CURRENT MICROBIOLOGY Vol. 40 (2000), pp.

69
DOI: 10.1007/s002849910002
An International Journal

R Springer-Verlag New York Inc. 2000

Properties of Chitosanase from Bacillus cereus S1

Masahiro Kurakake, Shou Yo-u, Kiyomi Nakagawa, Minako Sugihara, Toshiaki Komaki
Department of Food Science & Technology, Fukuyama University, Sanzou, Gakuenchou 1 banchi, Fukuyama, Hiroshima 729-0292, Japan
Received: 8 June 1999 / Accepted: 20 July 1999

Abstract. Chitosanase from Bacillus cereus S1 was purified, and the enzymatic properties were
investigated. The molecular weight was estimated to 45,000 on SDS-PAGE. Optimum pH was about 6,
and stable pH in the incubation at 40C for 60 min was 611. This chitosanase was stable in alkaline side.
Optimum temperature was around 60C, and enzyme activity was relatively stable below 60C. The
degradations of colloidal chitosan and carboxymethyl cellulose (CMC) were about 30 and 20% relative to
the value of soluble chitosan, respectively, but colloidal chitin and crystalline cellulose were not almost
hydrolyzed. On the other hand, S1 chitosanase adsorbed on colloidal chitin completely and by about 50%
also on crystalline cellulose, in contrast to colloidal chitosan, which it did not adsorb. S1 chitosanase
finally hydrolyzed 100% N-deacetylated chitosan (soluble state) to chitobiose (27.2%), chitotriose
(40.6%), and chitotetraose (32.2%). In the hydrolysis of various chitooligosaccharides, chitobiose and
chitotriose were not hydrolyzed, and chitotetraose was hydrolyzed to chitobiose. Chitobiose and
chitotriose were released from chitopentaose and chitohexaose. From this specificity, it was hypothesized
that the active site of S1 chitosanase recognized more than two glucosamine residues posited in both sides
against splitting point for glucosamine polymer.

Chitosan is one of the abundant resources, preparing from

chitin (crab or shrimp shell origin) through the chemical
N-deacetylation, and its biological properties like biocompatibility and antimicrobial activity are remarked. Actually, chitosan is applied widely to medical and food
materials. Especially, the chitooligosaccharides depolymerized from chitosan are also demanded because of the
high solubility in which the application is widened and
the absorption in vivo is improved. Enzymatic hydrolysis
by chitosanase is very useful for producing di-, tri-, and
tetra-chitooligosaccharides mainly. Some chitosanaseproducing microorganisms were separated from nature,
but all the action patterns to chitosan were almost the
same endo-type [13, 510].
Previously, we separated Bacillus cereus S1 from
soil in the screening with 0.2% colloidal chitosan plate
culture. However, chitosan was not an inducer for the
expression of chitosanase because the enzyme was also
intensely produced in a medium with glucose as a carbon
source. This chitosanase from B. cereus has been not
reported over the world, though the strain is well known

Correspondence to: M. Kurakake

in food poisoning. In this study, B. cereus S1 chitosanase

was purified, and the enzymatic properties were investigated.
Materials and Methods
Materials. Commercial chitosan (Kimitsuchitosan LLWP [7585%
N-deacetylated]; Kimitsu Chemical Industries Ltd., Tokyo) was used
for the culture medium and the measurement of chitosanase activity.
Perfectly N-deacetylated chitosan was supplied from Katakura Chikkarin Ltd., Tokyo. All other chemicals were of reagent grade.
Production of chitosanase by liquid culture. B. cereus S1 was
cultivated by 5 ml liquid medium containing 1% glucose, 0.5% yeast
extract, and 0.2% Na2HPO4 12H2O in 18 180 mm test tube at 30C
for 2024 h. Three milliliters of the precultured broth was inoculated
into 100 ml same liquid medium in a 500-ml flask and cultivated at
30C and 220 rpm for 48 h. The cultured broth of the seven duplications
were collected and centrifuged at 3,000 rpm for 10 min. The chitosanase
containing in the supernatant was salted out by adding ammonium
sulfate to make 90% saturation. The precipitated protein separated by
centrifugation was dissolved with 100 ml water and precipitated again
by adding cold acetone to be 70% v/v. The occurring precipitate was
separated by centrifugation, dissolving with water. The crude enzyme
solution was applied to column chromatography for the purification of
chitosanase.
Determination of chitosanase activity. One gram of chitosan (LLWP)
was dissolved in 80 ml water by adding 0.57 ml acetic acid and volume

