Preparation of Chitooligosaccharides Wit
Preparation of Chitooligosaccharides Wit
Preparation of Chitooligosaccharides Wit
Received 2 December 2004; received in revised form 1 April 2005; accepted 10 April 2005
Abstract
Chitosan was depolymerized either by HCl hydrolysis or enzymatic degradation with a commercial preparation Pectinex Ultra Spl. The
chitooligosaccharides released by both methods were selectively precipitated in methanol solutions and characterized using MALDI-TOF mass
spectrometry. Differences between the two methods were detected and concerned the degrees of polymerization of the fragments produced and
their acetylation. The enzymatic method yielded shorter fragments with a higher proportion of fully deacetylated chitooligomers. Conversely,
acid hydrolysis of the starting chitosan resulted in fragments with degrees of polymerization up to sixteen and more monoacetylated residues
than with the enzymatic procedure.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Chitooligosaccharides; Enzyme technology; MALDI-TOF-MS; Enzymatic hydrolysis; Acid hydrolysis; Viscosity
1369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2005.04.025
166 J.C. Cabrera, P. Van Cutsem / Biochemical Engineering Journal 25 (2005) 165–172
Table 1
Assigned ion composition of MALDI-TOF-MS spectra of chitooligosac-
charides prepared by 30 min of acid hydrolysis and isolated by selective
precipitation in 90% methanol
m/z Types Ion composition
524.2 [M + Na]+ (GlcN)3
540.1 [M + K]+
566.2 [M + Na]+ (GlcN)2 –GlcNAc
582.1 [M + K]+
684.9 [M + Na]+ (GlcN)4
701.2 [M + K]+
743.2 [M + K]+ (GlcN)3 –GlcNAc
846.3 [M + Na]+ (GlcN)5
862.3 [M + K]+
904.3 [M + K]+ (GlcN)4 –GlcNAc
1007.4 [M + Na]+ (GlcN)6
1023.4 [M + K]+
1065.4 [M + K]+ (GlcN)5 –GlcNAc
1168.4 [M + Na]+ (GlcN)7
1184.4 [M + K]+
1226.4 [M + K] + (GlcN)6 –GlcNAc
1329.5 [M + Na]+ (GlcN)8
1345.5 [M + K]+
1387.5 [M + K]+ (GlcN)7 –GlcNAc
1490.5 [M + Na]+ (GlcN)9
1506.5 [M + K]+
1548.5 [M + K]+ (GlcN)8 –GlcNAc
1651.6 [M + Na]+ (GlcN)10
1667.6 [M + K]+
1709.6 [M + K]+ (GlcN)9 –GlcNAc
1812.7 [M + Na]+ (GlcN)11
1828.7 [M + K]+
1870.7 [M + K]+ (GlcN)10 –GlcNAc
Fig. 2. Thin layer chromatography analysis of methanol soluble products
1973.7 [M + Na]+ (GlcN)12
resulting from chitosan hydrolysis by HCl. TLC performed on silica gel plate
1989.7 [M + K]+
in a solvent system composed of n-propanol–water–ammonia water (7:2:1,
2031.8 [M + K]+ (GlcN)11 –GlcNAc
v/v/v). The plate was developed by spraying ethanol containing 10% sulfuric
2150.8 [M + K]+ (GlcN)13
acid. To the left of the TLC, the degree of polymerization of corresponding
2192.9 [M + K]+ (GlcN)12 –GlcNAc
chitooligosaccharides is shown.
2311.9 [M + K]+ (GlcN)14
2473.0 [M + K]+
2634.1 [M + K]+ (GlcN)16
units. The analysis of our MS data show that the acid digests
contained a wide distribution of both fully deacetylated and
monoacetylated chitooligosaccharides. This pattern could be
related to a random distribution of the acetyl substituents in
the chitosan used for hydrolysis.
From these results, acid hydrolysis of chitosan combined
with a selective methanol precipitation appears to be a quick
and simple method to obtain good yields of chitooligosac-
charides with DPs up to 16 and little low molecular weight
oligomers.
Fig. 6. MALDI-TOF-MS of the chitooligosaccharides oligomers mixtures obtained during the chitosan hydrolysis with Pectinex Ultra Spl. The proportion of
low molecular weigth oligomers was reduced by precipitation in 90% MeOH. Identified peaks are labeled as dpAc , where dp indicate degree of polymerization
and Ac, the number of acetyl group. The hydrolysis time is labeled in each spectrum.
