Polymer Degradation and Stability 94 (2009) 1895–1899

Contents lists available at ScienceDirect

Polymer Degradation and Stability

journal homepage: www.elsevier.com/locate/polydegstab

Short note

Preparation of chitooligosaccharides by the enzymatic hydrolysis of chitosan

Yu Xie*, Jingang Hu, Ya Wei, Xiaowei Hong
College of Environment and Chemical Engineering, Nanchang Hangkong University, Nanchang, Jiangxi 330063, PR China

articleinfo
abstract
Article history:
Received 30 November 2008 Chitosan of 80% degree of deacetylation was depolymerised by the cellulase of Aspergillus niger to
Received in revised form prepare chitooligosaccharides. MALDI-TOF MS analysis of the enzymatic hydrolyzates indicated that the
24 May 2009 products were mainly chitooligosaccharides with degree of polymerization (DP) in the 3–11 range. By
Accepted 23 June 2009 fractionation of the hydrolyzates with acetone–water, products of DP below 6 and DP above 6 were
Available online 2 July 2009 separated in good yield.
Crown Copyright © 2009 Published by Elsevier Ltd. All rights reserved.
Keywords:
Chitoogliosaccharides
Cellulase
Hydrolysis
Chitosan

1. Introduction
chitohexaose has significant growth-inhibitory effects against
Chitosan, a linear polymer composed of b-1,4-linked D-glucos- sarcoma-180, MM-46, and Meth-A solid tumours in mice [7–9].
amine residues with various degrees of N-acetylated residues, is
Furthermore, chitohexaose has been found to enhance protective
a deacetylated derivative of chitin extracted from an abundant
effects against infection with some microorganisms in mice [10–
source, shellfish exoskeletons [1]. The polysaccharide and its
13]. Several investigations proved that chitooligosaccharide with
derivatives have found extensive applications in the chemical
degree of polymerization (DP) from 7 to 14 has been consid- ered as
industry, environmental protection, functional food, cosmetics and
phytoalexin inducer to prevent infection of plants by several fungi
pharmaceutical industries, biological medicine and biomaterials
[14–16].
[2]. However, the molecular weight of chitosan prepared by
With the emergence of these and other potential biomedical
deacetylation of chitin is very high and it is insoluble in common
and food applications for chitooligosaccharides, the development
solvents. It only can be dissolved in some acidic media, which very
of viable processes for the hydrolysis of chitosan is attracting
much limits the application of chitosan. On the other hand, many
growing interest. Chitooligosaccharides have been obtained by
studies have shown that the molecular weight has much effect on
sonic irradiation, hydrodynamic shearing, chemical hydrolysis of
the properties of chitosan [3–5]. The physicochemical properties of
chitosan and enzymatic hydrolysis [2,17–21]. However, yields of the
chitosan with different molecular weights vary greatly. Sometimes
chitooligosaccharides with DP 6–8 were low because of low effi-
it even takes on contrary traits [6]. Many special functions of chi-
ciency and random cleavage. Enzymatic degradation of chitosan
tosan are displayed when the molecular weight declines to some
seems to be a better way to prepare chitooligosaccharides [22,23].
extend.
Enzymatic processes have advantages over chemical reactions,
Chitooligosaccharides, oligomers of b-1,4-linked D-glucosamine, since the hydrolysis course and product distribution are subject to
are of special interest because they have a variety of biological and
more facile control. For commercial applications, the utility of
physiological activities. It has been reported that chitooligo-
chitinases and chitosanases is limited as they presently constitute
saccharides from the tetramer to heptamer display strong attract-
costly research reagents that are unavailable in bulk quantities.
ing responses to peritoneal exudate cells in BALB/c mice, and
Cellulase is a complex enzymatic system responsible for the
degradation of cellulosic substances. It occurs widely in various
* Corresponding author. College of Environment and Chemical Engineering, fungi, bacteria, insects and other lower animals. It has not been
Nanchang Hangkong University, No. 696 Fenghe Southern Road, Nanchang, mentioned very often in conjunction with chitosan. Recently, an
Jiangxi 330063, PR China. Tel.: þ86 791 3164118; fax: þ86 791 3953373.
unspecific hydrolytic action of cellulase on chitosan at optimum
E-mail address: xieyu_121@163.com (Y. Xie).
condition of pH 4.5 and 60 ○C was reported [24]. Xie et al. described
the unspecific hydrolysis of chitosan by a cellulase preparation from

