JOURNAL OF BIOSCIENCE AND BIOICNGINFERING

Vol. 93. No. 2, 2OlL206. 2002

Immobilization and Stabilization of Chitosanase bY

Multipoint Attachment to Agar Gel Support
SOSAKU ICHIKAWA,’ KAZUYA TAKANO,’ TAKASHI KUROIWA,’
OSAMU HIRUTA,’ SEIGO SATO,’ AND SUKEKUNI MUKATAKA’”

Institute qfApplied Biochemistry. Universi@ of Tsukuhu, lharaki 305-8572. Japan’ und Pharmaceutical Technoloa
Laboratories, Me[ji Seika Kaisha Ltd., 788 Kayama, Odowara. Kanagawa 2504852, Japan2

Received 16 July 20OliAccepted 27 November 2001

Highly stable chitosanase immobilized on an agar gel support was prepared by the multipoint
attachment method. The optimum pH range was broadened to between 4 and 6, whereas for free
chitosanase, the pH was only 5.6. The optimum temperature was also increased from 60°C to
80°C after the immobilization. The activity of immobilized chitosanase remained at 95% of its ini-
tial activity level after 225 h of incubation at 5O”C, whereas for free chitosanase, it decreased to
20% after 1 h of incubation. The immobilization markedly increased the thermostability of chito-
sanase. These changes in the reaction characteristics are favorable for the practical use of chito-
sanase in industrial processes. The effect of glycidol concentration in the activation of agar gel
was also examined. The surface density of the aldehyde residue increased with increasing glycidol
concentration. A maximal activity of 11.9 U/g-support was obtained when the glycidol concentra-
tion was 0.7 M. At concentrations higher than this, thermostability was almost the same. It was
therefore proven that the optimal glycidol concentration in this system is 0.7 M. The effects of gly-
cidol concentration on the activity and the thermostability of chitosanase are discussed in relation
to the number of covalent bonds between the chitosanase and its support. Chitosan oligosaccha-
rides were continuously produced using a column reactor packed with the immobilized chito-
sanase. The percentage of hydrolyzed chitosan after 28 reaction days was 44%. This was a slight
decrease from the 48% observed on the first day. The total concentration of pentamer and hex-
amer ranged from 1.3 mg/ml to 1.5 mg/ml during the 28 reaction days. This was approximately
30% of the chitosan concentration in the supplied substrate solution.

[Key words: chitosanase, immobilization, multipoint attachment, chitosan oligosaccharide]

Chitosan is part of the biomass that occupies large por-
tions of the earth. Although it would be desirable to utilize
chitosan extensively as a biomaterial (I), most chitosan is
not being utilized. Chitosan itself has various physiological
functions (2-S). It is expected to be used as a medical sup-
ply, as a functional food, and as a biologically active sub-
stance. In order to exploit use of chitosan, it is important to
use the substances derived from it. In particular, chitosan
oligosaccharides, which are produced by the partial hydrol-
ysis of chitosan, have physiological functions similar to
those of chitosan (2-5, 9-l 1). They are soluble in water and
easily digested because their molecular weights are lower
than that of chitosan.
Chitosan oligosaccharides have been produced by the
chemical hydrolysis of chitosan using acid (12). This chem-
ical hydrolysis yields large amounts of monosaccharides as
a byproduct, because of the difficulty of controlling the
progress of the reaction. This, in turn, causes a low yield of
oligosaccharides. However, it is known that the hydrolysis
by chitosanases from Bacillus sp. no. 7-M ( 13) and Bacillus
sp. BN-262 (14) does not produce monosaccharides. Fur-
* Corresponding author. e-mail: mukataka@sakura.cc.tsukuba.ac,ip
phone: +XI -(0)298-53-4607 [ax: +8 I -(0)298-53.4605
thermore, this enzymatic hydrolysis is carried out under
mild conditions, so it is easy to control the progress of the
reaction. Therefore, the method which involves the use of
chitosanase is favorable for the production of oligosaccha-
rides.
When the enzyme is used in industrial processes, it is
generally preferred that it be in the immobilized form for
the following reasons (15). The enzyme is more stable in
this form. The enzyme can be used for long-term produc-
tion, because it can be reused. Continuous operation be-
comes easier. Since it is easy to separate the enzyme from
the reaction, the process of separating products can be sim-
plified. In particular, in the case of chitosanase, immobiliza-
tion is highly advantageous from the viewpoint of operating
cost, because chitosanase is expensive. The immobilization
of chitosanase will enable its long-term use in producing
oligosaccharides. The preparation of immobilized chitosan-
ase by the adsorption method has been reported (16). How-
ever, long-term uses for stable immobilized chitosanase
have yet to be developed.
In this study, the immobilization of chitosanase by the
multipoint attachment method was investigated in order to
prepare stable immobilized chitosanase. In this method, the
enzyme is immobilized by contact with the support material

