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Applied and Environmental Microbiology, June 1999, p. 2762-2764, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Removal of Endotoxin during Purification of
Poly(3-Hydroxybutyrate) from Gram-Negative Bacteria
Sang Yup
Lee,1,*
Jong-il
Choi,1
Kyuboem
Han,2 and
Ji Yong
Song2
Department of Chemical Engineering and
BioProcess Engineering Research Center, Korea Advanced Institute of
Science and Technology, Yusong-gu, Taejon
305-701,1 and Biotech Research
Institute, LG Chem Research Park, Yusong-gu, Science Town, Taejon
305-380,2 Korea
Received 4 January 1999/Accepted 4 March 1999
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ABSTRACT |
Poly(3-hydroxybutyrate) (PHB) was produced by cultivating several
gram-negative bacteria, including Ralstonia eutropha,
Alcaligenes latus, and recombinant Escherichia
coli. PHB was recovered from these bacteria by two different
methods, and the endotoxin levels were determined. When PHB was
recovered by the chloroform extraction method, the endotoxin level was
less than 10 endotoxin units (EU) per g of PHB irrespective of the
bacterial strains employed and the PHB content in the cell. The NaOH
digestion method, which was particularly effective for the recovery of
PHB from recombinant E. coli, was also examined for
endotoxin removal. The endotoxin level present in PHB recovered by 0.2 N NaOH digestion for 1 h at 30°C was higher than 104
EU/g of PHB. Increasing the digestion time or NaOH concentration reduced the endotoxin level to less than 1 EU/g of PHB. It was concluded that PHB with a low endotoxin level, which can be used for
various biomedical applications, could be produced by chloroform extraction. Furthermore, PHB with a much lower endotoxin level could be
produced from recombinant E. coli by simple NaOH digestion.
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TEXT |
Poly(3-hydroxybutyrate) (PHB), the
best known member of the polyhydroxyalkanoates (PHA), is an energy
and/or carbon storage material synthesized and intracellularly
accumulated by numerous microorganisms, usually when an essential
nutritional element such as nitrogen, phosphorous, oxygen, sulfur, or
potassium is limited in the presence of excess carbon source (1,
9). PHB is a partially crystalline thermoplastic and possesses
material properties similar to those of polypropylene (6).
It has been drawing much attention as a good candidate for
biodegradable and/or biocompatible plastic material which can be
produced from renewable raw materials (10). Possible
applications of PHB include the following: packaging films and
containers, biodegradable carriers for controlled chemical and/or drug
release, disposable items, surgical pins and sutures, wound dressings,
and bone replacements (6, 9). For biomedical applications,
it should be noted that the degradation product of PHB,
D(
)-3-hydroxybutyrate, is a common intermediate
metabolite present in all higher animals, including humans
(14). Furthermore, a low-molecular-weight PHB consisting of
100 to 200 monomer units has also been detected in a relatively large
amount in human plasma (14). Therefore, it is highly
plausible that implanting highly purified PHB in mammalian tissues or
parenteral injection of PHB microspheres will not cause any problems.
Several gram-negative bacteria, including Ralstonia
eutropha, Alcaligenes latus, and recombinant
Escherichia coli, have been employed for the efficient
production of PHB (11). These gram-negative bacteria can
release endotoxin (pyrogen), which is in the form of
lipopolysaccharides, from the outer cell wall (12).
Endotoxin causes fever if introduced into the bloodstream of humans or
other animals. As we experienced with recombinant pharmaceutical
proteins, a safe parenteral drug (or material) should have an endotoxin level below a set limit. According to the U.S. Food and Drug
Administration guideline, the upper limit of the pyrogen level is 5.0 endotoxin units (EU)/kg (body weight) per injection. Since PHB is
mostly efficiently produced by gram-negative bacteria as described
above, the endotoxin levels present in the purified PHB should be
examined for biomedical applications. However, until now, there has
been no study of the removal of the endotoxins during the purification of PHB from bacteria. In the present study, PHB was produced by R. eutropha, A. latus, and recombinant E. coli, and the removal of the endotoxins during the purification of
PHB was examined. Two different methods were employed for the recovery
of PHB from these bacteria. Based on these results, the strategies for
producing PHB with a low endotoxin level are discussed.
R. eutropha NCIMB11599 (7), A. latus
DSM1123 (16), and recombinant E. coli strain
XL1-Blue(pJC4) harboring the A. latus PHA biosynthesis genes
(5) were used for the production of PHB. The bacteria were
grown for the production of PHB in a chemically defined medium with
their respective best carbon sources: glucose for R. eutropha (7) and recombinant E. coli
(5) and sucrose for A. latus
(16), as previously described. The PHB concentration was
determined by gas chromatography (HP5890; Hewlett-Packard, Wilmington,
Del.) with benzoic acid as an internal standard (2). PHB
content was defined as the percentage of the ratio of PHB to cell dry weight.
Two different methods, chloroform extraction (13) and
NaOH digestion (4), were used to recover PHB from the
bacterial cells. Endotoxin levels were semiquantitatively determined by the Limulus amebocyte lysate-gel clot method (Associates of
Cape Cod, Inc., Falmouth, Mass.). The endotoxin levels are reported as
the average values of three repeated experiments.