M. Kurakake et al.: Chitosanase from Bacillus cereus

up to 100 ml after adjusting pH to 6 (1% chitosan, 0.1 M acetic acid).
This chitosan solution (0.91%) was incubated with enzyme sample at
pH 6 and 40C for 10 min. The reaction was stopped by adding 0.2 ml of
1 M NaOH and the mixture was centrifuged at 3,000 rpm for 10 min.
The concentration of reducing sugar of the supernatant was determined
by 3,5-dinitrosalicylic acid method [4]. One unit was defined as the
amount of enzyme that could produce 1 mol reducing sugar (glucose
base) for 1 min.
Purification of chitosanase. Chitosanase was purified by the following
chromatography steps, which were done at 4C. Step 1: Crude enzyme
solution was applied to gel filtration on Sephadex G-25 column
(2.5 50 cm; Pharmacia Ltd., Uppsala) preequilibrated with 10 mM
sodium acetate buffer (pH 5). Proteins were eluted with the same buffer
at a flow rate of 1.3 ml/min. Fractions were collected every 3 min. Step
2: Fractions with chitosanase activity were pooled and applied to
anion-exchange chromatography on Super Q Toyopearl column
(1.0 50 cm; Toso Ltd., Tokyo) preequilibrated with 10 mM sodium
acetate buffer (pH 5). Proteins were eluted with a linear gradient of 0 to
0.6 M NaCl at a flow rate of 0.2 ml/min.

Fig. 1. Separation of chitosanase on gel chromatography on Sephadex

G-25 column. , chitosanase activity, solid line; absorbance at 280 nm.

Adsorption of chitosanase. S1 chitosanase (3.44 U/ml) was mixed

with 0.5% solid carbohydrates, colloidal chitosan, colloidal chitin, and
crystalline cellulose (Funacel; Funakoshi Ltd., Tokyo) and incubated at
40C for 20 min with stirring. After centrifuging (3,000 rpm, 10 min),
the chitosanase activity in the supernatant (unadsorbed enzyme) was
measured. Adsorbed enzyme (Eads) was calculated by the subtraction of
activity in supernatant from total activity (Et).
Analysis of sugars. Chitooligosaccharides were analyzed by highperformance liquid chromatography (HPLC) under the following
conditions: column, Amido 80 (Toso Ltd.); mobile phase, acetonitrile:
20 mM phosphate (45:55 v/v); flow rate, 1.0 ml/min; and detector,
Hitachi (Tokyo) model L-3300 differential refractive index monitor.
SDS-Polyacrylamide gel electrophoresis (SDS-PAGE). It was done
with Atto (Tokyo) model AE-6400 using 7.5% polyacrylamide gel (pH
8.8). SDS molecular weight marker (Sigma, Ltd., St. Louis, MO)
consisting of carbonic anhydrase (29,000), egg albumin (45,000),
bovine albumin (66,000), phosphorylase b (97,400), and -galactosidase (116,000) were used as standard proteins.
Amino acid sequencing. The N-terminal amino acid sequence of
chitosanase was analyzed by automatic Edman degradation using 491
protein sequencing system (Applied Biosystems Ltd., Foster City, CA)