201
202 ICHIKAWAETAL.
through multiple covalent bonds. This method enabled the
preparation of the highly stable immobilized enzyme for
penicillin G acylase (17), trypsin (1 S), a-chymotrypsin (19),
and thermophilic esterase (20). In those studies, an agarose
gel was used as the support material. However, we exam-
ined the use of a cheaper agar gel as a support because of
the cost advantage.
MATERIALS AND METHODS
Materials Chitosanase (EC 3.2.1.132) from BaciZZus pumilus
BN-262 was supplied by Meiji Seika Kaisha Ltd. (Tokyo). The
enzyme contained 13.2% protein as determined by the Bradford
method based on a bovine serum albumin as a standard protein.
Its molecular weight was approximately 3 1 kDa and its isoelectric
point was ca. 9.1. Chitosanase is an end-hydrase that splits the p-
I ,4-glycosidic linkages of GlcN-GlcN and GlcNAc-GlcN (GlcN,
D-gh_icosamine; GlcNAc, f%acetyl-D-glucosamine) (14). As a
substrate, 100% deacetylated chitosan from Funakoshi Co. Ltd.
(Tokyo) was used. Agar powder, glycidol (2,3-epoxypropanol),
sodium borohydrate, and other chemicals were purchased from
Wako Pure Chemical Industries Ltd. (Osaka).
Activation of agar gel support Glyoxyl agar gel (Agar-O-
CH,-CHO) as an activated support was prepared by etherification
of 6% agar gel with glycidol and further oxidation of the resulting
glyceryl agar gel (Agar-0-CH,-CHOH-CH,OH) by NaIO,. In a
typical activation, 20 g of agar gel cut into 1 mm3 cubes was ad-
mixed in 100 ml of 0.16 M NaOH containing 6 mg/ml NaBH, as
an antioxidant and then an adequate amount of glycidol was added
to the mixture. The mixture was stirred for 18 h at 25”C, then
washed with water through a Biichner funnel with suction. The
resulting glyceryl agar gel was suspended in 100 ml of 35 mM
NaIO,, and stirred for I h. The resulting activated agar gel was
washed with water and stored in distilled water at 4°C until use.
The aldehyde content of the activated agar gel support was de-
termined to be equivalent to the amount of periodate consumed
(I 7). The periodate that was not consumed in the activation of the
gel was measured by titration with KI.
Immobilization of chitosanase Fifteen g wet weight of acti-
vated gel support was suspended in 120 ml of 0.1 M borate buffer
(pH 10.0) containing I mg/ml enzyme, and stirred gently for 24 h
at 25°C. After a given contact time, 0.24g of solid NaBH, was
added and gently stirred for 30 min at 25°C. This borohydride
reduction constitutes a suitable end-point of the enzyme-support
interactions because this reduction produces two complementary
effects: stabilization of the enzyme-support attachments (Schiff’s
bases) already formed and reduction of unreacted aldehyde resi-
dues to inert hydroxyl ones (2 1). The gel support was washed with
0. I M phosphate buffer (pH 7.0) and distilled water. The resulting
immobilized chitosanase on the gel support was stored in distilled
water at 4°C until use. The percentage of immobilized chitosanase
was determined by a Bradford protein assay (22).
Activity measurement The substrate used to measure the
chitosanase activity was 100% deacetylated chitosan. The chitosan