0141-3910/$ – see front matter Crown Copyright © 2009 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.polymdegradstab.2009.06.021
1896 Y. Xie et al. / Polymer Degradation and Stability 94 (2009) 1895–1899

Aspergillus niger, which is an alternative economical way of

through sintered glass filter. The filtrate obtained has been desig-
obtaining low molecular weight chitosans and chitooligo-
nated as ‘‘culture filtrate’’. The enzyme preparation was obtained by
saccharides [25]. In this note, we report the preparation and frac-
precipitation using solid ammonium sulfate and centrifugation at
tionation of chitooligosaccharides, from the trimer to tetradecamer,
4000 rev min—1 for 30 min.
by the cellulase from A. niger.

2.3. Purification of chitosan

2. Experimental
Chitosan was further purified before use. Chitosan (20 g) was
2.1. Materials
dissolved in 1 L of 1%(v/v) acetic acid solution. After it was
filtered under diminished pressure, concentrated alkali (NaOH,
Chitosan was obtained from Qingdao Haisheng Bioengineering
1 M) was added into the filtrate to produce precipitate. The
Co., Ltd. It originated from crab shell and its degree of
precipitate was washed repeatedly with deionised water until the
deacetylation was 80%. Its apparent viscosity was 333 mPa s (2%
pH value of rinse water was about 7. Anhydrous alcohol (200
chitosan solution, measured by the DNJ-circumrotate viscometer).
mL) was then added to remove a little red pigment in the
Its viscosity-average
chitosan. At the end the precipitate was dried in a vacuum oven.
molecular weight was 5.18 × 105 (measured by the Ubbelohde
The purified chitosan was milled to power to be used for the
viscometer).
following experiment.

2.2. Preparation of cellulase

2.4. Enzymic hydrolysis of chitosan

Cellulase was prepared with liquid-state fermentation by our

Chitosan solution was prepared by dispersing 3.00 g of chitosan
lab using A. niger as the enzyme-producing strain. A. niger No.
in 20 mL of water, dissolving it with 12 mL of 5% acetic acid and
5.1 was provided by the Culture Collection and Research Centre
making up to 60 mL with water. The pH value of the chitosan
of Micro- biology, Beijing Normal University, China. It was
solution obtained was adjusted to w5.60 with NaOH. The cellulase
cultivated in the medium containing cellulose powder as the sole
solution (1%, 6 mL) was added and the mixture was incubated for
carbon source at
24 h at 50 ○C. The reaction was stopped by boiling for 10 min to
32 ○C. The medium was prepared by the method of Cappellini and
denature the enzyme which was then removed by filtration. The
Peterson [26]. An inoculum of spore suspension of the fungus con-
filtrate was concentrated to about one-twentieth with a rotary
taining 1.0 ×
107 spores was introduced into 50 mL culture solution.
evaporator under reduced pressure. Enough alcohol was then
After 5 days of growth at 120 rev min—1 the medium was filtered

Fig. 1. TOF-MS of products from hydrolysis of chitosan by cellulase.