Results
Purification of chitosanase. Crude enzyme solution
prepared by salting out with ammonium sulfate and
precipitating with acetone was applied to gel chromatography on Sephadex G-25 column. As shown in Fig. 1, two
chitosanase peaks of F1 (major fraction) and F2 (minor
fraction) were separated. F1 was applied to anionexchange chromatography on Toyopearl Super Q column, but the enzyme was not adsorbed on the resin and
passed out. Chitosanase of the eluted active fraction was
almost homogeneous on SDS-PAGE (see Fig. 2), as other
proteins adsorbed on the anion-exchange resin. The
molecular weight was estimated to 45,000. These purification steps from culture broth were summarized as
shown in Table 1. The N-terminal amino acid sequence of

Fig. 2. SDS-PAGE of purified S1 chitosanase. a: standard, b: purified S1

chitosanase.
Table 1. Purification of chitosanase from S1 strain

Purification steps
Culture filtrate
90% (NH3 )2SO4
70% acetone
Sephadex G-25
Super Q Toyopearl

Total
activity (U)

Yield (%)

Specific
activity (U/mg)

319.5
251.0
179.9
77.0
54.9

100
78.5
71.7
24.1
17.2

53
66
85
145
196

S1 chitosanase was GEKEMKPFPQQVNYA. Incidentally, F2 (minor fraction) had the same molecular weight
and reactivity for substrate as F1 (data not shown), except
for the adsorption on dextran of Sephadex G-25 resin.
Effect of pH and temperature on chitosanase. Chitosanase activity was measured at various pHs under the
stated condition, using phosphate-citrate (pH 2.28) and
glycine-NaCl-NaOH (pH 910) buffers. Figure 3A shows
the effects of pH on S1 chitosanase. Optimum pH was

CURRENT MICROBIOLOGY Vol. 40 (2000)

Table 2. Degradation of some carbohydrates by S1 chitosanase
Relative
degradation (%)

Carbohydrates
Soluble chitosan
Colloidal chitosan
Colloidal chitin
Carboxymethyl cellulose (CMC)
Crystalline cellulose (Funacel)

100
31.8
7.0
21.6
0.9

Each sample (0.5%) was incubated with chitosanase (0.108 U/ml) at pH

6 and 40C for 24 h.
Table 3. Adsorption of S1 chitosanase on some carbohydrates
Carbohydrates
Colloidal chitosan
Colloidal chitin
Crystalline cellulose

Eads /Et (%)

Eads (U/g)

0.0
100
46.4

0.0
688.2
319.4

Each sample (1%) was incubated with chitosanase (3.44 U/ml) at pH 6

and 40C for 20 min.
Table 4. Hydrolysis of chitooligosaccharides by S1 chitosanase

Fig. 3. Effects of pH (A) and temperature (B) on chitosanase activity.

Stability for pH was examined at 40C and at various pHs for 60 min,
and that for temperature was done at pH 5 and at various temperatures
for 30 min. , , pH and , , temperature for relative, residual
activities.

about 6, and stable pH in the incubation at 40C for 60

min was 611. This chitosanase was stable in alkaline
side. As shown in Fig. 3B, optimum temperature was
around 60C and enzyme activity was relatively stable
below 60C in the incubation at pH 5 for 30 min.
Reactivity for substrate. Purified chitosanase (0.12
U/ml) was incubated with various substrates (0.5%), such
as soluble chitosan, colloidal chitosan, colloidal chitin,
carboxymethyl cellulose (CMC), and crystalline cellulose (Funacel; Funakoshi Ltd.) at pH 6 and 40C for 24 h.
As shown in Table 2, the degradation of colloidal
chitosan was about 30% relative to the value of soluble
chitosan. In other carbohydrates with different structure
from chitosan, the relative degradation for CMC was
about 20%, but colloidal chitin and crystalline cellulose
were not almost hydrolyzed by S1 chitosanase.
Table 3 shows the adsorption of chitosanase on three
solid carbohydrates. Chitosanase adsorbed on colloidal

Substrates

Sugars after hydrolysis

Relative initial
rate (%)