was dissolved in 0.1 M acetic acid to a concentration of 0.5%
(w/v). The pH of the chitosan solution was adjusted using 5N
NaOH.
The activity of the free and immobilized chitosanases was mea-
sured by monitoring the increases in the amount of reducing sugar.
One ml of 40 &ml chitosanase solution, or 1 g wet weight of the
immobilized chitosanase was added to 100 ml of chitosan solution
at pH 5.6. The reaction mixture was incubated at 35°C. The
amount of reducing sugar formed by the hydrolysis reaction was
measured by the Schales method (23). One unit of activity of
chitosanase was defined as the amount of enzyme that liberated I
J. Brasc~. BIOENC;..
pmol equivalent of D-glucosamine in I min under the above condi-
tions.
Continuous production of chitosan oligosaccharides using a
packed bed reactor Immobilized chitosanase was prepared by
the method described above. The glycidol concentration in the ac-
tivation of the agar gel support was 0.7 M. Fifteen g wet weight of
activated gel was suspended in 120 ml of 0.1 M borate buffer con-
taining 0.01 mg/ml enzyme. immobilized enzymes (3.1 g) were
packed in a column (412.2x39.0 mm), and a 0.5% chitosan solu-
tion (pH 5.6) was continuously supplied to the packed bed reactor
at 35°C. The flow rate was adjusted to 5.16x IO-’ l/h. Concentra-
tions of pentamer and hexamer of chitosan oligosaccharides in the
permeate were measured by HPLC with a Capcell Pak NH, col-
umn ($4.6~250 mm; Shiseido Co. Ltd., Tokyo).
RESULTS AND DISCUSSION
Optimum pH of immobilized chitosanase In order to
confirm the effectiveness of the method of immobilization
by multipoint attachment to the agar gel support, the activity
of chitosanase was examined under various pH and temper-
ature conditions.
The effects of pH on the relative activity of free and im-
mobilized chitosanases are shown in Fig. 1. The optimum
pH for immobilized chitosanase was in the range of 4 to 6,
whereas for free chitosanase, it was 5.6. Hence, the opti-
mum pH range was broadened after the multipoint attach-
ment immobilization. This wider optimum pH range is most
likely attributable to the stabilization of the enzyme stmc-
ture by the multipoint linkages between the enzyme and the
support.
Optimum temperature of immobilized chitosanase
The effects of temperature on the relative activity of free
and immobilized chitosanases are shown in Fig. 2. The opti-
mum temperature of immobilized chitosanase was SOY,
20°C higher than that of free chitosanase. Under the high-
temperature condition, the enzyme was inactivated by ther-
mal denaturation. Even at a higher temperature, the immo-
bilized chitosanase could retain its active structure com-
pared to the free enzymes, because of the multipoint link-
ages to the gel support.
The immobilization of chitosanase by multipoint attach-
ment broadened the optimum pH range and increased the
0-
3 3.5 4 4.5 5 5.5 6 6.5
PH
FIG. I. Effect of pH on the activity of free (open circle) and im-
mobilized (closed circle) chitosanases. The concentration of rlycidol
in the activation of the agar gel support was 4.8 M. The acti&y was
measured at 35’C.
 IMMOBILlZATION AND STABILIZA-TION OF CHITOSANASE 203