Chloroform extraction has been widely used to recover PHB with a high
degree of purity without polymer degradation during the recovery
(13). Therefore, PHB samples were first recovered by
chloroform extraction of bacterial cells with different PHB contents.
Cells were collected by centrifugation at 4,000 × g for 20 min at 25°C and were washed with hot acetone for 20 min. After
being dried, the cells were mixed with 50 volumes of chloroform for
48 h at 30°C. A clear PHB solution was recovered by
centrifugation; this was followed by polishing filtration. Finally,
pure PHB was obtained by nonsolvent precipitation (five times the
volume of chloroform) and filtration. The nonsolvent used was a mixture of methanol and water (7:3 [vol/vol]). The endotoxin levels present in PHB recovered from R. eutropha, A. latus, and
recombinant E. coli were almost the same and were <10 EU/g
of PHB (Table 1). The PHB content of the
cells did not affect the removal of the endotoxin level during the
purification of PHB by chloroform extraction. From these data, the
chloroform extraction method was found to be efficient not only for the
recovery of PHB with a high degree of purity but also for the removal
of the endotoxins of gram-negative bacteria. However, large quantities
of toxic and/or volatile chloroform were required for the recovery of
PHB because the polymer solution containing more than 5% (wt/vol) PHB
was very viscous and the processing operation became difficult. To
overcome these problems, several other recovery methods have been
developed. One of these recovery methods, NaOH digestion, has several
advantages (4): (i) NaOH is inexpensive and much more
environmentally friendly, (ii) a high degree of purity (>98%) of PHB
can be obtained, and (iii) there is no degradation of PHB during
recovery.
In particular, PHB could be most efficiently recovered by NaOH
digestion from recombinant E. coli because these cells, upon accumulating a large amount of PHB, became fragile (4). Cell broth was washed with distilled water and centrifuged. Cells were resuspended in distilled water, and NaOH solutions were added. After
the cells were mixed with NaOH solution to digest non-PHB cellular
material, PHB granules were separated from the aqueous fraction
containing cell debris by centrifugation at 2,500 × g for 20 min. The PHB granules recovered were gently rinsed with distilled water, recentrifuged, and air dried. Right after the NaOH
digestion, the pH of the digestion solution was adjusted to pH 7.0 with
endotoxin-free HCl solution for the corrective determination of the
endotoxin level. Recombinant E. coli cells with a PHB
content of 69% were digested with 0.2 N NaOH at 30°C for 1 h,
and PHB with a purity of 97% was recovered. The endotoxin level
present in purified PHB was higher than 104 EU/g of PHB.
The original endotoxin level of E. coli cells was higher
than 107 EU/g of PHB. In order to possibly reduce the
endotoxin level, the effects of digestion time and NaOH concentration
were examined. The endotoxin level could be decreased to 1 EU/g of PHB
when the digestion time was increased to 5 h (Fig.
1). The purity of PHB recovered was also
increased to 98% by increasing the digestion time. The endotoxin
levels present in PHB recovered with NaOH solutions of different
concentrations are shown in Fig. 2. When the NaOH concentration was higher than 2 N, the endotoxin level present
in PHB was less than 1 EU/g of PHB. The endotoxin level present in PHB
obtained by NaOH digestion from recombinant E. coli was
lower than that in PHB recovered by chloroform extraction method. This
is consistent with the previous finding that NaOH was quite effective
for inactivating endotoxins (15).

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FIG. 1.
Endotoxin levels present in PHB recovered by 0.2 N NaOH
digestion at 30°C for various durations. Recombinant E. coli cells (50 g of cells [dry weight]/liter) with a PHB content
of 69% were used.
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FIG. 2.
Endotoxin levels present in PHB recovered by digestion
with various concentrations of NaOH at 30°C for 2 h. Recombinant
E. coli cells (50 g of cells [dry weight]/liter) with a
PHB content of 69% were used.
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One of the important validation issues to be considered for the
biomedical products made by recombinant E. coli and other gram-negative bacteria is the endotoxin removal (15). This
study showed for the first time that the endotoxin levels present in PHB, a completely biodegradable polymer produced by several
gram-negative bacteria, can be within the allowable limit when
purified by chloroform extraction. For small-scale application,
therefore, the chloroform extraction method will be useful. For the
large-scale production of endotoxin-free PHB, however, a new,
more-efficient recovery method must be used (3). In this
sense, NaOH digestion method can be efficiently used for the recovery
of endotoxin-free PHB produced by recombinant E. coli. Other
endotoxin-free PHAs, such as
poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (17) and
poly(3-hydroxyhexanoate-co-3-hydroxyoctanoate) (8) can also
be produced by using recombinant E. coli by NaOH digestion.
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ACKNOWLEDGMENTS |
This work was supported by the Ministry of Science and Technology
and by LG Chemicals, Ltd.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Chemical Engineering and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Korea. Phone: 82-42-869-3930. Fax: 82-42-869-8800. E-mail: leesy{at}sorak.kaist.ac.kr.
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Applied and Environmental Microbiology, June 1999, p. 2762-2764, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.