170 J.C. Cabrera, P. Van Cutsem / Biochemical Engineering Journal 25 (2005) 165–172
mixture and determining reducing sugar and glucosamine Enzymatically produced oligosaccharides with DPs >11
contents in the hydrolyzed chitosan fraction as a function of have been obtained from chitosans: Zhang and coworkers
time (Fig. 4). The enzyme treatment resulted in a fast and [15] using MALDI-TOF-MS detected chitooligosaccharides
substantial loss of viscosity of the solution during the initial with DPs between 3 and 17 released by a mixture of cellulase,
5 h. A 50% decrease of viscosity within the first 60 min was alpha amylase, and proteinase. They found that the peak in-
associated with a low reducing sugar and glucosamine con- tensities of non-acetylated fragments and of the mono-, di- or
tent in the degraded fraction, suggesting an endo mode of tri-acetylated forms of the same fragments were all similar.
action of the Pectinex Ultra Spl preparation on chitosan. It is very clear from Fig. 6 that the strongest m/z signals
The amount of degraded chitosan measured as glu- originate from oligomers of DPs 6–8 and that the m/z intensity
cosamine in the hydrolyzed fraction gradually increased profiles of these enzymatic digests do not change significantly
during the enzymatic hydrolysis (Fig. 5). However, the over time. This confirms that the Pectinex mixture constantly
glucosamine to reducing sugar ratio did not increase very releases short fragments of homogeneous size, which is to
much in solution and tended to stabilize at value of about 3 be compared to the MS spectra obtained from the acid hy-
after 1 day of enzymatic hydrolysis. This behavior suggests drolyzed chitosan precipitate (Fig. 3) that revealed the pres-
a low and relatively similar degree of polymerization ence of much longer chitooligomers. Another characteristic
of chitooligosaccharides released during the enzymatic of the Pectinex enzymes is the presence in the digestion prod-
degradation. ucts of fragments with one and/or three acetyl residues. The
In order to confirm this, the composition of the hydrolyzed MALDI-TOF analysis could not reveal the position of the
fractions produced by the enzymes was analyzed by MALDI- acetyl substituents, but in all cases the most abundant peak
TOF mass spectrometry. Since we are interested in longer did still correspond to fully desacetylated oligomers.
oligomers, most of the shorter DPs were eliminated with the From the comparison of the MS spectra, it seems that there
supernatant of a methanol selective precipitation. MALDI- were less acetylated oligomers in the enzymatically prepared
MS analysis obtained in positive-ion mode of 90% methanol samples than in the acid hydrolyzed ones. Since no chitin
insoluble chitooligosaccharides in the Pectinex Ultra Spl di- acetylesterase activity could be detected in Pectinex Ultra Spl
gests after 4, 12, 24 and 72 h are shown on Fig. 6. More infor- (unpublished results), this suggests that the enzymatic prepa-
mation about the assigned structure of each signal at different ration preferentially attacks close to acetylated residues on
times of hydrolysis is given in Table 2. Chitosan fragments chitosan chains. This is in agreement with previous obser-
were detected mainly as sodium and/or potassium adduct ions vations showing an increased degrading activity of Pectinex
and, under the conditions used, only chitooligosaccharides up enzymes with an increased degree of acetylation of the chi-
to 12 mers were observed. tosan sample [28].
Table 2
Assigned ion composition of MALDI-TOF-MS spectra of chitooligosaccharides with degree of polymerization higher than 6 prepared by enzymatic hydrolysis
for the indicated time and fractionated by selective precipitation in 90% methanol
m/z Ion composition Types Hydrolysis time (h)
2 4 8 12 16 24 48 72
1007.4 (GlcN)6 [M + Na]+ X X X X X X X X
1023.4 [M + K]+ X X X X X X X
1049.9 (GlcN)5 –GlcNAc [M + Na]+ X X X X X X X
1065.4 [M + K]+ X X
1109.9 (GlcN)3 –(GlcNAc)3 [M + H]+ X X X X X X X
1168.4 (GlcN)7 [M + Na]+ X X X X X X X X
1184.4 [M + K]+ X X X X X X X X
1210.7 (GlcN)6 –GlcNAc [M + Na]+ X X X X X X X
1226.4 [M + K]+ X X X
1270.7 (GlcN)4 –(GlcNAc)3 [M + H]+ X X X X X X X
1329.5 (GlcN)8 [M + Na]+ X X X X X X X X
1345.5 [M + K]+ X X X X X X X X
1371.8 (GlcN)7 –GlcNAc [M + Na]+ X X X X X X X
1387.5 [M + K]+ X X X X
1490.5 (GlcN)9 [M + Na]+ X X X X X X X X
1506.5 [M + K]+ X X X X X X
1532.8 (GlcN)8 –GlcNAc [M + Na]+ X X X X X X X
1548.5 [M + K]+ X X
1651.6 (GlcN)10 [M + Na]+ X X X X X X
1667.6 [M + K]+ X
1812.7 (GlcN)11 [M + Na]+ X X X X X
1973.7 (GlcN)12 [M + Na]+ X X
J.C. Cabrera, P. Van Cutsem / Biochemical Engineering Journal 25 (2005) 165–172 171
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