High yield production of monomer-free chitosan oligosaccharides by pepsin catalyzed hydrolysis of a high deacetylation degree chitosan

Article abstract of DOI:10.1016/j.carres.2007.08.023

The high molecular weight of chitosan, which results in a poor solubility at neutral pH values and high viscosity aqueous solutions, limits its potential uses in the fields of food, health and agriculture. However, most of these limitations are overcome by chitosan oligosaccharides obtained by enzymatic hydrolysis of the polymer. Several commercial enzymes with different original specificities were assayed for their ability to hydrolyze a 93% deacetylation degree chitosan and compared with a chitosanase. According to the patterns of viscosity decrease and reducing end formation, three enzymes-cellulase, pepsin and lipase A-were found to be particularly suitable for hydrolyzing chitosan at a level comparable to that achieved by chitosanase. Unlike the appreciable levels of both 2-amino-2-deoxy-d-glucose and 2-acetamido-2-deoxy-d-glucose monomers released from chitosan by the other enzymes after a 20 h-hydrolysis (4.6-9.1% of the total product weight), no monomer could be detected following pepsin cleavage. As a result, pepsin produced a higher yield of chitosan oligosaccharides than the other enzymes: 52% versus as much as 46%, respectively. Low molecular weight chitosans accounted for the remaining 48% of hydrolysis products. The calculated average polymerization degree of the products released by pepsin was around 16 units after 20 h of hydrolysis. This product pattern and yield are proposed to be related to the bond cleavage specificity of pepsin and the high deacetylation degree of chitosan used as substrate. The optimal reaction conditions for hydrolysis of chitosan by pepsin were 40 °C and pH 4.5, and an enzyme/substrate ratio of 1:100 (w/w) for reactions longer than 1 h.

Full text of DOI:10.1016/j.carres.2007.08.023

Available online at www.sciencedirect.com
Carbohydrate Research 342 (2007) 2750–2756
High yield production of monomer-free chitosan oligosaccharides by
pepsin catalyzed hydrolysis of a high deacetylation degree chitosan
a,
b,
b
*
´
´
Tomas Roncal, Alberto Oviedo, Iratxe Lopez de Armentia,
b
b
´
´
Laura Fernandez and M. Carmen Villaran
a
´
Unit of Sustainable Chemistry, Fundacio´n LEIA C. D. T., Parque Tecnolo´gico de Alava, Leonardo da Vinci 11,
´
01510 Min˜ano (Alava), Spain
b
´
Unit of Food Science, Fundacio´n LEIA C. D. T., Parque Tecnolo´gico de Alava, Leonardo da Vinci 11, 01510 Min˜ano (Alava), Spain
´
Received 1 June 2007; received in revised form 28 August 2007; accepted 29 August 2007
Available online 6 September 2007
Abstract—The high molecular weight of chitosan, which results in a poor solubility at neutral pH values and high viscosity aqueous
solutions, limits its potential uses in the fields of food, health and agriculture. However, most of these limitations are overcome by
chitosan oligosaccharides obtained by enzymatic hydrolysis of the polymer. Several commercial enzymes with different original spec-
ificities were assayed for their ability to hydrolyze a 93% deacetylation degree chitosan and compared with a chitosanase. According
to the patterns of viscosity decrease and reducing end formation, three enzymes—cellulase, pepsin and lipase A—were found to be
particularly suitable for hydrolyzing chitosan at a level comparable to that achieved by chitosanase. Unlike the appreciable levels of
both 2-amino-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-glucose monomers released from chitosan by the other enzymes after a
20 h-hydrolysis (4.6–9.1% of the total product weight), no monomer could be detected following pepsin cleavage. As a result, pepsin
produced a higher yield of chitosan oligosaccharides than the other enzymes: 52% versus as much as 46%, respectively. Low mole-
cular weight chitosans accounted for the remaining 48% of hydrolysis products. The calculated average polymerization degree of the
products released by pepsin was around 16 units after 20 h of hydrolysis. This product pattern and yield are proposed to be related
to the bond cleavage specificity of pepsin and the high deacetylation degree of chitosan used as substrate. The optimal reaction con-
ditions for hydrolysis of chitosan by pepsin were 40 ꢀC and pH 4.5, and an enzyme/substrate ratio of 1:100 (w/w) for reactions
longer than 1 h.
