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Previous Article | Next Article 
Applied and Environmental Microbiology, September 2001, p. 3970-3975, Vol. 67, No. 9
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.9.3970-3975.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Direct Incorporation of Glucosamine and
N-Acetylglucosamine into Exopolymers by
Gluconacetobacter xylinus (=Acetobacter xylinum)
ATCC 10245: Production of Chitosan-Cellulose and
Chitin-Cellulose Exopolymers
Jin W.
Lee,1
Fang
Deng,2
Walter G.
Yeomans,3
Alfred L.
Allen,3
Richard A.
Gross,4,* and
David
L.
Kaplan5,*
Dong-A University, Hadan 2-dong, Sha-gu, Pusan 604-714, Korea1; Department of Chemistry,
University of Massachusetts, Lowell, Massachusetts
018542; Biotechnology Division, U.S.
Army Natick RD & E Center, Natick, Massachusetts
017603; Polytechnic University,
Brooklyn, New York4; and Department of
Chemical & Biological Engineering, Biotechnology Center, Tufts
University, Medford, Massachusetts 021555
Received 19 April 2001/Accepted 17 June 2001
 |
ABSTRACT |
Gluconacetobacter xylinus (=Acetobacter
xylinum) ATCC 10245 incorporated
2-amino-2-deoxy-D-glucose (glucosamine) and
2-acetamido-2-deoxy-D-glucose (N-acetylglucosamine), but not
3-O-methyl-D-glucose or
2-deoxy-D-glucose into exopolymers. Incorporation
was confirmed by gas chromatography with and without mass spectrometry,
Fourier transform infrared, and 1H nuclear magnetic
resonance. The average molar percentage of glucosamine and
N-acetylglucosamine in the exopolymers was about 18%.
| |
INTRODUCTION |
Cellulose,
(1-4)-linked-
-D-glucan, is a major structural component
of the cell walls of higher plants (8) and also generated as an exopolymer by some microorganisms (5, 6,
21). Structurally related polysaccharides such as chitin
[(1-4)-linked 2-acetamino-2-deoxy--D-glucose] occur as
a major cuticular or skeletal component in all arthropods, some
invertebrata, and some fungi (11). Chitosan
(2-acetamido-2-deoxy--D-glucopyranose) is the fully or
partially deacetylated form of chitin and is found in the cell walls of
some fungi, such as Mucor rouxi (2-4, 13).
The biosynthesis of polysaccharides has traditionally been studied
using unmodified simple sugars such as glucose and sucrose or complex
carbon sources such as wheat gluten and molasses (14). Alternatively, microbial mutants have been used to manipulate biopolymer molecular weight, yield, and main chain or branch
composition (12, 22). Glucose-rich polysaccharides such as
cellulose and curdlan have been postbiosynthetically derivatized by
nonspecific chemical means to change physical or biological properties
(18, 23) and by selective chemical modification under
homogeneous conditions (19). Disadvantages to these
approaches can include include low yields, side reactions, the use of
toxic solvents, and purification requirements. Therefore, it was
desirable to explore direct, in vivo incorporation of simple sugar
analogs as building blocks for polysaccharides.
Glucose derivatives may be particularly effective as novel carbon
sources because glucose is a main component in many polysaccharides and
therefore a logical target for direct polymerization by the microorganism. We previously reported the direct incorporation of
glucose-related sugars 3-O-methyl-D-glucose
(3-O-methylglucose) and
2-acetamido-2-deoxy-D-glucose
(N-acetylglucosamine) into curdlan (15), the
direct incorporation of specific fatty acid pendant groups on a main
chain polysaccharide such as emulsan (9, 10, 24), and the
modification of metabolic pathways involved in exopolymer
synthesis in the case of pullulan (17).
In the present work, investigations were conducted by selective feeding
experiments to determine the flexibility of the polymerization system
in Gluconacetobacter xylinum ATCC 10245. Specifically, we
report on the ability of this strain to incorporate glucosamine and
N-acetylglucosamine into exopolymers.
| |
MATERIALS AND METHODS |
Bacterial strains and culture conditions.