(GlcN)2
(GlcN)3
(GlcN)4
(GlcN)5
(GlcN)6

(GlcN)2
(GlcN)3
(GlcN)2
(GlcN)2 , (GlcN)3
(GlcN)2 , (GlcN)3 , 5(GlcN)4 )6

0
0
1.95
44.4
100

Each chitooligosaccharide (0.45%) was incubated with chitosanase

(0.25 U/ml) at pH 6 and 40C for 22 h.

chitin completely and by about 50% also on crystalline

cellulose. However, the adsorption on colloidal chitosan
was not observed at all, in contrast to that on colloidal
chitin.
S1 chitosanase finally hydrolyzed 100% N-deacetylated chitosan (soluble state) to chitobiose (27.2%),
chitotriose (40.6%), and chitotetraose (32.2%) in the
reaction for 24 h. Chitotetraose and chitopentaose were
released at an early reaction period, hydrolyzing to lower
molecular sugars during the reaction. In the hydrolysis of
7585% deacetylated chitosan, the products were three
chitooligosaccharides as well as in that of 100% deacetylated chitosan. Table 4 show hydrolyzing action for
various chitooligosaccharides. Chitobiose and chitotriose
were not hydrolyzed by S1 chitosanase, and chitotetraose
was hydrolyzed to chitobiose. Chitobiose and chitotriose
were released from chitopentaose and chitohexaose,
where small amount of chitotetraose was residual in the

M. Kurakake et al.: Chitosanase from Bacillus cereus

hydrolysis of chitohexaose. The larger the molecular

weight among tetra-, penta-, and hexa-saccharides, the
larger the reactivity for S1 chitosanase.

Discussion
Chitosanase from B. cereus S1 was more stable under the
alkaline condition, than that from Bacillus P1-7S (pH
3.37.4) and Bacillus circulans MH-K1 (pH 49) [6, 10].
The N-terminal amino acid sequence was different from
those of other chitosanases according to DAD and
SWISS-PROT databases. There was a feature also in the
reactivity for CMC. S1 chitosanase hydrolyzed CMC by
about 20% relative to chitosan, though most chitosanases
do not show the reactivity for CMC [2, 5, 6, 10]. It seems
that the enzyme does not recognized so severely amino
group of C2 position in glucosamine residue when
enzyme-substrate complex was formed. This finding
could be associated with the property that some cellulase
can hydrolyze chitosan.
From the finding in Table 3, the adsorption of S1
chitosanase depends on the type of functional group of
C2 position in the glycoside residue. The degree of
adsorption for enzyme was larger for acetoamido
(-NHCOCH3), hydroxyl (OH), and amino group
(NH2) in turn. It seems that the colloidal chitin binds
with chitosanase as a competitive inhibitor because of the
similar structure. On the other hand, in the adsorption on
colloidal chitosan, the adsorption site would be decreased
because of the following hydrolysis process. Anyway, it
was found that S1 chitosanase had specificity on the
adsorption.
Main products from chitosan by many chitosanase
were di-, tri-, and tetra-chitooligosaccharides. However
their action patterns to chitosan were different on the
origin. Although Bacillus P1-7S chitosanase hydrolyze
chitotetraose to glucosamine and chitotriose, and chitotriose to chitobiose and glucosamine [6], S1 chitosanase
does not hydrolyze chitotriose and hydrolyze chitotetraose to chitobiose as well as those from Bacillus
licheniformis UTK, Amycolatopsis CsO-2, and B. circulans MH-K1 [5, 9, 10]. As shown in Table 4, a glu-

cosamine monomer was not released from chitopentaose

and chitohexaose. This means that the active site of S1
chitosanase recognizes more than two glucosamine residues posited in both sides against a splitting point for the
glucosamine polymer. The subsites for four glucosamine
residues in all were not satisfied with chitobiose and
chitotriose (two and three glucosamine residues). Chitotetraose was just fitted to the subsites and split in the
middle glycosidic bond. In the same manner, two sugars
of chitobiose and chitotriose were released from chitopentaose, and three sugars of chitobiose, chitotriose, and
chitotetraose were released from chitohexaose, where
chitotetraose was successively hydrolyzed to chitobiose.
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