ꢁ 2007 Elsevier Ltd. All rights reserved.
Keywords: Chitosan hydrolysis; Chitosan oligosaccharides; D-Glucosamine; N-Acetyl-D-glucosamine; Pepsin; Deacetylation degree
1. Introduction
Chitosan is a polysaccharide with linear structure con-
stituted by a copolymer of b-(1!4)-linked 2-amino-2-
deoxy-D-glucose (GlcN) and 2-acetamido-2-deoxy-D-
glucose (GlcNAc) residues. It is obtained chiefly by
homogeneous deacetylation of chitin with strong bases,
rendering chitosans of different acetyl content or deacet-
ylation degrees. Chitosan is a non-toxic, biodegradable
biopolymer of high molecular weight with a fiber-like
structure similar to that of cellulose. As a result of these
properties, chitosan solutions show high viscosity. Con-
trary to chitin and owing to its acetyl groups removal,
chitosan is readily dissolved in dilute acidic aqueous
solutions below pH 6.0. Obviously, chitosan solubility
depends on its deacetylation degree: the higher the
The field of bioactive oligosaccharides, compounds hav-
ing interesting functional properties and many uses, and
their production from different raw materials is gaining
considerable attention in recent years.1 So far, the main
interest has been focused on a few oligosaccharides, such
as galactooligosaccharides, lactulose and fructooligosac-
charides, and considerably less attention has been paid
to the rest, including chitosan oligosaccharides.
*
Corresponding author. Fax: +34 945 298217; e-mail: tomasr@leia.es
Present address: Hematopoietic Division, CIEMAT, Avda. Complu-
tense 22, Ed.07-P.01-D.12, 28040 Madrid, Spain.
0008-6215/$ - see front matter ꢁ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.08.023

T. Roncal et al. / Carbohydrate Research 342 (2007) 2750–2756
2751
deacetylation degree, the higher the solubility. Above
pH 7.0, chitosan solubility is low and precipitation or
gelation tends to occur.
b-(1!3)(1!4)-glucanases, proteases, lysozyme and lip-
ases.9–13 These findings have opened the possibility of
developing novel efficient and economically feasible
industrial processes for hydrolyzing chitosan.
Although chitosan shows a number of interesting
functional properties in different areas, the above men-
tioned features regarding its high molecular weight
and viscosity of aqueous solutions, and the special con-
ditions (acidic media) required to achieve solubilization
in water, limit the best part of its potential uses.2 In this
respect, chitosan oligosaccharides, because of their
shorter chain length, display a reduced viscosity and
are soluble in aqueous media at pH values close to neu-
trality, which increases their bioavailability and opens a
wide range of new potential applications.
Chitosan oligosaccharides are bioactive compounds
with many uses in the fields of food, health and agricul-
ture. They have been claimed to have a great number of
effects and activities, including among others: prebiotic,
antimicrobial, antitumoural, tissue recovery stimulation,
The use of chitosanases and some of the above
enzymes in the production of chitosan oligosaccharides
has been assessed at laboratory scale, using enzyme
reactors both in batch and in continuous configura-
tions.8,14–17 In most cases, the resulting products show
high molecular weight (>10 kDa) and, when low poly-
merization degree oligosaccharides have been obtained,
their yield has been poor and with predominance of the
smallest sized species (2–4 residues) and monomers.