G. xylinum
(=Acetobacter xylinum) ATCC 10245 was obtained from the American
Type Culture Collection. Glucose (>99.5% purity), 3-O-methyl-D-glucose, glucosamine (>99.0%
purity), N-acetylglucosamine, 2-deoxy-D-glucose
(2-deoxyglucose), and t-butanol (>99.0% purity) were all
purchased from Sigma Chemical Co. (St. Louis, Mo.). Methanol (>99.9%
purity) was purchased from J. T. Baker Co. (Phillipsburg, N.J.);
pyridine (>99.9% purity) was purchased from Aldrich Chemical Co.
(Milwaukee, Wis.); acetic anhydride (>97.0% purity) and acetyl chloride (98% purity) were purchased from Fisher Scientific (Fair Lawn, N.J.); and Tri-Sil Z (25% [vol/vol]
N-trimethylsilyimidazole in pyridine) for silylation was
purchased from Pierce (Rockford, Ill.). The medium (Y3-3) for the
production of cellulose was based on that of Johnson and Neogi
(13a). Carbon sources included glucose, 3-O-methylglucose, glucosamine,
N-acetylglucosamine, and 2-deoxyglucose. Cultures were
incubated for 7 to 14 days with either glucose or one of the glucose
analogs under the same conditions as for the starter cultures.
Purification and characterization of expolymers.
Cell
cultures of G. xylinum with the formed exopolymer
were centrifuged at 12,000 × g for 40 min at 4°C
(Fig. 1). After removing the supernatant,
the precipitate was added to an equivalent volume of distilled water,
and the mixture was stirred for 10 min at room temperature. The
resulting viscous solution was centrifuged at 12,000 × g for 40 min at 4°C. After removing the supernatant, the
precipitate was added to an equivalent volume of distilled water, the
mixture was stirred for 10 min and sonicated for 5 min at a sonication
level of 5 using a Cell Disruptor 350 (Branson Sonic Power Co.,
Danbury, Conn.) at room temperature. The resulting solution was
centrifuged at 12,000 × g for 40 min at 4°C. After removing the supernatant, the precipitate was added to an equivalent volume of distilled water, and the mixture was stirred for 10 min. The
resulting viscous solution was centrifuged at 12,000 × g for 40 min at 4°C. After removing the supernatant, the
precipitate was repeatedly washed with water, acetone, and ether to
remove contaminants. The washed precipitate was freeze-dried to obtain the total exopolymer produced by G. xylinum ATCC
10245.
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FIG. 1.
Procedure to purify and fractionate exopolymers
synthesized with glucosamine. AIGGCP, acetic acid-insoluble
glucose-coglucosamine copolymer; ASGGCP, acetic acid-soluble
glucose-coglucosamine copolymer; DW, distilled water.
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Further fractionation was accomplished by the addition of 150 ml of
aqueous acetic acid (10%, vol/vol) to 300 mg of the exopolymer synthesized with glucosamine. The resulting aqucous acetic acid solutions were centrifuged at 6,000 × g for 20 min.
The precipitate was washed with distilled water and freeze-dried. This
fraction was termed exopolymer AmG-1 and represented the acetic
acid-insoluble glucose-coglucosamine copolymer. The supernatant was
neutralized with sodium hydroxide and centrifuged at 15,000 × g for 30 min. The precipitate was also washed with distilled
water and freeze-dried. This fraction was termed exopolymer
AmG-2 and represented the acetic acid-soluble
glucose-coglucosamine copolymer.
Further fractionation was accomplished by addition of 450 ml of
distilled water to 300 mg of the exopolymer synthesized with N-acetylglucosamine. The resulting solutions were
centrifuged at 6,000 × g for 20 min. The precipitate
was washed with distilled water and freeze-dried. This fraction was
termed exopolymer AcG-1 and represented the water-insoluble
glucose-co-N-acetylglucosamine copolymer. The
supernatant was dialyzed against deionized water using dialysis tubing
with a molecular weight cutoff of 3,000 and then lyophilized. This
fraction was termed exopolymer AcG-2 and represented the
water-soluble glucose-coglucosamne copolymer.
The yield of the exopolymer by G. xylinum was
determined by direct weighing after purification of
exopolymers. Gas chromatographic (GC) analysis after
methanolysis of exopolymers and subsequent trimethylsilylation
was used to determine the composition of carbohydrates in the
exopolymers (7). Samples (approximately 0.5 mg)
were dried over phosphorous pentoxide under vacuum in 600-µl
Reacti-Vials (Pierce). Methanolic hydrogen chloride (300 µl) prepared
by mixing methanol and acetyl chloride in a ratio of 20:1 (vol/vol) was added, and the tubes were sealed with Teflon-lined septum caps. The
contents were vortexed and heated at 70°C for 24 h with
stirring. t-Butyl alcohol (30 µl) was then added to each
tube before evaporating the contents using a stream of dry oxygen-free
nitrogen at room temperature.