 Y. Xie et al. / Polymer Degradation and Stability 94 (2009) 1895–1899
added to form a precipitate, which was dried under diminished
pressure.
2.5. Fractionation with acetone–water
The filtrate from the hydrolysis was concentrated by evapora-
tion under reduced pressure until the final volume was w10%
that of the initial solution. Then it was mixed with an equal
volume of
acetone to give a precipitate. The precipitate was collected by
centrifugation at 4000 rev min—1 for 30 min, washed with aq 50%
acetone several times, and dried in a vacuum dry box (Fraction A).
The residual solution was again concentrated to w5% of the initial
volume by evaporation under reduced pressure, and acetone (9 vol)
was added to effect precipitation. The precipitate formed after
thorough mixing was collected by centrifugation, washed with 90%
acetone, and dried under reduced pressure (Fraction B).
2.6. Product analysis
Mass spectra were obtained with a matrix-assisted laser
desorption–ionization time-of-flight mass spectrometer (MALDI-
TOF MS, Bruker, American).
3. Results and discussion
We degraded chitosan for 24 h in the conditions of 50 ○C, pH
5.0 and cellulase to chitosan ratio of 1:5, and determined the
intrinsic viscosities and thus the viscosity-average molecular
weights. The results showed that the average molecular weights
of chitosan
Fig. 2. TOF-MS of products (Fraction B) separated from the reaction mixture by fractionation with acetone–water.
1897
declined to 1.13 × 103, compared with chitosan with a viscosity-
average molecular weight of 5.18 × 105 before enzymatic hydro-
lysis; the cellulase preparation thus had rather apparent effect on
depolymerization of chitosan.
After chitosan had been degraded by the cellulase preparation
for 24 h, a portion of the mixture was taken out and then concen-
trated alkali was added into it with stirring. No precipitate
was formed, indicating that chitosan had been depolymerised
into small oligomers. In the mass spectrum of the hydrolyzates
(Fig. 1) peaks were detected corresponding to the mass numbers
of (M þ Na)þ of trimer to tetradecasaccharide. The products
were mainly composed of chitooligosaccharides, especially of DP 3–
11.
In order to obtain the chitooligosaccharides with narrower
distribution range, the enzymic hydrolyzate was separated by
fractionation with acetone–water from the reaction mixture on
the basis of the differential solubilities of the oligosaccharides in
acetone–water. Two main fractions were obtained in high yield.
The mass spectra of the two fractions are shown in Figs. 2 and 3.
They indicate peaks mainly for heterooligosaccharides from trimer
to pentamer (Fig. 2) and peaks from hexamer to unidecamer
(Fig. 3).
4. Conclusions
These results show that the cellulase preparation from A. niger
can hydrolyze chitosan to yield chitooligosaccharides with DP 3–11,
especially with DP 6–8 in good yield, and it is cheaper than such
enzymes specific as chitinase, chitosanase and lysozyme.
1898
Y. Xie et al. / Polymer Degradation and Stability 94 (2009) 1895–1899
Fig. 3. TOF-MS of products (Fraction A) separated from the reaction mixture by fractionation with acetone–water.
Chitinase, chitosanase, and lysozyme can hydrolyze partially N-
acetylated chitosans, but N-acetylation of the enzymatic hydro-
lyzates produces mainly the dimer, trimer and tetramer of N-acetyl-
D-glucosamine. Chitosan can also be degraded by other non-specific
enzymes such as lipase, papain, pectinase and proteinase. The rate
of decrease in viscosity of chitosan is fast, but the average
molecular weight of the products is about 10,000. For this result,
the hydro- lysis of chitosan by the cellulase from A. niger is a very
useful procedure to prepare chitooligosaccharides with DP 3–11,
and it was preferred over chitosanase because of its economy.
