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J. Li et al. / Carbohydrate Research 342 (2007) 1030–1033
4 ꢁC for 20 min. The precipitate was dissolved in
50 mL 50 mmol/L phosphate buffer (pH 7.0) and the
crude enzyme was used in agarose degradation. Agarase
activity was determined by measuring the increase in the
concentration of reducing sugar as described by Von
Borel et al.16 One unit (U) of agarase activity was
defined as the amount of enzyme that released 1 lmol
of D-galactose per minute.
3.2. Preparation of neoagarosaccharides
Figure 3. 13C NMR spectrum of neoagarohexaose prepared with
AgaA from agarose. Resonances at about 97 and 93 ppm correspond
to neoagaro-oligosaccharides. G represents the 3-O-linked b-D-galac-
topyranose; A represents the 4-O-linked 3,6-anhydro-a-L-galactopyr-
anose; r and nr denote residues at the reducing and non-reducing end,
respectively; a/b for anomer.
Agarose (200 mg) was dissolved in 100 mL hot water
(99 ꢁC). The soln was cooled down in a 40 ꢁC water
bath, mixed with 0.1 mL crude agarase and incubated
at 30 ꢁC and 150 rpm for 12 h. The hydrolytic reaction
was stopped by heating the mixture in a boiling water
bath for 10 min. After being cooled down to 4 ꢁC, the
hydrolyzate was centrifuged at 12,000g and 4 ꢁC for
10 min with the supernatant concentrated to 2 mL
through rotary evaporation at 45 ꢁC.
The signal at 90.72 ppm, characteristic of agaro-oligo-
saccharides, the main products of a-agarase degradation
of agarose was not found.14 The spectra for the other
neoagaro-oligosaccharides showed similar patterns, but
with different intensities (data not shown). Therefore,
the obtained neoagaro-oligosaccharides had their struc-
ture maintained during the various steps of preparation.
The two new b-agarases, AgaA and AgaB, are highly
active and specific. Furthermore, gel-filtration can sepa-
rate neoagaro-oligosaccharides with a good resolution.
These advantages allowed us to successfully prepare five
neoagaro-oligosaccharides. FACE is a useful tool for
carbohydrate analysis.15 To our knowledge, it is the first
time that FACE is used in the analysis of neutral neoag-
aro-oligosaccharides.
The entire procedure including pre-cultivation of the
b-agarases producing strain can be completed in one
week. The real working time is less than 30 h since most
of the steps, such as degradation, chromatography, and
lyophilization, do not require constant attention. Pre-
parative scales over 10 mg can be easily reached in one
experiment and each step of the method can be scaled
up to some degree. The establishment of this simple
and fast method will open a new avenue for the research
on structure–function relationship of neoagaro-oligo-
saccharides and their industrial preparation.
3.3. Purification of neoagarosaccharides
The neoagaro-oligosaccharides solns (2 mL) were
loaded onto a Bio-gel P2 or P6 column (fine, 95 cm ·
1.5 cm, Bio-Rad Laboratories, USA). NH4HCO3
(0.5 mol/L) was used as eluent at a flow rate of 7 mL/
h. Detection used the classical phenol–H2SO4 method.
Fractions (1.7 mL) were collected. The salt (NH4HCO3)
was removed by repeated evaporation under diminished
pressure at 50 ꢁC. After a second column chromatogra-
phy using the same conditions, the recovered neoagaro-
oligosaccharides were lyophilized.
3.4. FACE
The oligosaccharide was modified by mixing 5 lL carbo-
hydrate (10 lg) with 5 lL 0.2 mol/L AGA (dissolved in
3:17 AcOH–water) and 5 lL 1 mol/L NaCNBH3 fol-
lowed by incubation at 45 ꢁC overnight (>8 h) and then
vortexing and brief centrifugation. The modified labeled
oligosaccharide was mixed with an equal vol of 50%
sucrose and separated on polyacrylamide gel buffered with
the discontinuous Tris–glycine/Tris–HCl system. Elec-
trophoresis was performed at 300 V for 120 min using
a Hofer miniVE apparatus (Amersham Phamarcia Bio-
tech, Sweden). The gel was photographed and analyzed
with a Bio Imaging System (Syngene, Cambridge, UK).
3. Experimental
3.1. Preparation of recombinant b-agarase
We ourselves prepared the recombinant b-agarases,
AgaA and AgaB. Briefly, Escherichia coli BL21 (DE3)
carrying the AgaA or AgaB encoding gene in the plas-
mid pET24a (+), respectively, were cultivated. One liter
of cell free supernatant was mixed with solid ammonium
sulfate till 70% saturation was reached. The mixture was
stirred at 4 ꢁC for 1 h and centrifuged at 12,000g and
3.5. MALDI-TOF mass spectrometry
It was performed on a Biflex III equipment (Bruker Dal-
tonics, Inc., Billerica, MA) set in positive-ion mode with
2,5-dihydroxybenzoic acid (DHB) as the matrix. All
detected oligosaccharide ions were observed as pseudo-
molecular ions, [M+Na]+ and [M+K]+.