A simple method of preparing diverse neoagaro-oligosaccharides with β-agarase

Article abstract of DOI:10.1016/j.carres.2007.02.008

In order to prepare pure and well-defined oligosaccharides from agarose in a rapid and simple manner, an enzymatic degradation method was developed, which includes degradation with either recombinant β-agarase (EC 3.2.1.81) AgaA or AgaB and gel permeation chromatography. Agarose was degraded with AgaA at the optimized conditions, yielding 47% and 45% of neoagarotetraose and neoagarohexaose, respectively. These neoagaro-oligosaccharides were conveniently separated by consecutive column chromatography on Bio-Gel P2 or P6 and were identified by FACE. The structure of these neoagaro-oligosaccharides was confirmed by MALDI-TOF MS and ¹³C NMR spectroscopy.

Full text of DOI:10.1016/j.carres.2007.02.008

Carbohydrate Research 342 (2007) 1030–1033
A simple method of preparing diverse neoagaro-oligosaccharides
with b-agarase
Jingbao Li, Feng Han, Xinzhi Lu, Xiaoyan Fu, Cuiping Ma, Yan Chu and Wengong Yu*
Marine Drug and Food Institute, Ocean University of China, 5 Yushan Road, Qingdao 266003, China
Received 27 November 2006; received in revised form 9 February 2007; accepted 9 February 2007
Available online 16 February 2007
Abstract—In order to prepare pure and well-defined oligosaccharides from agarose in a rapid and simple manner, an enzymatic
degradation method was developed, which includes degradation with either recombinant b-agarase (EC 3.2.1.81) AgaA or AgaB
and gel permeation chromatography. Agarose was degraded with AgaA at the optimized conditions, yielding 47% and 45% of neo-
agarotetraose and neoagarohexaose, respectively. These neoagaro-oligosaccharides were conveniently separated by consecutive col-
umn chromatography on Bio-Gel P2 or P6 and were identified by FACE. The structure of these neoagaro-oligosaccharides was
confirmed by MALDI-TOF MS and 13C NMR spectroscopy.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Agarose; Enzymatic hydrolysis; Neoagaro-oligosaccharides; Recombinant b-agarase; AgaA; AgaB
1. Introduction
Agaro-oligosaccharides can be prepared conveniently
through acid hydrolysis of agar. However, the hydro-
lysis product is not homogenous and the hydrolyzing
reaction is not easily controllable. Therefore, acid hydro-
lysis is not appropriate for large-scale preparation of oli-
gosaccharides with high purity. Enzymatic degradation
of polysaccharides should be a promising alternative
to acid hydrolysis; the enzyme degrades polysaccharides
with high specificity and under mild conditions. So far,
several types of b-agarase have been identified in Pseu-
domonas, Alteromonas, Pseudoalteromonas, Vibrio, and
Bacillus.4–10 These b-agarases degrade agarose, usually
generating neoagarobiose, neoagarotetraose, or neoag-
arohexaose as the main products. Recently, two novel
b-agarase genes, agaA (GenBank access number:
AY150179) and agaB (GenBank access number:
AY293310), have been cloned from Pseudoaltermonas
sp. CY24. AgaA is a new member of glycoside hydrolase
family 16, and it degrades agarose to generate neoagaro-
tetraose and neoagarohexaose as the main products.
AgaB does not belong to any existing glycoside hydro-
lase family, and appears to represent a new family of
glycoside hydrolases. AgaB decomposes agarose to gen-
erate mainly neoagaro-octaose and neoagarodecaose.11
Agarose, an important marine polysaccharide present in
the cell walls of some red algae, is a linear chain com-
posed of alternately arranged 3-O-linked b-D-galacto-
pyranose and 4-O-linked 3,6-anhydro-a-L-galacto-
pyranose residues.1 Owing to its gelling ability, stabiliz-
ing properties, and high viscosity, agarose and its
derivatives are widely used in food, cosmetics, and
pharmaceutical industries. a-Agarase hydrolyzes a-
(1!3) linkages of agarose, producing agaro-oligosac-
charides, while b-agarase hydrolyzes b-(1!4) linkages,
yielding neoagaro-oligosaccharides. Neoagaro-oligosac-
charides inhibit the growth of bacteria and slow down
the degradation of starch. As additives, they can reduce
the caloric value of food. Neoagarobiose is a rare agent
with moisturizing and whitening effects on melanoma
cells.2 Recently, the prebiotic effectiveness of neoagaro-
oligosaccharides has been investigated both in vivo
and in vitro.3
*
Corresponding author. Tel.: +86 532 82031680; fax: +86 532
82033054; e-mail: yuwg66@ouc.edu.cn
0008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.02.008