In this paper, an efficient procedure for the produc-
tion of low- and medium-size chitosan oligosaccharides,
in high yields and almost free of monomers, starting
from a high deacetylation degree chitosan as substrate
and using pepsin, a low cost commercial enzyme, as
catalyst is described. The products resulting from
chitosan hydrolysis are expected to be suitable for most
of the reported chitosan applications, where the large
molecular weight of the polymer limits its use.
antidiabetic,
immunostimulant,
antiinflammatory,
calcium absorption acceleration, antimutagenic, antioxi-
dant and activator of plant resistance towards insect and
pathogen attack.2,3
Chitosan oligosaccharides can be obtained by chemi-
cal or enzymatic hydrolysis of the chitosan chains.
Chemical hydrolysis is carried out by two alternative
methods: acid hydrolysis with concentrated acids4 or
oxidative degradation with hydrogen peroxide.5 Both
methods have been applied successfully to chitosan
degradation, which occurs almost quantitatively, but
show some drawbacks,2 including the difficulty to obtain
low polymerization degree oligosaccharides because
high polydispersity mixtures predominate, and to con-
trol the extent of hydrolysis, which frequently results
in hydrolysates containing a high ratio of monosaccha-
rides. In addition, the harsh reaction conditions
required, such as elevated temperatures and high reagent
concentrations, may cause environmental problems and
often result in the formation of chemically modified
oligosaccharides.
Alternatively to the aggressive chemical hydrolysis,
chitosan may also be hydrolyzed in a milder way using
enzymes. Enzyme catalyzed chitosan hydrolysis is more
specific and allows a greater control of the extent of
reaction and, therefore, of the product size. The specific
enzymes intended to catalyze chitosan hydrolysis would
be chitosanases.6–8 These enzymes, however, show a
reduced commercial availability and, as a consequence,
are rather expensive, which limit their industrial use.
In the search for alternative enzymes to chitosanases,
showing original specificities different from chitosan
hydrolysis, but able to catalyze the hydrolysis of this
polysaccharide and, most important, commercially
available in great quantities and at reduced cost,
several enzymes have been found to fulfil these require-
ments, including cellulases, hemicellulases, pectinases,
2. Results and discussion
2.1. Hydrolysis of chitosan by commercial enzymes
In an attempt to develop an efficient process for the pro-
duction of chitosan oligosaccharides on a large scale,
that is, with a good yield of low- and medium-chain
length species and reduced levels of monomers, we
studied the hydrolysis of chitosan catalyzed by several
commercial enzymes previously reported to do it. The
enzymes assayed included cellulase, hemicellulase,
papain, bromelain, pepsin, protease type XIV from
Streptomyces griseus, lysozyme and lipase A, with
chitosanase as control. Unlike most of the previous stud-
ies of this kind, which used chitosan with deacetylation
degrees ranging from 70% to 85% and resulted in high
levels of dimers and monomers, our rationale was to
use a very high deacetylation degree (93%) chitosan in
order to avoid a too extensive hydrolysis of the polymer.
Except hemicellulase, all the enzymes assayed showed
an appreciable activity under the standard reaction con-
ditions, as determined by viscosimetry and reducing end
assay. Results are shown in Table 1.
According to the viscosimetry assays, all enzymes,
apart from lysozyme and papain, showed a chitosan
hydrolyzing activity comparable, and even greater, to
that of chitosanase, reaching a viscosity decrease higher
than 80–85% in 20 h. The effects of enzyme action could
be observed early, occurring the greatest viscosity
decreases in the first hour of hydrolysis.
