the
intracellular PHA degradation is a function of the NMR microstructure
of the inclusion polyesters. A NMR structure parameter D is
defined as the ratio
FBBFVV/FBVFVB for 3HB-3HV polymers, where FBB is the fraction
of the dyad sequence 3HB-3HB and the other Fij
values are defined similarly (2, 4, 5). Each value for the fraction
Fij was determined by measuring the intensity of the
related split 13C NMR signal and normalizing it in terms of
the total summed area for the carbon under consideration. Similarly,
for 3HB-4HB polymers, D is defined as the ratio
F33F44/F34F43,
where F34 is the fraction of the dyad sequence
3HB-4HB. Before degradation, the initial D values for the
two blend-type polymers, 3HB/4HB and 3HB/3HV, were 35.7 and 56.6, respectively. A random copolymer has the D value of 1.0 (2,
4, 5). The high D value for each blend-type polymer means
that the polymer in cells was a mixture of the corresponding homopolymer and copolymers (e.g. P(3HB) and 4HB-rich
P(3HB-co-4HB) or 3HV-rich P(3HB-co-3HV)). Inhomogeneity of the
blend-type polymers in cells was also ascertained from the significant
change in monomer composition before and after degradation as well as
from the significant difference in the disappearance rate
(k1) of the two comprising monomer units (Table
I). Considering the low D values close to unity, the two
P(3HB-co-3HV) copolymers were thought to be random copolyesters. The
randomness of comonomer distribution is also indicated by the little
change in their monomer composition before and after degradation and
the similar apparent k1 values of the two
monomer units getting close to each other. However, with the 3HB-4HB
copolymer having the initial comonomer ratio of 41 mol % 3HB:59 mol % 4HB, the calculated D value was 3.7 for the sample before
degradation. A remarkable change in monomer composition was also
observed after degradation of the copolymer. The large difference in
apparent k1 value for the two comonomers, 3HB
and 4HB, is another indication of the heterogeneity of the polymer.
Even after degradation, the remaining polymer exhibited a high
D value of 3.3, reflecting still a heterogeneous state of
the polymer. A careful examination of the DSC trace for the polymer
before degradation reveals at least two phase structures associated
with an sharp endotherm at around 50 °C and a broad endotherm around
130 °C (Fig. 7). Thus, the parameter D can be used for the
determination of the structural heterogeneity of bacterial
polyesters.
Determination of Relative PHA Depolymerase Specificity by in Situ
Degradation Kinetics Analysis--
It is interesting to note the fact
that the degradation of the inclusion polymers (e.g. P(3HB))
in the cells occurs by first order kinetics in the presence of
additional ammonium sulfate (1.0 g/liter). The little degradation in
the absence of additional nitrogen may be due to the metabolic
suppression which prevents the flow of carbon derived from P(3HB)
or/and a limited number of the depolymerase. The addition of
nitrogen thus releases the suppression by synthesizing proteins, etc.
Actually, the content of total protein in cells increased up to almost
twice the initial amount when the added ammonium (1.0 g/liter) was
nearly consumed after 20 h of incubation (Fig. 1), whereas without
additional ammonium, the protein content was relatively constant
through the incubation (data not shown).
Little is known about the mechanism of the intracellular PHA
degradation (6). The degradation rate may presumably be governed by the
concentration of a polymer substrate as well as the substrate specificity and concentration of the PHA depolymerase(s). Although it remains to be proved, the depolymerization of PHA by the
depolymerase is considered to be the rate-determining step in the
intracellular PHA degradation pathway. The proteins such as PHA
synthases, PHA depolymerases, phasins (16-18), and other
granule-associated proteins are known to be mostly located on the
surface of intracellular PHA granules (16-19). The enzyme proteins
constitute only a relatively small amount of the granule-associated
proteins, whereas the phasin protein, contributing approximately 3-5%
(w/w) of the total cellular protein, is the predominant protein present
on the surface of the granules. Thus, PHA granules are covered with the
granule-associated proteins and phospholipids. The internal part of the
granules is almost free from proteins (19). The polymer chains within granules are in an amorphous state but slightly ordered (9). Thus, the
native amorphous granules are stabilized by the presence of the surface
layer that protects the granules from aggregating. So, during
degradation, the granules kept the original spherical shape unaltered,
which was confirmed in our electron microscopic investigation (data not
shown), and polymer chains are not expected to be solubilized into the
cytoplasm because of their high hydrophobicity. Furthermore, the
measurement of molecular weight during degradation of P(3HB) in cells
showed that the molecular weight of P(3HB) was 983,000, 512,000, and
577,000 at 0, 10, and 20 h of incubation, respectively. As seen in
Fig. 1, 10 and 20 h of incubation correspond to 64 and 82% P(3HB)
degradation, respectively. Polydispersity of P(3HB) polymer in
Ralstonia eutropha is known to be constant during
degradation (20). The modest (less than 2-fold) decrease in the
molecular weight of P(3HB) and the constant polydispersity of P(3HB)
during degradation may thus imply that the depolymerization is an
exo-type reaction (20). Therefore, the degradation started from the
surface and continued without significant perturbation of the overall
granular structure.