For incubation on N-acetylglucosamine, complete
N-acetylation of amino sugars was ensured by re-N-acetylation by
addition of methanol (150 µl), pyridine (15 µl), and acetic
anhydride (15 µl) to the above tubes. After standing at room
temperature for 30 min, the solutions were evaporated to dryness by the
use of a dry oxygen-free nitrogen stream at room temperature, followed by vacuum over phosphorous pentoxide. After this thorough drying, Tri-Sil Z (100 µl) was added, and each mixture was stirred for 2 h at room temperature. GC analyses were performed on a Hewlett Packard
(HP) gas chromatograph, model 5890 series II, equipped with a flame
ionization detector and HP model 7673 injector. The column was a 30-m
by 0.32-mm inner diameter fused silica with cross-linked 0.25-µm 5%
phenylmethyl silicon liquid phase (Supelco). Dry oxygen-free nitrogen
(2.9 ml/min flow rate) was used as the carrier gas at 10 lb/iu2 head pressure using a temperature program (140°C
for 2 min, then increasing at 8°C per min up to 260°C). The
injector was purged for 0.8 min after injection.
To quantitate the repeat unit composition of products, response factors
were generated from the relative values of GC peak areas using an
equimolar mixture of pure sugars and myoinositol as the internal
standard. GC/mass spectrometry (GC/MS) analyses were performed on an HP
gas chromatograph, model 5890 series II, also equipped with HP model
7673 injector and coupled to a mass selective detector (HP 5971 series). The capillary column was a cross-linked 5% phenylmethyl
silicone-fused silica (HP Ultra MS 5; 30 m by 0.25 mm; film
thickness, 0.33 µm). Dry oxygen-free helium (0.8 ml/min flow rate)
was used as the carrier gas using a temperature program (140°C for 2 min, then increasing at 8°C per min up to 260°C). Sample volumes of
1 µl were injected, and the injector was purged for 0.6 min after
injection. Mass spectra were compared based on computer HP computer
database, W search version 1/10/99C, and compared with data from methyl
esters of known structures. Fourier transform infrared (FTIR) spectra
were recorded with a Perkin-Elmer 1720 spectrometer (16 scans;
resolution, 2 cm
1) over KBr pellet. The polysaccharide
sample (2 mg), which was dried previously at 50°C under reduced
pressure, was manually well blended with 100 mg of KBr powder. The
powder mixture of the polysaccharide sample and potassium bromide was
then desiccated overnight at 50°C under reduced pressure prior to
FTIR measurement. Proton nuclear magnetic resonance
(1H-NMR) spectra were recorded at 250 MHz using a Bruker
model AMX-250. Chemical shifts in parts per million (ppm) were reported
with trace amount of acetone (
= ppm) as an internal reference.
The polysaccharide sample (2%, wt/vol) was dissolved in phosphoric acid-d3 (85 wt.% solution in D2O,
99+ atom% D; Aldrich) at room temperature for a minimum of 4 days
prior to NMR measurement. The instrumental parameters were as follows:
temperature, 300 K; pulse width, 7.8 µs; 32,000 data points,
3.18-s acquisition time; 1-s relaxation delay; and 32 transients.
| |
RESULTS |
Incorporation of monosaccharides into exopolymers.
Glucose as the main carbon source resulted in the highest production of
exopolymer among the carbon sources used under the conditions
of this experiment (Table 1).
3-O-Methylglucose and 2-deoxyglucose as carbon sources were
not utilized effectively by G. xylinum ATCC 10245. The yield
of exopolymers purified from the culture with
glucosamine and N-acetylglucosamine was not
as high as with glucose, but higher than that obtained from either 3-O-methylglucose or 2-deoxyglucose. To determine the
composition of exopolymers purified from cultures, samples were
depolymerized and derivatized as described earlier. The
exopolymers fractionated by acetic acid (10%, vol/vol) and
distilled water were also analyzed. GC chromatograms corresponding to
exopolymer purified from cultures with glucose,
glucosamine, and N-acetylglucosamine are
shown in Fig. 2. For all the products,
the chromatograms show the major component to be glucose, identified by
the peak retention times and peak area ratios of the 
and anomers (Fig. 2).