Reducing end formation was slower than viscosity
decrease and, in general, paralleled to it, although both

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T. Roncal et al. / Carbohydrate Research 342 (2007) 2750–2756
Table 1. Chitosan hydrolysis catalyzed by different commercial
2.50
2.00
1.50
1.00
0.50
0.00
enzymesa
Pepsin
Cellulase
Lipase A
Chitosanase
Enzyme
Viscosity decrease
(%)
Reducing ends
(mM)
t = 1 h
t = 20 h
t = 1 h
t = 20 h
Bromelain
Lysozyme
Cellulase
61
28
69
80
82
42
37
65
86
41
85
89
97
70
82
96
—
—
0.34
0.43
0.26
—
0.51
0.20
3.11
3.32
4.69
0.28
0.42
5.46
Pepsin
Lipase Ab
Papain
Protease type XIV
Chitosanase
—
0.41
0
1
2
3
4
5
6
7
8
9
a Enzyme activity was measured by the resulting decrease in viscosity
and by reducing end formation, according to the standard method
described in the text.
b Lipase A assays were carried out at pH 3.0, instead of standard pH
(4.5).
Time (h)
Figure 2. Time course of the reducing end formation from a 1% (w/v)
chitosan solution treated with different commercial enzymes. All the
enzymes were assayed according to the standard procedure described
in the text, except lipase A, which was assayed at pH 3.0. Reducing
ends are expressed as D-glucosamine equivalents.
measurements were not directly related in all the cases.
The most notorious example of this was bromelain that,
causing a viscosity decrease comparable to that of the
most active enzymes, showed a significantly lower reduc-
ing end formation. After the first hour of hydrolysis,
reducing end formation could only be detected for four
of the assayed enzymes: cellulase, pepsin, lipase A and
the control chitosanase. Again, these enzymes showed
activities similar to that of chitosanase.
As a result of the fast viscosity decrease occurring at
the beginning of hydrolysis, the change of this parame-
ter with time was monitored in the first hour of reaction
(Fig. 1). Most active enzymes were bromelain, cellulase,
pepsin, lipase A and chitosanase. By far, pepsin showed
the initial higher rates, reaching a viscosity decrease
greater than 60% in only 10 min. All the enzymes
showed similar reaction kinetics, with the main viscosity
decrease taking place in the first 10–20 min.
These kinetics results from the predominant endo-type
action of the enzymes: the hydrolysis of the initially
large chitosan chains cause greater viscosity decreases
than the subsequent degradation of their shorter hydro-
lysis products.
The four enzymes that showed the highest chitosan
hydrolyzing activities (cellulase, pepsin, lipase A and
chitosanase) were also studied for the formation of reduc-
ing ends throughout time (Fig. 2). In the first hour a
relatively fast generation of reducing ends occurred,
decreasing their formation rates in the following 3 h.
Afterwards, hydrolysis rates increased again and were
maintained rather constant up to 8 h. This behaviour
was common for all the enzymes, with lipase A and chito-
sanase showing a higher reducing end releasing activity.
The initial fast generation of reducing end during the
first hour of enzymatic hydrolysis of chitosan could be
explained taking into account the four different types
of glycosidic linkages found in chitosan and the cleavage
specificities of the enzymes (see Section 2.2). An enzyme
able to cleave two or more types of these linkages would
likely show different affinities for each of them, so result-
ing in different cleavage rates. As a result, the initial fast
generation of reducing ends would represent the prefer-
ential cleavage of bonds towards which enzymes show a
higher affinity.
100
Bromelain
80
Lysozyme
Cellulase
Pepsin
60
40
20
0
Lipase A
Papain
Protease
Chitosanase
No enzyme
Therefore, three of the commercial enzymes assayed—
cellulase, pepsin and lipase A—, when compared to
chitosanase, were particularly suitable for hydrolyzing
chitosan, as judged by their ability to reduce the viscos-
ity of chitosan solutions and to release reducing ends
from the polymer.
0
10
20
30
40
50
60
70
Time (min)
Figure 1. Time course of the viscosity decrease of a 1% (w/v) chitosan
solution resulting from the chitosanase activity of different commercial
enzymes. All the enzymes were assayed according to the standard
procedure described in the text, except lipase A, which was assayed at
pH 3.0. Chitosan solution viscosity is expressed relative to the starting
value. A control reporting the viscosity variations in the absence of
enzymes has also been included.