By fractionation of the hydrolyzates with acetone–water,
prod- ucts of DP below 6 and DP above 6 were separated out.
The frac- tionation with acetone–water can yield two fractions of
chitooligosaccharides with DP 3–5 and DP 6–11. The yields for
DP 3–5 and DP 6–11 were estimated to be 30% and 55%,
respectively. The hydrolysis of chitosan by cellulase from A.
niger, together with the separation by fractionation with
acetone–water, is thus a useful procedure for preparing
chitooligosaccharides and N-acetylchi- tooligosaccharides (by N-
acetylation of the hydrolyzate) with DP values less than 11 in
good yield.
This work indicates that the cellulase preparation from A. niger,
a low-cost commercial enzyme and widely accepted in the food and
medicine industries, could be a valid alternative to chitinase and
chitosanase which are expensive and unavailable in bulk quantity
for the production of chitooligosaccharides. It may serve as an
effective approach of industrialized production of chitooligo-
saccharides because the speed of unspecific enzymatic hydrolysis is
fast, the production condition is mild and easy to control, the
complicated producing equipment is not demanded and the cost of
production is low.
Acknowledgements
The authors thank Jiangxi Provincial Department of Education
(2007168) and Natural Science Foundation of Jiangxi Province
(0620071) for their financial support for this work. In addition, we
thank Center of Chromatographic Analysis, Institute of Chemistry,
Chinese Academy of Sciences, for MS analysis.
References
[1] Cheng CY, Li YK. Biotechnol Appl Biochem 2000;32(3):197–203.
[2] Choi WS, Ahn KJ, Lee DW, Byun MW, Park HJ. Polym Degrad Stab 2002;
78(3):533–8.
[3] Skiak-Braek G, Anthonsen T, Sandford P. (Eds.) (1989). Chitin and chitosan.
New York: Elsevier Applied Science.
[4] Li J, Du YM, Yang JH, Feng T, Li AH, Chen P. Polym Degrad Stab 2005;87(3): 441–
8.
[5] Nah JW, Jang MK. J Polym Sci Part A: Polym Chem 2002;40:3796–803.
[6] Yang YM, Shu RG, Shao J, Xu GF, Gu XX. Eur Food Res Technol 2006;222(1–2):
36–40.
[7] Semeˇnuk T, Krist P, Pavl´ıcˇek J, Bezousˇka K, Kuzma M, Nova˘k P, et al.
Glyco- conjugate J 2001;18(10):817–26.
[8] Kulikov SN, Chirkov SN, Il’ina AV, Lopatin SA, Varlamov VP. Appl Biochem
Microbiol 2006;42(2):200–3.
[9] Sun T, Zhou DX, Xie JL, Mao F. Eur Food Res Technol 2007;225:451–6.
[10] You Y, Park WH, Ko BM, Byung-Moo M. J Mater Sci: Mater Med
2004;15(3):297–301.
 Y. Xie et al. / Polymer Degradation and Stability 94 (2009) 1895–1899
[11] Saltykova ES, Ben’kovskaya GB, Poskryakov AV, Sukhorukova OV,
Nikolenko AG. J Evolut Biochem Physiol 2003;39(4):288–93.
[12] Kadokura K, Rokutani A, Yamamoto M, Ikegami T, Sugita H, Itoi S, et al. Appl
Microbiol Biotechnol 2007;75(2):357–65.
[13] Lien TS, Yu ST, Wu ST, Too JR. Biotechnol Bioprocess Eng 2007;12(6):610–7.
[14] Qin CQ, Du YM, Xiao L, Li Z, Gao XH. Wuhan Univ J Natural Sci 2002;7(2):231–6.
[15] Moon JS, Kim HK, Koo HC, Joo YS, Nam HM, Park YH, et al. Appl Microbiol
Biotechnol 2007;75(5):989–98.
[16] Qin CQ, Du YM, Xiao L, Li Z, Gao XH. Int J Biol Macromol 2002;31(1–3):111–7.
[17] Chebotok EN, Novikov VY, Konovalova IN. Russ J Appl Chem 2006;79(7):1172–
6.
[18] Qin CQ, Du YM, Xiao L. Polym Degrad Stab 2002;76:211–8.
1899
[19] Kittur FS, Kumar ABV, Gowda LR, Tharanathan RN. Carbohydr Polym
2003;53(2):191–196.
[20] Lin H, Wang H, Xue C, Ye M. Enzyme Microb Technol 2002;31:588–92.
[21] Qin CQ, Zhou B, Zeng LT, Zhang ZH, Liu Y, Du YM, et al. Food Chem
2004;84(1):107–15.
[22] Kittur FS, Kumar ABV, Tharanathan RN. Carbohydr Res 2003;338(12):1283–90.
[23] Shakhbazau AV, Kartel’ NA. Russ J Genet 2008;44(8):881–9.
[24] Li J, Du YM, Liang HB. J Appl Polym Sci 2006;102(22):1098–105.
[25] Xie Y, Wei Y, Hu JG. Appl Biochem Biotechnol 2009. doi:10.1007/s12010-009-
8559-2.
[26] Cappellini RA, Peterson JL. Mycologia 1965;57:962–6.