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FIG. 2.
GC chromatograms of trimethylsilylated (TMS) sugar
components of (A) exopolymer made with glucose, (B) 10% acetic
acid-insoluble fraction of exopolymer made with
glucosamine (the molar ratio of glucose to glucosamine
was 6.0 to 1.0), (C) 10% acetic acid-soluble fraction of
exopolymer made with glucosamine (the molar ratio of
glucose to glucosamine was 0.6 to 1.0), (D) water-insoluble
fraction of exopolymer made with
N-acetylglucosamine (the molar ratio of glucose to
N-acetylglucosamine was 6.2 to 1.0), and (E)
water-soluble fraction of exopolymer made with
N-acetylglucosamine (the molar ratio of glucose to
N-acetylglucosamine was 0.8 to 1.0). Glu, glucose;
N-AcGlu, N-acetylglucosamine.
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|
Initial GC identification of glucosamine found in the
exopolymers purified from cultures grown on glucosamine
and fractionated by acetic acid was based on the elution time of
N-acetylglucosamine due to re-N-acetylation of amino
sugars during sample preparation for GC (13.05 to 13.16, Fig. 2B and C)
(7). The average molar percentage of glucosamine
in the exopolymer synthesized with glucosamine was
19%. After fractionation of the glucosamine-incorporated
exopolymer, the molar ratios of glucose to
glucosamine in the acetic acid-insoluble exopolymer
(Fig. 2B) and that in the acetic acid-soluble exopolymer (Fig.
2C) were 6.0:1.0 and 0.6:1.0, respectively (the molar percentages of
glucosamine in these exopolymers were 14 and 63%,
respectively). GC identification of
N-acetylglucosamine found in the exopolymers purified from cultures grown on N-acetylglucosamine
and fractionated by distilled water (DW) was also based on the
elution time (12.99 to 13.05 min) (Fig. 2D and E). The
average molar percentage of N-acetylglucosamine
repeat units in the exopolymer synthesized with
N-acetylglucosamine was 18%. After fractionation of
the N-acetylglucosamine-incorporated exopolymer by
DW, the molar ratios of glucose to
N-acetylglucosamine in the
water-insoluble exopolymer (Fig. 2D) and that in the
water-soluble exopolymer (Fig. 2E) were 6.2:1.0 and 0.8:1.0,
respectively (the molar percentages of
N-acetylglucosamine in these exopolymers were 14 and 56%, respectively).
The presence of the trimethylsilyl (TMS)
N-acetylglucosamine derivative in the GC/MS
chromatogram of exopolymers with glucosamine and
N-acetylglucosamine was confirmed by positive
identification with reference spectra in the GC/MS data bank (Fig. 3A
and B). The FTIR spectra of
polysaccharides derived from different carbon sources are shown in Fig.
4. As can be seen, the FTIR spectra of
10% acetic acid-soluble fraction of exopolymer synthesized with glucosamine as the carbon source and water-soluble
fraction of exopolymer synthesized with
N-acetylglucosamine as the carbon source exhibited
similar features as in the chitosan and chitin spectra
(17). Specially, the absorbance bands of the amide I (1,650 cm1) and amide II (1,550 cm1)
signals were clearly observed (Fig. 4D and E). The strong absorption at
1,650 cm1 in Fig. 4D indicated that the acetic
acid-soluble fraction of exopolymer isolated from the culture
grown on glucosamine as the main carbon source had some
glucosamine repeating units acetylated. Evidence for the
production of chitosan- and chitin-like exopolymers from this
cellulose-producing system also came from another related experiment in
which the water-soluble fraction of exopolymer synthesized with
N-acetylglucosamine as the carbon source was treated
with 40% (wt/vol) aqueous sodium hydroxide solution at 25°C for 4 days. FTIR spectra of the chitin-like exopolymer before (Fig.
4A) and after alkaline treatment (Fig. 4B) also showed the expected
reduction of the acetyl group content after treatment. This result
correlated well with the decreased absorption signal intensity of the
amide I (1,650 cm1) and amide II (1,550 cm1) bands, although quantitative comparisons were
difficult.