2.2. Analysis of hydrolysis products
The hydrolysis products resulting from the enzyme
action upon chitosan were first divided into an insoluble

T. Roncal et al. / Carbohydrate Research 342 (2007) 2750–2756
2753
fraction and a soluble one following neutralization of
the reaction mixture, as described elsewhere.13 The
insoluble fraction, containing low molecular weight
chitosan, was separated and weighted after lyophyliza-
tion. The soluble fraction, composed of a mixture of
oligosaccharides and monomers, was separated by
HPLC and the percent yield of monomers (GlcN and
GlcNAc) and chitosan oligosaccharides was calculated
based on their corresponding peak area. The calculated
percent yield of each of these fractions is showed in
Table 2.
Typical chromatograms of the soluble fraction
showed, in addition to monomer peaks where present,
three major peaks that could not be assigned to specific
oligosaccharide species because they were composed of
several unresolved peaks. However, a fraction obtained
after passing the whole hydrolysate through an ultrafil-
tration membrane having a molecular weight cut-off of
5 kDa, corresponding to a polymerization degree of
around 27–28 units, showed an HPLC profile similar
to that of the soluble fraction, with the same three main
peaks but the third extending to higher elution times
(higher polymerization degrees). This would mean that
the soluble fraction would probably contain a mixture
of chitosan oligosaccharides with polymerization de-
grees of up to around 20 units.
The product distribution after a 20 h-hydrolysis
showed some interesting features. Probably the most
important was that both GlcN and GlcNAc monomers
were not detected in pepsin-derived hydrolysates, in con-
trast with those of the other enzymes, which released
appreciable levels of these species, ranging from 4.6%
to 9.1% of the total product weight. This particular prop-
erty of pepsin allowed it to produce a significantly higher
quantity of chitosan oligosaccharides than the other
enzymes: nearly 52% of the total products from pepsin
hydrolysis were oligosaccharides, whereas the quantities
released by rest of the enzymes did not exceed 46%.
Finally, the yield of low molecular weight chitosans
generated by the four enzymes ranged from 45% to 50%.
In order to add new elements to the above hydrolysis
product analysis, through a careful examination of the
results shown in Table 1, it was possible to make an
approximate calculation of the average polymerization
degree of the hydrolysis products taking into account
three factors: the total weight of chitosan in the reaction
mixture, the concentration of the reducing ends released,
and the average molecular weight of the monomers
(considering a molar ratio of 93% GlcN and 7% Glc-
NAc). According to this approach, after 20 h of hydro-
lysis, the average polymerization degree of chitosan
degradation products would be 16.6, 17.6, 11.8 and
10.0, for pepsin, cellulase, lipase A and chitosanase,
respectively.
All these results as a whole point out that the most
suitable enzyme for hydrolyzing a high deacetylation
degree chitosan in order to obtain oligosaccharides is
pepsin, because it causes one of the greatest (and the
fastest) viscosity decreases and reducing end releases,
and produces the highest quantities of short- and
medium-chain oligosaccharides, without hardly mono-
mer generation. In addition, pepsin is the cheapest
among the enzymes used in this study, which is very
important for development of an economically feasible
industrial process.