0 ‘.~“..~.~.~.~~~~~~‘....I”“~‘~“‘..~.
20 30 40 50 60 70 80 90 100
0 50 100 150 200 250 300
Temperature [“Cl Incubation trme at 50°C [h]
FIG; 2. Effect of the reaction temperature on the activity of free FIG. 4. Thermostabilities of free (open circle) and immobilized
(open circle) and immobilized (closed circle) chitosanases. The con- (closed circle) chitosanases. The concentration of glycidol in the acti-
centration ofglycidol in the activation of the agar gel support was 4.8 vation of the agar gel support was 4.8 M.
M.

backbone, showed a markedly enhanced thermostability

optimum temperature. These effects are favorable for the
application of chitosanase to industrial processes.
Repeated use of immobilized chitosanase To exam-
ine the stability of immobilized chitosanase, we employed
the batch hydrolysis of chitosan at 35°C using the same im-
mobilized chitosanase three times. After each reaction, the
agar gel was washed with a large amount of distilled water,
then stored in distilled water at 4°C until the next repetition
of the batch reaction. The results are shown in Fig. 3. The
three time courses of hydrolysis were almost identical. This
means that the activity of immobilized chitosanase did not
change during the three batch reactions.
Thermostability of immobilized chitosanase Free and
immobilized chitosanases were incubated in distilled water
at 50°C. Figure 4 shows the changes in their residual activ-
ity with incubation time. The residual activity of free chito-
sanase decreased markedly to 20% after 1 h of incubation.
On the other hand, for the immobilized chitosanase, it re-
mained at 95% of the starting activity level after 225 h of
incubation at 50°C. Thus, the immobilization by the multi-
point attachment method significantly increased the thermo-
stability of chitosanase. It was reported that P-glucosidase,
when stabilized by a high number of links with the dextran
(24). The multipoint linkages between chitosanase and the
gel support would help to maintain the active configuration
of chitosanase, even under high-temperature conditions.
These results showed that the multipoint attachment
method using agar gel as a support enables preparation of a
highly stable immobilized chitosanase.
Effect of glycidol concentration The effects of changes
in glycidol concentration in the activation of the agar gel
support were examined in order to optimize glycidol con-
centration. Figure 5 shows the effect of glycidol concentra-
tion on the surface density of aldehyde residues in the acti-
vated gel support. The surface density of the aldehyde resi-
due increased with increasing glycidol concentration. This
result shows that the surface density of aldehyde residue can
be controlled by adjusting the concentration of glycidol in
the activation of the agar support.
Figure 6 shows the effect of glycidol concentration on the
percentage of immobilized chitosanase on the activated agar
support. The concentration of chitosanase in the immobili-
zation step was held constant at 1 mgiml. The percentage of
immobilized chitosanase was 85% when the glycidol con-
centration was in the range of 0.7 to 4.8 M, but decreased to
62.5% at a glycidol concentration of 0.25 M. The surface
density of the aldehyde residue decreased with decreasing
glycidol concentration, as shown in Fig. 5. Therefore, it can

20 :

10 '.

OC"',"'.,..,""","' L...~~~~~(....~
FIG. 3. Repeated use of immobilized chitosanase for the hydroly- 012345678
sis ofchitosan at 35°C. The same immobilized chitosanase was used in
Glycidol concentration [M]
the I st (closed square), 2nd (closed diamond), and 3rd (closed circle)
reactions. The concentration of glycidol in the activation of the agar FIG. 5. Effect of glycidol concentration on the surface density of
gel support was 4.8 M. aldehyde residues in activated gel supports.
204 ICHIKAWA ET AL J. BIOSU BIOENG.,

Glycidol concentration [M]

0 50 100 150 200 250 300
FIG. 6. Effect of glycidol concentration on the percentage of im-
mobilized chitosanase. Incubation time at 60°C [h]

