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Immobilization and Stabilization of Chitosanase Multipoint Attachment To Agar Gel Support
Immobilization and Stabilization of Chitosanase Multipoint Attachment To Agar Gel Support
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
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.
Chitosan is part of the biomass that occupies large por- thermore, this enzymatic hydrolysis is carried out under
tions of the earth. Although it would be desirable to utilize mild conditions, so it is easy to control the progress of the
chitosan extensively as a biomaterial (I), most chitosan is reaction. Therefore, the method which involves the use of
not being utilized. Chitosan itself has various physiological chitosanase is favorable for the production of oligosaccha-
functions (2-S). It is expected to be used as a medical sup- rides.
ply, as a functional food, and as a biologically active sub- When the enzyme is used in industrial processes, it is
stance. In order to exploit use of chitosan, it is important to generally preferred that it be in the immobilized form for
use the substances derived from it. In particular, chitosan the following reasons (15). The enzyme is more stable in
oligosaccharides, which are produced by the partial hydrol- this form. The enzyme can be used for long-term produc-
ysis of chitosan, have physiological functions similar to tion, because it can be reused. Continuous operation be-
those of chitosan (2-5, 9-l 1). They are soluble in water and comes easier. Since it is easy to separate the enzyme from
easily digested because their molecular weights are lower the reaction, the process of separating products can be sim-
than that of chitosan. plified. In particular, in the case of chitosanase, immobiliza-
Chitosan oligosaccharides have been produced by the tion is highly advantageous from the viewpoint of operating
chemical hydrolysis of chitosan using acid (12). This chem- cost, because chitosanase is expensive. The immobilization
ical hydrolysis yields large amounts of monosaccharides as of chitosanase will enable its long-term use in producing
a byproduct, because of the difficulty of controlling the oligosaccharides. The preparation of immobilized chitosan-
progress of the reaction. This, in turn, causes a low yield of ase by the adsorption method has been reported (16). How-
oligosaccharides. However, it is known that the hydrolysis ever, long-term uses for stable immobilized chitosanase
by chitosanases from Bacillus sp. no. 7-M ( 13) and Bacillus have yet to be developed.
sp. BN-262 (14) does not produce monosaccharides. Fur- In this study, the immobilization of chitosanase by the
multipoint attachment method was investigated in order to
* Corresponding author. e-mail: mukataka@sakura.cc.tsukuba.ac,ip prepare stable immobilized chitosanase. In this method, the
phone: +XI -(0)298-53-4607 [ax: +8 I -(0)298-53.4605 enzyme is immobilized by contact with the support material
201
202 ICHIKAWAETAL. J. Brasc~. BIOENC;..
through multiple covalent bonds. This method enabled the pmol equivalent of D-glucosamine in I min under the above condi-
preparation of the highly stable immobilized enzyme for tions.
penicillin G acylase (17), trypsin (1 S), a-chymotrypsin (19), Continuous production of chitosan oligosaccharides using a
and thermophilic esterase (20). In those studies, an agarose packed bed reactor Immobilized chitosanase was prepared by
the method described above. The glycidol concentration in the ac-
gel was used as the support material. However, we exam-
tivation of the agar gel support was 0.7 M. Fifteen g wet weight of
ined the use of a cheaper agar gel as a support because of
activated gel was suspended in 120 ml of 0.1 M borate buffer con-
the cost advantage. 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-
MATERIALS AND METHODS 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-
Materials Chitosanase (EC 3.2.1.132) from BaciZZus pumilus tions of pentamer and hexamer of chitosan oligosaccharides in the
BN-262 was supplied by Meiji Seika Kaisha Ltd. (Tokyo). The permeate were measured by HPLC with a Capcell Pak NH, col-
enzyme contained 13.2% protein as determined by the Bradford umn ($4.6~250 mm; Shiseido Co. Ltd., Tokyo).
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- RESULTS AND DISCUSSION
I ,4-glycosidic linkages of GlcN-GlcN and GlcNAc-GlcN (GlcN,
D-gh_icosamine; GlcNAc, f%acetyl-D-glucosamine) (14). As a Optimum pH of immobilized chitosanase In order to
substrate, 100% deacetylated chitosan from Funakoshi Co. Ltd. confirm the effectiveness of the method of immobilization
(Tokyo) was used. Agar powder, glycidol (2,3-epoxypropanol), by multipoint attachment to the agar gel support, the activity
sodium borohydrate, and other chemicals were purchased from of chitosanase was examined under various pH and temper-
Wako Pure Chemical Industries Ltd. (Osaka). ature conditions.