Unlike the results presented in this paper, in previous
studies dealing with pepsin catalyzed chitosan hydrolysis
the production of relatively high levels of monomers was
reported.13,18 The key that explains this difference is the
high degree of deacetylation (93%) of chitosan substrate
used in this work, which contrasts with the ꢀ74%
deacetylation degree chitosan employed in the other
studies. Chitosan may contain four types of glycosidic
linkages (-GlcN-GlcN-, -GlcN-GlcNAc-, -GlcNAc-
GlcN- and -GlcNAc-GlcNAc-), the probability of the
occurrence of each of these bonds depending on the
deacetylation degree of the polymer. On the other hand,
chitosan obtained by homogeneous deacetylation of
chitin (such as chitosan used in this work) appears to
contain its N-acetyl groups randomly distributed along
the polysaccharide chains.19 It is also known that pepsin
is able to hydrolyze chitin,20 which means that it acts on
-GlcNAc-GlcNAc- bonds, and that chitosans with lower
deacetylation degrees are more prone to hydrolysis by
pepsin.21 Furthermore, from the hydrolysis products
pattern and its susceptibility to the action of hexosamin-
idase, which specifically releases GlcNAc from the
non-reducing end, it has been suggested that pepsin
cleaves chitosan at -GlcN-GlcNAc- and -GlcNAc-Glc-
NAc- linkages, resulting in products with GlcNAc in
the non-reducing end.13 Finally, the presence of mono-
mers in the hydrolysis products has been also considered
indicative of an exo-type action of pepsin,13 complemen-
tary to its endo-type activity.
Table 2. Product yield from enzymatic hydrolysis of chitosan for 20 ha
Enzyme
Monomersb (%)
COSc (%)
LMWCd (%)
Cellulase
Pepsin
Lipase Af
Chitosanase
4.6
46.1
52.2
42.5
46.3
49.3
47.8
50.4
44.6
n.d.e
7.1
9.1
a The percent yield of each of the fractions was measured after 20 h of
hydrolysis according to the procedure described in the text.
b Monomers: sum of GlcN and GlcNAc.
c COS: chitosan oligosaccharides.
d LMWC: low molecular weight chitosan.
e n.d.: not detected.
f Lipase A assays were carried out at pH 3.0, instead of standard pH
(4.5).
Taking in mind the above considerations, a likely
explanation of the absence of monomers in the hydroly-
sis products obtained in this work can be found. As a
result of the high deacetylation degree of the chitosan

2754
T. Roncal et al. / Carbohydrate Research 342 (2007) 2750–2756
used, in which only 7% of the glycose residues is
GlcNAc, the probability of the occurrence of
-GlcNAc-GlcNAc- pairs in the polymer is very low
(<0.5%) and, therefore, the vast majority of GlcNAc
present is linked to GlcN. This means that almost all
the hydrolytic events should occur at -GlcN-GlcNAc-
linkages. In addition, following cleavage of this latter
linkages, the reducing ends of the resulting products
are not susceptible to further exo-type attack by pepsin
and no GlcN is released. The only case where the mono-
mer of GlcN could be obtained is by cleavage of a termi-
nal GlcN-GlcNAc- pair present in the non-reducing end
of the starting chitosan chains, a possibility present in a
statistically very minoritary number of the relatively few
initial chitosan chains, so that the amount of GlcN
monomer potentially released would be extremely low.
The absence of the other monomer, GlcNAc, at detect-
able levels can be explained by the same kind of reason-
ing. There are only two ways to generate GlcNAc
monomers. One of them is similar to the case explained
above: the exo-type cleavage of a terminal -GlcN-Glc-
NAc pair present in the reducing end of the starting
chitosan chains. The other requires the presence of at
least two contiguous GlcNAc residues anywhere in the
chitosan chains. Again, both cases have, as we have
previously explained, a low probability to occur. In
conclusion, the high deacetylation degree of chitosan
used in this work and the cleavage specificity of pepsin
explain the extremely low levels of monomers released,
that even remained under the detection threshold of
the HPLC detector employed.
The hydrolytic action of pepsin on the high deacetyl-
ation degree chitosan resulted in a great yield of oligo-
saccharides (ꢀ52%). The average polymerization
degree of the hydrolysis products was around 16.6 units
after 20 h of hydrolysis and this value is probably close
to the minimum value reachable after the complete
hydrolysis of the substrate. This statement is in good
accordance with the theoretical value of around 14.3
units calculated taking into account the proposed
specificity cleavage of pepsin and the degree of deacetyl-
ation of the chitosan substrate.