FIG. 8. Effect of glycidol concentration on the thermostability of

be considered that the immobilization percentage decreases
because the surface density of the aldehyde residue is too
low to immobilize chitosanase. At the same time, a 16%
immobilization percentage was obtained without glycidol.
Under this condition, the agar support was not activated.
Therefore, chitosanase would only be immobilized by phys-
ical adsorption to the agar gel support.
The effect of glycidol concentration on the activity of
the immobilized chitosanase was examined. The results are
shown in Fig. 7. When the glycidol concentration was 0.7
M, the immobilized chitosanase showed the highest activity,
11.9 U/g-support. Below this glycidol concentration, the
activity decreased. This could be due to the decrease in
the number of immobilized chitosanases on the support, as
shown in Fig. 6. At glycidol concentrations above 0.7 M,
the activity gradually decreased with increasing glycidol
concentration. The increase in the number of covalent bonds
between the chitosanase and the support seems to be the
reason for this decrease in activity. The number of aldehyde
residues on the support increased with the glycidol concen-
tration, as shown in Fig. 5, whereas the number of immobi-
lized chitosanase on the support was constant, as shown in
Fig. 6. Therefore, the number of covalent bonds between the
chitosanase and the support should increase with increasing
glycidol concentration. This would affect the configuration
of immobilized chitosanase, and then cause the activity of
the immobilized enzyme to gradually decrease.
l
2 4 6 6
Glycldol concentration [M]
FIG. 7. Effect of glycidol concentration on the activity
lized chitosanase.
immobilized chitosanase.
The effects of changes in glycidol concentration on the
thermostability of immobilized chitosanase were also exam-
ined. Immobilized chitosanase was incubated in distilled
water at 60°C. Changes in the residual activity with incu-
bation time are shown in Fig. 8. When the immobilized
chitosanase was prepared at a glycidol concentration of 0.25
M, activity decreased faster than those under higher glyci-
do1 conditions. Under this low-glycidol-concentration con-
dition, since the surface density of aldehyde residues is low,
the number of covalent bonds between the chitosanase and
the support should be relatively small. This would decrease
the thermostability. Above a glycidol concentration of 0.7
M, thermostability was almost the same because residual
activity decreased similarly.
These results proved that a concentration of 0.7 M opti-
mizes the activity level of immobilized chitosanase. On the
other hand, the concentrations above 0.7 M were preferable
for thermostability. From these results, it can be concluded
that the optimal glycidol concentration in the gel activation
is 0.7 M in this system.
Continuous production of chitosan oligosaccharides
using a packed bed reactor In order to examine long-
term stability, immobilized chitosanase was used in a
packed bed reactor for the continuous hydrolysis of chito-
san. On the basis of the results discussed above, the agar gel
support was activated with 0.7 M glycidol. Under this im-
mobilization condition, the relative specific activity of im-
mobilized chitosanase was 19.6%. Chitosan solution (OS%,
pH 5.6) was continuously supplied to the packed bed reactor
at 35°C at a constant flow rate of 5.16x lO~‘llh during 28
reaction days. The time course for the determination of the
percentage of hydrolyzed chitosan in the permeate is shown
in Fig. 9. Chitosan was hydrolyzed continuously and stably
through the packed bed reactor. Initially, the percentage of
hydrolyzed chitosan was 48%. After 28 d of continuous re-
action, the percentage was 44%, a slight decrease from that
observed on the first day. This result clearly shows that the
chitosanase immobilized by the multipoint attachment
method is stable in the reactor.
of immobi-
Chitosan oligosaccharides, particularly pentamer and hex-
amer, are expected to be used as a medical supply, as a
