Chitosan-Soyprotein Interaction as Determined

by Thermal Unfolding Experiments

Tomoko TAKEUCHI,1 Kazuhisa MORITA,1 Tsutomu SAITO,2

Wataru KUGIMIYA,2 and Tamo FUKAMIZO1;y

1Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan2Food Science Research Institute, Tsukuba R&D Center, Fuji Oil Co., Ltd., 4-3 Kinunodai,

Yawara, Tsukuba-gun, Ibaraki 300-2497, Japan

Received January 10, 2006; Accepted February 9, 2006; Online Publication, July 23, 2006

[doi:10.1271/bbb.60015]

Chitosan interaction with soybean �-conglycinin �3

was investigated by thermal unfolding experiments

using CD spectroscopy. The negative ellipticity of the

protein was enhanced with rising solution temperature.

The transition temperature of thermal unfolding of the

protein (Tm) was 63.4�C at pH 3.0 (0.15M KCl). When

chitosan was added to the protein solution, the Tm value

was elevated by 7.7 �C, whereas the Tm elevation upon

addition of chitosan hexamer (GlcN)6 was 2.2�C. These

carbohydrates appear to interact with the protein

stabilizing the protein structure, and the interaction

ability could be evaluated from the Tm elevation.

Similar experiments were conducted at various pHs

from 2.0 to 3.5, and the Tm elevation was found to be

enhanced in the higher pH region. We conclude that

chitosan interacts with �-conglycinin through electro-

static interactions between the positive charges of the

chitosan polysaccharide and the negative charges of the

protein surface.

Key words: chitosan; �-conglycinin; protein-carbohy-

drate interaction; thermal unfolding

Chitosan is a �-1,4-linked polysaccharide of 2-amino-2-deoxy-D-glucopyranose (glucosamine, GlcN), and ithas been used as a food ingredient due to its variousbiological functions, such as antibacterial, antifungal,and hypocholesterolemic activities.1–3) When chitosan isintroduced into certain foods, the polysaccharide inter-acts with various molecules, which play important rolesin food processing and functional properties. Foodproteins are the most probable candidates to interactwith chitosan, because electrostatic interactions arehighly possible between the polyamines of chitosanand the negative charges of food proteins. In addition, asmall amount of N-acetylglucosamine (GlcNAc) resi-

dues, which occur in the chitosan polysaccharide chain,possibly interacts with GlcNAc-recognizing proteins,such as lysozymes, chitinases, and lectins.4,5) Theseinteractions might significantly affect the processing andfunctional properties of the food proteins. Nevertheless,the chitosan–protein interaction has not yet been fullyunderstood.On the other hand, �-conglycinin contributes signifi-

cantly to the food processing properties of soybean, dueto its gel-forming and emulsifying abilities. This proteinalso attracts public attention because of its hypocholes-terolemic effect.6) The nutritional and physicochemicalproperties and the crystal structure of �-conglycininhave been studied by several investigators.7–9) Mixingthe soyprotein with chitosan might produce a new foodmaterial possessing an integrated function and improvethe food processing properties. Before producing such anew material, however, it is important to elucidate themolecular mechanism of chitosan-soyprotein interac-tion. In recent years, we have been working withchitosan-degrading enzymes, such as endochitosanaseand exo-�-glucosaminidase.10–12) In the course of thesestudies, we have developed several strategies for ex-amining chitosan–protein interaction.13–15) Among thesestrategies, thermal unfolding experiments are mostconvenient for simple evaluation of the ability ofchitosan interaction with the enzyme protein.13) In thisstudy, we attempted to evaluate chitosan interactionability with the soyprotein �-conglycinin by thermalunfolding experiments. The experiments were conduct-ed at various pHs to achieve insight into the interactionmechanism.The chitosan used for the experiments was obtained

by nitrous acid degradation16) of commercial chitosan(Chitosan 10B, Funakoshi Co.), of which the degree ofN-acetylation was less than 1% as determined by 1H-

y To whom correspondence should be addressed. Tel: +81-742-43-8237; Fax: +81-742-43-8976; E-mail: fukamizo@nara.kindai.ac.jp

Abbreviations: GlcN, 2-amino-2-deoxy-D-glucopyranose; GlcNAc, 2-acetamido-2-deoxy-D-glucopyranose; (GlcN)6, �-1,4-linked hexasaccharide

of GlcN; CD, circular dichroism

Biosci. Biotechnol. Biochem., 70 (7), 1786–1789, 2006

Note

NMR spectroscopy. First, soybean �-conglycinin �3

prepared by the method of Morita et al.17) was employedfor far-UV CD measurements at 20 �C and 70 �C.Typical CD spectra are shown in Fig. 1. At highertemperatures, the negative ellipticity was enhanced inthe spectral region above 205 nm. The thermal unfoldingtransition appears to result in a conformational change

of the protein, similar to the change induced byethanol.8) After annealing the protein solution to 20 �C,the spectrum did not recover to that in the folded/nativestate. Thus the thermal unfolding transition was found tobe irreversible.To obtain the thermal unfolding curve of the protein,

the CD value at 210 nm was monitored, raising thesolution temperature at a rate of 1 �C/min. To facilitatecomparison between the unfolding curves obtained, theexperimental data were normalized as follows: Fractionsof unfolded protein at individual temperatures werecalculated from the CD value by linearly extrapolatingthe pre- and post-transition baselines into the transitionzone, and plotted against the temperature. The resultsare shown in Fig. 2. At pH 3.0, the transition temper-ature of thermal unfolding of the protein (Tm) was63.4 �C. When chitosan (1.0 mmoles in GlcN monomerunits) was added to the protein solution (1.7 nmoles),the Tm value was elevated by 7.7 �C, even though noappreciable change was observed in the CD spectrum.The Tm elevation (�Tm) was 2.2 �C when chitosanhexamer (GlcN)6 (1.0 mmoles in GlcN monomer units)was added instead of chitosan polysaccharide. Thesecarbohydrates appear to interact with the protein,stabilizing the protein structure. Much larger effectswere observed by Tremblay et al.,14) who examined thechitosan interaction in Streptomyces sp. N174 chitosa-nase. They found that the chitosanase Tm value waselevated by 11.3 �C and 14.8 �C upon the addition of