Activation of agar gel support Glyoxyl agar gel (Agar-O- The effects of pH on the relative activity of free and im-
CH,-CHO) as an activated support was prepared by etherification mobilized chitosanases are shown in Fig. 1. The optimum
of 6% agar gel with glycidol and further oxidation of the resulting pH for immobilized chitosanase was in the range of 4 to 6,
glyceryl agar gel (Agar-0-CH,-CHOH-CH,OH) by NaIO,. In a whereas for free chitosanase, it was 5.6. Hence, the opti-
typical activation, 20 g of agar gel cut into 1 mm3 cubes was ad-
mum pH range was broadened after the multipoint attach-
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
ment immobilization. This wider optimum pH range is most
to the mixture. The mixture was stirred for 18 h at 25”C, then likely attributable to the stabilization of the enzyme stmc-
washed with water through a Biichner funnel with suction. The ture by the multipoint linkages between the enzyme and the
resulting glyceryl agar gel was suspended in 100 ml of 35 mM support.
NaIO,, and stirred for I h. The resulting activated agar gel was Optimum temperature of immobilized chitosanase
washed with water and stored in distilled water at 4°C until use. The effects of temperature on the relative activity of free
The aldehyde content of the activated agar gel support was de- and immobilized chitosanases are shown in Fig. 2. The opti-
termined to be equivalent to the amount of periodate consumed mum temperature of immobilized chitosanase was SOY,
(I 7). The periodate that was not consumed in the activation of the 20°C higher than that of free chitosanase. Under the high-
gel was measured by titration with KI.
temperature condition, the enzyme was inactivated by ther-
Immobilization of chitosanase Fifteen g wet weight of acti-
mal denaturation. Even at a higher temperature, the immo-
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 bilized chitosanase could retain its active structure com-
at 25°C. After a given contact time, 0.24g of solid NaBH, was pared to the free enzymes, because of the multipoint link-
added and gently stirred for 30 min at 25°C. This borohydride ages to the gel support.
reduction constitutes a suitable end-point of the enzyme-support The immobilization of chitosanase by multipoint attach-
interactions because this reduction produces two complementary ment broadened the optimum pH range and increased the
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. 0-
3 3.5 4 4.5 5 5.5 6 6.5
One ml of 40 &ml chitosanase solution, or 1 g wet weight of the
immobilized chitosanase was added to 100 ml of chitosan solution PH
at pH 5.6. The reaction mixture was incubated at 35°C. The FIG. I. Effect of pH on the activity of free (open circle) and im-
amount of reducing sugar formed by the hydrolysis reaction was mobilized (closed circle) chitosanases. The concentration of rlycidol
measured by the Schales method (23). One unit of activity of in the activation of the agar gel support was 4.8 M. The acti&y was
chitosanase was defined as the amount of enzyme that liberated I 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.
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.,
14. Fukamizo, T., Ohkawa, T., Ikeda, Y., and Goto, S.: Speci- agarose gels. Biotechnol. Bioeng., 38, 1144-l 152 (199 1).
fic&v of chitosanase from Bacillus uumilus. Biochim. Bio- 20. Fernandez-Laufente, R., Cowan, D.A., and Wood, A.N.
phy;. Acta, 1205, 183-188 (1994). 1 P.: Hyperstabilization of a thermophilic esterase by multi-
15. Bailey, J. E. and Ollis, D. F.: Biochemical engineering fun- point covalent attachment. Enzyme Microb. Technol., 17,
damentals, p. 180. McGraw-Hill, New York (I 986). 366372 (1995).
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oligosaccharides using a dual reactor system. Process Bio- by multiple-point attachment to aldehyde-agarose gels. Ann.
them., 35,623-632 (2000). N.Y. Acad. Sci., 501,67-72 (1987).
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bilization of enzymes. Enzyme Microb. Technol., 10, 3755 titation of microgram quantities of protein utilizing the princi-
382 (1988). ple of the protein-dye binding. Anal. Biochem., 72, 248-254
18. Blanco, R. Y., Calvette, J. J., and Guisan, J. M.: Immo- (1976).
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intensity of the trypsin (amine)-agarose (aldehyde) multipoint of lysozyme. Agric. Biol. Chem., 35, 1154-I I56 (I 971).
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19. Guisau, J. M., Bastida, A., Cuesta, C., Fernandez- methodologies for obtaining beta-glucosidase immobilized on
Laufente, R., and Resell, C. M.: Immobilization-stabiliza- dextran-modified silica. Enzyme Microb. Technol., 19, 601-
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