In previously published studies that used chitosans of
lower deacetylation degrees, in addition to an apprecia-
ble yield of monomers, the average molecular weight of
the main products after 5 h of hydrolysis was reported
to be 4.6 kDa,13 corresponding to a polymerization
degree of around 25 units. This value can be compared
with that obtained in the present study, where by this
time the average size of the products was close to 68
units (value calculated from data shown in Figure 2,
as previously explained), using in both cases the same
enzyme/substrate ratio (1:100). Taking into account all
these results and the fact that they used a chitosan
having a deacetylation degree of 74%, it is likely that
the complete hydrolysis of their substrate (surely not
reached by 5 h; see Fig. 2) would result in a hydrolysate
with an average size significantly lower than that
obtained in the present work.
2.3. Temperature and pH profiles of pepsin catalyzed
chitosan hydrolysis
The effect of temperature and pH on chitosan hydrolysis
by pepsin are depicted in Figure 3a and b, respectively.
Activity increased with rising temperatures reaching a
maximum at around 40–45 ꢀC, and decreasing after-
wards. This temperature optimum is in accordance with
previously published data.13,18,21 The enzyme showed a
high thermal stability up to 45 ꢀC (results not shown),
where no losses in enzyme activity were observed after
1 h of incubation. At 50 ꢀC the activity of pepsin
decreased by 30% in 1 h. In addition, the peak activity
of pepsin in chitosan degradation was measured at pH
4.5 which, again, agrees with reported values.13,18,21
Activity was not measured at pH values higher that
5.0 as a result of the low solubility of chitosan and the
reported irreversible inactivation of the proteolytic
activity of pepsin occurring at pH values >6.0.22 The
a
100
80
60
40
20
0
25
30
35
40
45
50
55
Temperature (ºC)
b
100
80
60
40
20
0
2.5
3.0
3.5
4.0
pH
4.5
5.0
5.5
Figure 3. Chitosan hydrolyzing activity of pepsin as a function of
incubation temperature (a) and pH (b). Activity was measured as the
release of reducing ends after a 1 h-hydrolysis according to the
standard procedure described in the text, and expressed in terms of
relative activity with respect to the highest activity measured, which
takes the value of 100.

T. Roncal et al. / Carbohydrate Research 342 (2007) 2750–2756
2755
optimum pH of the chitosanolytic activity of pepsin
markedly differed from that of its proteolytic activity
which although presenting some variability depending
on the particular protein substrate and its native or
denatured state, is generally accepted to be close to
pH 2.0.23
Consequently, a high yield of low- and medium-size
chitosan oligosaccharides, with barely any monomer,
can be produced by hydrolysis of chitosan catalyzed
by a low cost commercial enzyme such as pepsin, by
simply selecting a chitosan substrate with a high deacet-
ylation degree (>90%). For optimal results, pepsin cata-
lyzed hydrolysis should be carried out a 40 ꢀC and pH
4.5, and at an enzyme/substrate ratio not greater than
1:100 for reactions longer than 1 h. These results open,
therefore, the possibility of developing a feasible indus-
trial process aimed at producing chitosan oligosaccha-
rides on a large scale and useful for many applications.
2.4. Influence of the enzyme/substrate ratio on pepsin
catalyzed chitosan hydrolysis
All the experiments so far carried out were performed at
an enzyme/substrate ratio of 1:100 (w/w), which was the
standard value adopted in order to compare the different
enzymes tested. Apart from other parameters, the reac-
tion rate depends on the relative concentration of
enzyme (pepsin) and substrate (chitosan). The chitosan
concentration was set at a maximum of 10 g Lꢁ1
throughout the study because of the extremely high
viscosity of more concentrated chitosan solutions.
Therefore, the reaction dependence on the enzyme/
substrate ratio was determined by simply altering the
pepsin concentration in the reaction medium at a fixed
10 g Lꢁ1 chitosan concentration.