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FIG. 3.
GC chromatograms of trimethylsilylated (TMS) sugar
components of (A) 10% acetic acid-soluble fraction of
exopolymer made with glucosamine (the molar ratio of
glucose to glucosamine was 0.6 to 1.0) and (B) water-soluble
fraction of exopolymer made with
N-acetylglucosamine (the molar ratio of glucose to
N-acetylglucosamine was 0.8 to 1.0). Glu, glucose;
N-AcGlu, N-acetylglucosamine.
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FIG. 4.
FTIR spectra of (A) cellulose, (B) chitin, (C) chitosan
(DS = 0.1, measured by 1H-NMR), (D) 10% acetic
acid-soluble fraction of exopolymer made with
glucosamine as the carbon source, and (E) water-soluble
fraction of exopolymer made with
N-acetylglucosamine as the carbon source.
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A number of solvents used to dissolve cellulose, chitin, or chitosan
were tested for their suitability as an NMR solvent for the
samples, including CF3COOD/D2O,
DC1/D2O, NaOD/D2O, D2O, dimethyl sulfoxide-d6, DCOOD, 10% (wt/wt)
CD3COOD/D2O, 50% (wt/wt)
N-methyl morpholine oxide/dimethyl
sulfoxide-d6, and 85% (wt/wt)
D3PO4/D2O. An 85% (wt/wt)
D3PO4/D2O was found to be
able to dissolve the chitosan- or chitin-like exopolymers at
room temperature. To confirm the incorporation of glucosamine
and N-acetylglucosamine into exopolymers,
1H-NMR spectra were recorded for cellulose, chitin, 10%
acetic acid-soluble fraction of exopolymer made with
glucosamine as the carbon source, and water-soluble fraction of
exopolymer made with N-acetylglucosamine as
the carbon source. For the spectrum of each sample, the signals between
5.0 and 3.0 ppm were assigned to the sugar hydrogens based on previous
work on a related structure and the electron-withdrawing effect of the
ring oxygen (19). The signals around 2.0 ppm, which
correspond to the acetyl group (21), were found in the
spectra of chitin and the water-soluble fraction of exopolymer
synthesized with N-acetylglucosamine.
The production of exopolymers with glucose,
glucosamine, and N-acetylglucosamine was
observed as a function of time up to 14 days (Fig.
5). The production of exopolymer
with glucose increased over 14 days and reached 4.98 mg/ml., while with
glucosamine and N-acetylglucosime this level was low
throughout, likely reflecting the lack of cell growth. The maximum
yields of exopolymer found with glucosamine and
N-acetylglucosamine were 0.50 and 0.90 mg/ml, respectively. To increase the productivity of
glucosamine-incorporated exopolymer, mixtures
(2% total) of glucose and glucosamine were used as the carbon
source (Table 2). The data illustrate
that the higher the relative amount of glucose in the mixture, the higher the production of exopolymer and the lower the mole
percent of glucosamine. On the basis of GC analysis, the
average mole percent of glucosamine in the
exopolymer was 17% and yield of exopolymer was 1.60 mg/ml when the carbon source was the mixture of glucose (0.5%,
vol/vol) and glucosamine (1.5%, vol/vol).
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FIG. 5.
Production of exopolymers with glucose ( ),
glucosamine ( ), and N-acetylglucosamine
( ) as a function of cultivation time of G. xylinum ATCC
10245. Each point represents the average of three samples; standard
deviations ranged from 0.002 to 0.005 for the glucosamine and
N-acetylglucosamine samples and 0.01 to 0.05 for the
glucose samples.