vol.. 93. 2002
FIG. 9. Time course for the determination of percentage of hydro-
lyzed chitosan (closed circle) and total concentration of pentamer and
hexamer (open circle) in the continuous hydrol) sis of chitosan using a
packed bed reactor with immobilized chitosanase.
functional food, and as a biologically active substance that
shows antibacterial activity, antitumor action, and other
physiological activities (2, 5, 9-1 I). The total concentration
of pentamer and hexamer in the permeate is also shown in
Fig. 9. On the first day, the concentration was 1.5 mgiml.
This was 3 1% of the chitosan concentration in the supplied
substrate solution. Even after 28 reaction days, the concen-
tration was 1.3 mgiml. This demonstrated that chitosan oli-
gosaccharides were continuously produced using a column
reactor packed with immobilized chitosanase prepared by
the multipoint attachment method.
Conclusions The immobilization of chitosanase
multipoint attachment to agar gel support was investigated
in order to prepare stable immobilized chitosanase. The im-
mobilization ofchitosanase affected its reaction characteris-
tics in the hydrolysis reaction of chitosan. The optimum pH
range was broadened to between 4 and 6, whereas for free
chitosanase, it was only pH 5.6. The optimum temperature
increased from 60°C to 80°C. Using the same immobilized
chitosanase, the batch hydrolysis of chitosan was repeated
three times. Since all three hydrolysis profiles were almost
identical, the activity did not change throughout the three
batch reactions. The residual activity of the immobilized
chitosanase remained at 95% after 225 h of incubation at
5O”C, whereas for free chitosanase, it decreased to 20%
after 1 h of incubation. The immobilization markedly in-
creased the thermostability of chitosanase. These changes in
the reaction characteristics are favorable for the practical
use of chitosanase in industrial processes. These results
proved that highly stable immobilized chitosanase can be
prepared by the multipoint attachment method.
The effect of changes in glycidol concentration in the ac-
tivation of agar gel support was examined. The surface den-
sity of aldehyde residues increased with increasing glycidol
concentration. The percentage of immobilized chitosanase
increased with increasing glycidol concentration, then lev-
eled off at 85% for glycidol concentrations above 0.7 M.
The maximal activity of immobilized chitosanase was 11.9
U/g-support at a glycidol concentration of 0.7 M. The effect
of changes in glycidol concentration on the chitosanase ac-
tivity was discussed in relation to the number of covalent
bonds between the chitosanase and the support. The thermo-
IMMOBILIZA-I‘ION AND STABILIZATION OF CHITOSANASE 205
stability of chitosanase was almost the same at glycidol con-
centrations above 0.7 M. This showed that the optimal glyc-
idol concentration is 0.7 M in this system.
Long-term continuous production of chitosan oligosac-
charides was accomplished using a column reactor packed
with immobilized chitosanase. The percentage of hydro-
lyzed chitosan after 28 reaction days was 44%. This was a
slight decrease from the 48% observed on the first day. The
total concentration of pentamer and hexamer in the perme-
ate ranged from 1.3 mg/ml to 1.5 mgiml during the 28 reac-
tion days. Thus, chitosan oligosaccharides can be continu-
ously produced using a column reactor packed with immo-
bilized chitosanase.
REFERENCES
I. Shigemasa, Y., Morimoto, M., Saimoto, H., Okamoto, Y.,
and Minami, S.: Applications of chitin and chitosan for bio-
materials, p. 47-54. In Chen, R. H. and Chen, H.-C. (ed.), Ad-
vances in chitin science, vol. 3 (Proc. of the 3rd Asia-Pacific
chitin and chitosan symposium). National Taiwan Ocean Uni-
versity, Keelung (1998).
2. Kendra, D. F. and Hadwiger, L. A.: Characterization of the
smallest chitosan oligomer that is maximally antifungal to
Fusurium soluni and elicits pisatin formation in Pisum safi-
vum. Exp. Mycol., 8,27628 1 ( 1984).