-1200

-1000

-800

-600

-400

-200

0

200

400

208 216 224 232 240 248 256

[θ]

(deg

.cm

2 .dm

ol-1

)

Wavelength (nm)

20oC(after annealing)

70oC

20oC(before unfolding)

Fig. 1. Far-Ultraviolet CD Spectra of �-Conglycinin �3.

The spectra were recorded in 0.15M KCl solution, in which the

pH was adjusted to 3.0 with concentrated HCl using a Jasco J-720

spectropolarimeter. The protein concentration was 10mM.

0 3020Temperature (oC)

Fra

ctio

n un

fold

ed

0

0.2

0.4

0.6

0.8

1.0

0 3020

Fra

ctio

n un

fold

ed

Temperature (oC)

0

0.2

0.4

0.6

0.8

1.0

0

0.2

0.4

0.6

0.8

1.0

0 3020Temperature (oC)

0

0.2

0.4

0.6

0.8

1.0

0 40 50 60 70 803020Temperature (oC)

pH 2.0

pH 3.0 pH 3.5

Fra

ctio

n un

fold

ed

Fra

ctio

n un

fold

ed

pH 2.5

40 50 60 70 80

40 50 60 70 80

40 50 60 70 80

Fig. 2. Thermal Unfolding Curves Obtained from CD Spectroscopy at Various pHs.

The solution conditions were the same as in Fig. 1. , �-conglycinin �3; , �-conglycinin �3 + (GlcN)6; , �-conglycinin �3 + chitosan.

Chitosan-Soyprotein Interaction 1787

(GlcN)6 and chitosan, respectively. The enzyme speci-ficity might result in such a larger �Tm. In a lower pHregion (pH 2.5), �Tm was found to be suppressed inboth (GlcN)6 and chitosan. The Tm value was notelevated at pH 2.0. On the other hand, in a higher pHregion (pH 3.5), �Tm induced by chitosan polysaccha-ride was slightly enhanced (�Tm ¼ 8:8 �C). In this case,the unfolding transition could not be monitored com-pletely (Fig. 2), due to a limitation of a thermocontrolunit. Thus the Tm value was obtained only from the firsthalf of the transition zone by assuming that theellipticity in the unfolded state is identical to thatobtained without chitosan. All of the Tm and �Tm valuesobtained are summarized in Table 1. Thermodynamicparameters could not be obtained, because of theirreversibility of the unfolding transition.

As is evident from the table, the �Tm value becamegreater with pH elevation. Since the isoelectric point of�-conglycinin monomer has been reported to be 5.7–6.0,18) the positive charges are predominant in theprotein surface, and most of the carboxylates are ionizedat pH 2.0. This situation rather makes an electrostaticrepulsion between the soyprotein and the positivecharges of chitosan. With pH elevation, however, thecarboxylates gradually become deprotonated and neg-atively charged. The chitosan comes to interact electro-statically with the soyprotein, and this results in thegreater �Tm values. Hence we conclude that thechitosan-�-conglycinin interaction is caused mainly byelectrostatic interaction between the positive charges ofthe chitosan polyamines and the negative charges of theprotein surface of �-conglycinin �3. The effect was moreintense in chitosan polysaccharide than in the hexamer.This is due to the difference in the number of positivecharges per chitosan molecule. This information mightcontribute to advances in food processing technologyusing chitosan polysaccharide.

References

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Table 1. �Tm Induced by Chitosan and Chitosan Hexamer Inter-

action with �-Conglycinin �3

pH Tm �Tm

2.0 �-conglycinin 57.6 —

+ chitosan hexamer 57.5 �0:1

+ chitosan 56.4 �1:2

2.5 �-conglycinin 61.4 —

+ chitosan hexamer 62.7 1.3

+ chitosan 66.0 4.6

3.0 �-conglycinin 63.4 —

+ chitosan hexamer 65.6 2.2

+ chitosan 71.1 7.7

3.5 �-conglycinin 70.3 —

+ chitosan hexamer 72.0 1.7

+ chitosan 79.1� 8.8

�The unfolding transition could not be monitored completely. The Tm values

were obtained using data only from the first half of the transition zone, on

the assumption that the ellipticity in the unfolded state is identical to that

obtained without chitosan.

1788 T. TAKEUCHI et al.

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Brzezinski, R., and Fukamizo, T., Role of acidic aminoacid residues in chitooligosaccharide-binding to Strepto-myces sp. N174 chitosanase. Biochem. Biophys. Res.Commun., 338, 1839–1844 (2005).

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18) Thanh, V. H., and Shibasaki, K., �-conglycinin fromsoybean proteins: isolation and immunological andphysicochemical properties of the monomeric forms.Biochim. Biophys. Acta, 490, 370–384 (1977).

Chitosan-Soyprotein Interaction 1789