Upon pepsin addition chitosan solution viscosity de-
creased at a rate directly related to enzyme concentra-
tion (Fig. 4). At 60 min, for enzyme/substrate ratios of
1:100 and higher the resulting viscosity was independent
on this parameter, being stabilized in a value close to
20% of the initial one. At enzyme/substrate ratios lower
than 1:100, the effect was not so pronounced and the
viscosity continued to decrease slowly after 60 min.
Therefore, from the above results it can be stated that
for chitosan hydrolysis times higher than 1 h an
enzyme/substrate ratio of 1:100 is enough to reach the
maximum degree of hydrolysis.
3. Experimental
3.1. Enzymes
The following enzymes were purchased from Sigma–
Aldrich and used without further purification: chitosan-
ase from S. griseus, cellulase from Trichoderma viride,
hemicellulase from Aspergillus niger, papain from
papaya latex, bromelain from pineapple stem, pepsin
from porcine gastric mucosa, protease type XIV from
S. griseus, lysozyme from chicken egg white and lipase
A (Amano) from A. niger.
3.2. Chitosan hydrolyzing activity assay
The reaction mixture of the standard assay contained
1% (w/v) chitosan (Sigma–Aldrich; deacetylation
degree, 93%, according to the supplier) dissolved in
100 mM sodium acetate buffer, pH 4.5, and the corre-
sponding enzyme in an enzyme/substrate ratio of 1:100
(w/w). The extent of chitosan hydrolysis, which was car-
ried out at 40 ꢀC, was measured by viscosimetry and/or
reducing end formation assay.
E/S 0.1/100
3.3. Viscosimetry
100
E/S 0.5/100
E/S 1/100
E/S 5/100
E/S 10/100
80
The enzyme-catalyzed viscosity decrease of the highly
viscous chitosan solutions along the reaction time was
measured continuously with a Brookfield DV-II +
viscosimeter. Measured values were normalized and
plotted as the relative viscosity with respect to the initial
value, taking an enzyme-free chitosan solution as
control.
60
40
20
0
3.4. Reducing end assay
0
10
20
30
40
50
60
Time (min)
The concentration of reducing ends following chitosan
hydrolysis was measured according to the method of
Schales and Schales,24 with a few modifications. After
suitable time intervals, samples were withdrawn and
the reaction stopped by heat inactivation of the enzyme
in a boiling water bath for 5 min. Then, to a 1.5 mL
Figure 4. Influence of the enzyme/substrate ratio on pepsin catalyzed
chitosan hydrolysis. Relative activity was measured as the decrease in
viscosity of the chitosan solution, according to the standard procedure.
Chitosan solution viscosity is expressed relative to the starting viscosity
value. The enzyme/substrate ratio values assayed are shown in the
inset.

2756
T. Roncal et al. / Carbohydrate Research 342 (2007) 2750–2756
sample 2 mL of a 0.5 g Lꢁ1 potassium ferricyanide solu-
tion in 0.5 M sodium carbonate were added. Samples
were then protected from light by covering the test tubes
with aluminium foil, and heated in boiling water for
15 min. After cooling at room temperature and centri-
fuging to remove precipitated chitosan, 2 mL of the
supernatant were diluted with 1 mL of water, and the
absorbance of the resulting solution was measured at
420 nm. The concentration of reducing ends was
expressed as the concentration of reducing sugars
equivalent to glucosamine from a calibration curve
prepared with this monosaccharide as standard.
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Acknowledgements
We thank the Spanish Ministry of Industry, Tourism
and Trade for financial support of this project through
the PROFIT program (FIT-060000-2005-362 and FIT-
060000-2006-63). Funding to T.R. under the Torres
Quevedo program of the Spanish Ministry of Education
and Science and the European Social Fund is also
acknowledged (PTQ2004-0945).
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