Purification of EPS.
The isolation and purification of EPS from
were carried out as previously described.
strain Rm 1021 was grown in a glutamate mannitol salt (GMS) medium for 5 d at 30 ℃. Cells were removed by centrifugation, and the supernatant was concentrated to one-fifth of its original volume using a rotary evaporator. After adding 3 volumes of ethanol, the EPS turned into a gelatinous precipitate and was then collected by centrifugation. The EPS was dialyzed (MWCO ＞ 10000) against distilled water and then lyophilized. EA: C 38.56％, H 6.35％.
Synthesis of Li-dimsyl.
In a round bottom flask dried by heating under nitrogen, 1.6 M methyllithium (5％ solution in diethyl ether) was added to an equal volume of DMSO through a septum. A needle was inserted into the septum to release methane and evaporate diethyl ether. The mixture was stirred under nitrogen for 90 min and freshly prepared Li-dimsyl was used immediately.
Synthesis of Pentynyl EPS.
Pentynylation of the EPS was carried out according to the procedure described for dextran pentynylation.
Purified EPS (7.62 g, 47 mmol) was dissolved in 300 mL DMSO. After formation of a clear solution, Li-dimsyl (143 mL, 1.5 eq./OH) was added. After 1 h stirring at room temperature, 5-chloro-1-pentyne was slowly added under ice cooling and stirring was continued for 48 h. The product was purified by dialysis against demineralized water, during which time the water was changed 10 times for 5 d. The product was subsequently freeze-dried (8.80 g, whitish solid).
Click Reaction of Pentynyl EPS with Benzylazide.
Py-EPS (5 g) was dissolved in DMSO/H
O (4:1 v/v, 150 mL). After formation of a clear solution, benzylazide (2 Eq./alkynyl group) followed by freshly prepared 1 M aqueous solution of Na-L-ascorbate (20 mol ％ per alkynyl group), and CuSO
O (5 mol ％ per alkynyl group) was added. The reaction mixture was stirred at room temperature for 96 h. The product was purified by dialysis against deionized water and freeze-dried (6.98 g).
Preparation of Phenyl EPS Self Aggregates.
Phenyl EPS (5 mg/mL) was suspended and swollen in 10 mM phosphate buffer under stirring for 2 d to produce a milky suspension.
The resulting suspension was probe sonicated for 10 min using a Sonics VC-505 instrument at 40％ of its maximum power. The procedure was repeated twice. The resulting solution was filtered through a 0.8 mm filter (Satorius, Minisart).
Nuclear Magnetic Resonance (NMR) Spectroscopy.
A Bruker Avance 500 spectrometer was used to record the
H–NMR spectra. NMR analyses were performed in
-DMSO at room temperature.
Scanning Electron Microscopy (SEM).
Samples were mounted onto stubs using double-sided adhesive tape and then made electrically conductive by coating with a thin layer of gold. The surface morphologies of the materials were examined under a scanning electron microscope (Jeol, JSM 6380, Tokyo, Japan).
Transmission Electron Microscopy (TEM).
The phenyl EPS aggregates were absorbed onto Formvar-coated copper grids (200 mesh) and air-dried for 5 min. For negative staining, 2％ uranyl acetate solution was used. TEM images of the self-assembled structures were obtained using a JEM 1010 microscope (JEOL, Tokyo, Japan) operating at 80 kV.
Dynamic Light Scattering (DLS).
DLS measurements were carried out with a Wyatt Technology DynaPro Plate Reader at constant room temperature.
The self-aggregation property of phenyl EPS was determined using fluorescence spectroscopy with pyrene as a fluorescent probe. Fluorescence emission spectra of pyrene probe were recorded using a fluorescence spectrophotometer (SIMADZU, RF-5310PC) at room temperature. The probe was excited at 335 nm, and the emission spectra were obtained in the range of 350-550 nm. The excitation and emission slits had a width of 3.0 and 1.5 nm, respectively. A small amount of pyrene solution in acetone was added into each sample giving a final concentration of 1.0 mM in solution. Acetone was not removed, and its final content was 0.1％, v/v. All samples were sonicated for 15 min and left for 17 h at 25 ℃ before measurement.
This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2013R1A1A2012568 and NRF-2011-619-E0002) and supported by the Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012-0006686). SDG
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