J. Li et al. / Carbohydrate Research 342 (2007) 1030–1033
1031
In this study, a simple method of preparing diverse
neoagaro-oligosaccharides was developed, in which aga-
rose was degraded with two recombinant b-agarases,
AgaA and AgaB, yielding neoagarotetraose, neoagaro-
hexaose, neoagaro-octaose, neoagarodecaose, and neo-
agarododecaose, which were then purified through gel-
filtration chromatography and identified using fluoro-
phore-assisted carbohydrate electrophoresis (FACE),
matrix-assisted laser desorption ionization time-of-flight
mass spectrometry (MALDI-TOF MS) and 13C NMR
spectroscopy.
Figure 2. FACE analysis of the purified neoagaro-oligosaccharides.
Lines 1 and 7, the ladder of neoagaro-oligosaccharides with different
DP (4–14); Lanes 2–6, neoagaro-oligosaccharides with DP12, 10, 8, 6,
and 4, respectively.
2. Results and discussion
again with P2 or P6 columns. More than 90% of the oli-
gosaccharides were recovered after two chromatography
steps. After the second gel-filtration, the purity of the
neoagaro-oligosaccharides reached 99% as this was
determined by FACE analysis (Fig. 2). In order to deter-
mine whether Bio-gel column chromatography can be
used for the preparation of neoagaro-oligosaccharides
on a large scale or not, the AgaB hydrolyzate of 2 g
agarose was applied on an enlarged P6 column
(95 cm · 4.5 cm). It was found that this column could
purify diverse oligosaccharides with similar recovery
rates and resolutions comparable to the smaller
column.
MS and NMR spectroscopy were used for the struc-
ture determination of the different oligosaccharides.
MALDI-TOF MS confirmed the molecular weights of
630, 936, 1242, 1548, and 1854 Da for neoagarotetraose,
neoagarohexaose, neoagaro-octaose, neoagarodecaose,
and neoagarododecaose, respectively. The 13C NMR
spectrum of neoagarohexaose showed the typical pat-
tern for neoagaro-oligosaccharides (Fig. 3). Resonances
at about 97 and 93 ppm were characteristic of b and a
anomeric forms of galactose residues at the reducing
end of the neoagaro-oligosaccharides, respectively.13
In order to prepare pure and well-defined neoagaro-
oligosaccharides, an orthogonal design was applied to
optimize the enzymatic degradation conditions, which
are as follows: agarose (0.2% in water) was hydrolyzed
with recombinant b-agarase AgaA (15 U/100 mL) or
AgaB (20 U/100 mL) at 30 ꢁC for 12 h. Eighty-one per-
cent and 90% of agarose was degraded into oligosaccha-
rides by AgaA and AgaB, respectively, under these
conditions. The remaining agarose was removed easily
with centrifugation at 4 ꢁC because the polymer will
gel at low temperature. The supernatant of AgaA degra-
dation was concentrated by rotary evaporation and then
purified with a Bio-Gel P2 column with detection by
phenol–sulfuric acid analysis.12 As shown in Figure 1a,
most of the products of AgaA degradation were neoag-
arohexaose (45%) and neoagarotetraose (47%). On the
other hand, the products of AgaB degradation were
mainly neoagaro-octaose (36.9%) and neoagarodecaose
(32.0%) with a small amount of neoagarododecaose
(16.7%), neoagarohexaose (9.7%), and neoagarotetraose
(3.5%). These neoagaro-oligosaccharides were eluted as
five peaks from a Bio-gel P6 column (Fig. 1b). They
were collected and concentrated separately and purified
Figure 1. Gel-filtration on Bio-gel P2 or P6 of the neoagaro-oligosaccharides derived from crude agarase degradation. (a) Neoagaro-oligosaccharides
derived from AgaA degradation (separated with a P2 column). DP6, neoagarohexaose, DP4, neoagarotetraose; (b) neoagaro-oligosaccharides
derived from AgaB degradation (separated with a P6 column). DP12, neoagarododecaose, DP10, neoagarodecaose, and DP8, neoagaro-octaose.

1032
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]+.

J. Li et al. / Carbohydrate Research 342 (2007) 1030–1033
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3.6. 13C NMR spectroscopy
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This work was supported by the National High Tech-
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