3. Jeon, Y. J. and Kim, S. K.: Production of chitooligosaccha-
rides using an ultrafiltration membrane reactor and their anti-
bacterial activity. Carbohydr. Polym., 41, 133-141 (2000).
4. Jeon, Y. J., Park, P. J., and Kim, S. K.: Antimicrobial effect
of chitooligosaccharides produced by bioreactor. Carbohydr.
by Polym., 44,7 l-76 (200 I ).
5. Uchida, Y., Izume, M., and Ohtakara, A.: Preparation of
chitosan oligomers with purified chitosanase and its applica-
tion, p. 373-382. Proc. 4th Int. Conf. chitin/chitosan. Elsevier
Applied Science, UK (I 989).
6. Tokuyasu, K.: Utilization of chitin and chitosan in food in-
dustry. Nippon Shokuhin Kagaku Kogaku Kaishi, 46, 356-
360 (I 999). (in Japanese)
7. Maezaki, Y., Tsuji, K., Nakagawa, Y., Kawai, Y., Akimoto,
M., Tsugita, T., Takekawa, W., Terada, A., Hara, H., and
Mitsuoka, T.: Hypocholesterolemic effect of chitosan in
adult males. Biosci. Biotech. Biochem., 57, 1439-1444
(1993).
8. Tokura, S. and Tamura, H.: Chitin and its derivatives as bio-
medical materials, p. 55-71. In Chen, R. H. and Chen, H.-C.
(ed.), Advances in chitin science, vol. 3 (Proc. of the 3rd
Asia-Pacific chitin and chitosan symposium). National Taiwan
Ocean University, Keelung (I 998).
9. Tokoro, A., Tatewaki, N., Suzuki, K., Mikami, T., and
Suzuki, S.: Growth-inhibitory effect of hexa-N-acetylchito-
hexaose and chitohexaose against Meth-A solid tumor. Chem.
Pharm. Bull., 36,784-790 (1988).
IO. Suzuki, K., Mikami, T., Okawa, Y., Tokoro, A., Suzuki, S.,
and Suzuki, M.: Antitumor effect of hexa-Wacetylchito-
hexaose and chitohexaose. Carbohydr. Res., 151, 403-408
(1986).
II. Hirano, S., Iwata, M., Yamanaka, K., Tanaka, H., Toda,
T., and Inui, H.: Enhancement of serum lysozyme activity by
injecting a mixture of chitosan oligosaccharid& intravenous&
in rabbits. Aeric. Biol. Chem.. 55.2623-2625 I 1991 j.
12. Horowitz, STT., Roseman, s., and Blumenthal, G. J.: The
preparation of glucosamine oligosaccharides. I. Separation. J.
Amer. Chem. Sot., 79,5046-5049 (I 957).
13. Izume, M. and Ohtakara, A.: Preparation of D-glucosamine
oligosaccharides by the enzymatic hydrolysis of chitosan.
Agric. Biol. Chem., 51. 1189-1191 (1987).
206 ICHIKAWA ET AL.
14. Fukamizo, T., Ohkawa, T., Ikeda, Y., and Goto, S.: Speci-
fic&v of chitosanase from Bacillus uumilus. Biochim. Bio- 20.
phy;. Acta, 1205, 183-188 (1994). 1
15. Bailey, J. E. and Ollis, D. F.: Biochemical engineering fun-
damentals, p. 180. McGraw-Hill, New York (I 986).
16. Jeon Y. J. and Kim S. K.: Continuous production of chito- 21.
oligosaccharides using a dual reactor system. Process Bio-
them., 35,623-632 (2000).
17. Guisan, J. M.: Aldehyde gels as activated support for immo- 22.
bilization of enzymes. Enzyme Microb. Technol., 10, 3755
382 (1988).
18. Blanco, R. Y., Calvette, J. J., and Guisan, J. M.: Immo-
bilization-stabilization of enzymes; variables that control the 23.
intensity of the trypsin (amine)-agarose (aldehyde) multipoint
attachment. Enzyme Microb. Technol., 11, 353-359 (1989). 24.
19. Guisau, J. M., Bastida, A., Cuesta, C., Fernandez-
Laufente, R., and Resell, C. M.: Immobilization-stabiliza-
tion of cc-chymotrypsin by covalent attachment to aldehyde-
J. Broscr. BIOENG.,
agarose gels. Biotechnol. Bioeng., 38, 1144-l 152 (199 1).
Fernandez-Laufente, R., Cowan, D.A., and Wood, A.N.
P.: Hyperstabilization of a thermophilic esterase by multi-
point covalent attachment. Enzyme Microb. Technol., 17,
366372 (1995).
Guisan, J. M. and Blanco, R. M.: Stabilization of trypsin
by multiple-point attachment to aldehyde-agarose gels. Ann.
N.Y. Acad. Sci., 501,67-72 (1987).
Bradford, M. M.: A rapid and sensitive method for the quan-
titation of microgram quantities of protein utilizing the princi-
ple of the protein-dye binding. Anal. Biochem., 72, 248-254
(1976).
Imoto, T. and Yagishita, K.: A simple activity measurement
of lysozyme. Agric. Biol. Chem., 35, 1154-I I56 (I 971).
Matthijs, G. and Schacht, E.: Comparative study of
methodologies for obtaining beta-glucosidase immobilized on
dextran-modified silica. Enzyme Microb. Technol., 19, 601-
605 (1996).