It was reported that P(3HB) depolymerase activity in R. eutropha increased during P(3HB) synthesis (21). It was suggested that phasins might control the amount of the catalytically active proteins bound to the granules (16). The first order dependence of the
degradation reaction, with respect to the concentration of polymer
substrate, may suggest a relatively constant number density of the
depolymerase on the granular surface throughout the degradation, thus
implying apparently zero order with respect to the concentration of the
depolymerase. Only some of the depolymerase, additionally expressed
in the presence of additional ammonium, is expected to be initially
bound to the surface of granules, and the "excess" soluble
depolymerase in the cytosol, if any, might have a minor contribution to
the degradation of granular PHAs.
Depending on the type of polymer (a copolymer or blend), the first
order degradation rate constant for each monomer unit has a
characteristic value (Table I). The calculation was carried out by two
procedures, the steady-state method for the case in which a
steady-state was reached and the initial rate method for the case where
the degradation was still going on even after 60 h. Generally, the
steady-state method resulted in higher k1 values than the initial rate method. Intracellular PHA degradation can be
inhibited by various limiting factors in a cell such as a shortage of
some limiting nutrients, encapsulation of degradable PHA in outer
nondegradable PHA (e.g. P(3HB) core surrounded with outer P(4HB) layer) in blend-type polymers (22). Thus, the limiting factor
inhibiting PHA degradation could be different from one type of polymer
in cells to another type of polymer. In addition, the number density of
the depolymerase on the surface of PHA inclusions may depend on the
specific type of polymer. Therefore, the comparison of apparent
k1 values between different types of polymers is
meaningless, especially for the case in which they were determined by
the steady-state method. This ambiguity is further clarified when we
consider the percentage of PHA remaining after 60 h of degradation
(Table I). But, the comparison between the k1
values in a cellular polymer system is meaningful because they were
determined under the same degradation and limiting conditions.
Furthermore, for all types of blend-type polymers or copolymers, the
relative magnitude of k1 values in a cellular
polymer system agree well with the degradability of each local sequence
determined from the NMR analysis. Therefore, the relative
k1 value determined for a cellular polymer
system could be used as a measure for the determination of relative
substrate specificity of the depolymerase. In addition, the
dissimilarity in k1 values of the two different
monomer units in a cellular copolymer discloses its microstructural
heterogeneity (see above). Therefore, without isolation and
purification of the depolymerase, it has been possible to measure the
relative specificity of the enzyme as well as the microstructural
heterogeneity of polyesters in cells by applying a first order
degradation kinetics analysis to intracellular PHA degradation.
Local Sequence-dependent Intracellular PHA
Degradation--
It is well known that the sequence distribution of
comonomers in bacterial PHA is close to a statistically random
distribution (4, 5). Analysis of all 13C NMR signals for
the polymers in this study showed that the decrease and increase in the
intensities of the signals assigned to every local sequence that
occurred after degradation were internally consistent with one another
as well as among different carbons (data not shown) (23). This strongly
supports the view that the sequence distribution is random. All these
may imply that the supplying level of comonomers in the form of CoA is
more critical than the substrate specificity of PHA synthase in PHA
synthesis (24).
The local sequence-dependent degradation suggests that the
depolymerization step is the rate-determining one in intracellular PHA
degradation. In other words, the depolymerization rate is governed by
the specificity of the enzyme toward the local monomer sequence such as
dyad, triad, and tetrad. At present, we do not know anything about the
mechanism by which the local sequences are recognized and how they are
processed in the catalytic site. Such a local sequence dependence only
suggests that the H. pseudoflava intracellular PHA
depolymerases are not the processive enzymes that cleave the polymer
chain sequentially. In addition, from the lack of intracellular
degradability of P(4HB) or 4HB-rich P(3HB-co-4HB) we can surmise that
the presence of a chiral carbon in the ester backbone and/or the
oxidation position located three bonds away from the carbonyl is
essential for the hydrolysis reaction by the depolymerase (11). In
conclusion, if any two different monomers are copolymerized in a cell
and the 13C NMR signals of the copolymer synthesized
exhibit splittings because of the neighboring monomer units, the
relative specificity of the intracellular depolymerase against the
local sequences could be determined by analyzing PHA before and after
degradation using 13C NMR spectroscopy without purification
of the enzyme and native substrates.
and
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Tel.: 82-591-751-5942;
Fax: 82-591-759-0187; E-mail: scyoon@nongae.gsnu.ac.kr.