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| |
DISCUSSION |
The major components of exopolymers purified from cultures
of G. xylinum ATCC 10245 with glucose and its analogs
as the main carbon sources were confirmed by GC and GC/MS. In
this study, exopolymers generated with glucose,
3-O-methylglucose, and 2-deoxyglucose were confirmed
to contain only glucose as the major repeat unit. The
exopolymers synthesized with glucosamine or
N-acetylglucosamine as the main carbon source were
found to contain glucose and glucosamine or
Nacetylglucosamine. GC chromatograms of the
fractionated exopolymers synthesized with
glucosamine or N-acetylglucosamine showed
they were a mixture of polymers with different molar ratios of glucose to glucosamine or
N-acetylglucosamine. The polymerization of
glucosamine and N-acetylglucosamine by
G. xylinum ATCC 10245 to form glucose-coglucosamine copolymers and glucose-co-N-acetylglucosamine
copolymers, respectively, is one possibility based on these
results. This would suggest that the cellulose synthase and other
enzymes involved in cellulose synthesis have broader specificities. It
is unlikely that the sugars were modified postpolymerization by the
synthase, since such an enzyme has not been reported in G. xylinum. Furthermore, no evidence for modified glucose was found
in cellulose formed when glucose was the carbon source. The molar ratio
of glucose to glucosamine or
N-acetylglucosamine in the purified
exopolymers after fractionation ranged from 6.0:1.0 to 0.6:1.0
and from 6.2 to 0.8:1.0, respectively. This means that a variety of
glucose-coglucosamine copolymers and
glucose-co-N-acetylglucosamine copolymers with different contents of glucosamine and
N-acetylglucosamine can be generated. Further work is
ongoing to investigate the physiological conditions that affect
incorporation rates of these sugars as well as optimal production
conditions for the modified exopolymers.
The acetic acid-insoluble glucose-coglucosamine copolymer (the
molar ratio of glucose to glucosamine was 6.0 to 1.0) was
insoluble in distilled water. The acetic acid-soluble
glucose-coglucosamine copolymer (the molar ratio of
glucose to glucosamine was 0.6 to 1.0) exhibited a solubility
similar to that of chitosan, which is insoluble in distilled water but
soluble in acetic acid. The water-soluble
glucose-co-N-acetylglucosamine copolymer (the molar ratio of glucose to glucosamine was 0.8 to 1.0) seems to be
soluble in distilled water under certain conditions, such as a high
degree of acetylated exopolymer and low concentration of
solute. This observation may be explained by the fact that partially
acetylated chitosan can be soluble, depending on the level of
acetylation in chitosan and the pH of the chitosan solution
(1). The glucose analogs used in this study were highly
soluble in water (>20%, wt/vol). Therefore, the procedure used to
isolate and purify exopolymers from the cultures also supports
the absence of contamination by monosaccharides as well as
contaminating lipids and proteins.
Preliminary analysis of the fibers (cellulose and the new copolymers)
by environmental scanning electron microscopy suggested similar gross
morphology (e.g., diameter and surface smoothness). The formation of
chitin-like and chitosan-like polymers by direct bacterial
incorporation of glucosamine and
N-acetylglucosamine suggests new options in the
synthesis and purification of materials that may bridge the properties
of the respective homopolymers, cellulose and chitin/chitosan. Control
of the level of glucosamine or
N-acetylglucosamine incorporated into cellulose
would provide new options in tailorability in terms of solubility and
reactivity. For example, cellulose with a significant
glucosamine content would become soluble in dilute acid,
potentially leading to new processing options while maintaining
cellulose-like properties, in contrast to cellulose, which has severe
processing limitations due to low solubility in most solvents.
Cellulose with a significant content of
N-acetylglucosamine could be susceptible to lysozyme hydrolysis in the body, leading to new biodegradable biomaterials based
on cellulose. This would overcome current limitations with cellulose-based biomaterials that cannot be degraded in the body, thus
necessitating the use of lower molecular polymers that can be cleared
without degradation. New copolymers of cellulose with glucosamine would generate materials with reactive amine groups on the polymer surface, leading to simple cross-linking schemes and
surface modification reactions
e.g., covalent immobilization of dyes,
surface treatments to reduce hydrophilicity in pulp and paper, or
surface coupling of peptides for biomedical needs to control protein
adsorption and cell interactions.
| |
ACKNOWLEDGMENT |
Thanks are extended to the NSF (D.K.) for recent support of this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address for Richard A. Gross: Department of Chemistry, Polytechnic University, Six Metrotech Center, Brooklyn, NY 11201. E-mail: gros{at}poly.edu.
Mailing address for David L. Kaplan: Department of Chemical & Biological Engineering, Biotechnology Center, Tufts University, 4 Colby
Street, Medford, MA 02155. Phone: (617) 627-3251. Fax: (617) 627-3991. E-mail: david.kaplan{at}tufts.edu.
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Applied and Environmental Microbiology, September 2001, p. 3970-3975, Vol. 67, No. 9
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.9.3970-3975.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
