J. Biomedical Science and Engineering, 2011, 4, 692-698
doi:10.4236/jbise.2011.411086 Published Online November 2011 (http://www.SciRP.org/journal/jbise/
JBiSE
).
Published Online November 2011 in SciRes. http://www.scirp.org/journal/JBiSE
Increase in thermal stability of proteins adsorbed on biomass
charcoal powder prepared from plant biomass wastes
Hidetaka Noritomi1, Ryotaro Kai1, Daiki Iwai1, Hirotaka Tanaka1, Reo Kamiya1, Masahiko Tanaka2,
Kohichiroh Muneki3, Satoru Kato1
1Department of Applied Chemistry, Tokyo Metropolitan University, Minami-Ohsawa, Hachioji, Tokyo, Japan;
2EEN Co., Ltd., 2-1-2 Koishikawa Bunkyo-ku, Tokyo, Japan;
3Industry-Academic-Public Cooperation Center, Tokyo Metropolitan University, Minami-Ohsawa, Hachioji, Tokyo, Japan.
Email: noritomi@tmu.ac.jp
Received 2 August 2011; 6 September 2011; accepted 9 October 2011.
ABSTRACT
Thermal stability of lysozyme adsorbed on biomass
charcoal powder (BCP), which was prepared from
plant biomass wastes such as dumped adzuki bean,
bamboo, and wood by pyrolysis without combustion
under nitrogen atmosphere and comminution with a
jet mill, was examined. Adsorbing lysozyme on BCP
could sufficiently prevent proteins from denaturing
and aggregating in an aqueous solution at high tem-
peratures, and enhanced the refolding of thermally
denatured proteins by cooling treatment. The re-
maining activities of lysozyme adsorbed on BCP of
adzuki bean exhibited 51% by cooling treatment af-
ter the heat treatment at 90˚C for 30 min, although
that of native lysozyme was almost lost under the
same experimental conditions. The thermostabiliza-
tion effect of BCP on the remaining activity of ad-
sorbed lysozyme was markedly dependent upon the
kind of plant biomass wastes.
Keywords: Adsorption; Biomass Charcoal Powder; Ly-
sozyme; Refolding; Remaining Activity; Thermal Stabil-
ity
1. INTRODUCTION
Proteins are biomolecules of great importance in the
medical, pharmaceutical, and food fields, since they ex-
hibit their outstanding biological activities under mild
condition. However, most of proteins dissolved in an
aqueous solution are immediately denatured and inacti-
vated at high temperatures due to the disruption of weak
interactions, including ionic bonds, hydrogen bonds, and
hydrophobic interactions, which are prime determinants
of protein tertiary structures [1,2]. In particular, protein
aggregation easily occurs upon the exposure of the hy-
drophobic surfaces of a denatured protein, and this phe-
nomenon becomes the major problem because of the
irreversible inactivation. Thermal denaturation of pro-
teins is a serious problem not only in the separation and
storage of proteins but also in the processes of biotrans-
formation, biosensing, drug production, and food manu-
facturing. Several strategies have so far been proposed in
order to prevent thermal denaturation of proteins. They
include chemical modification, immobilization, genetic
modification, and addition of stabilizing agents. The
addition of stabilizing agents is one of the most conven-
ient methods for minimizing thermal denaturation [3-11].
It has been reported that inorganic salts, polyols, sugars,
amino acids, amino acid derivatives, chaotropic reagents,
and water-miscible organic solvents are available for im-
proving protein stability. However, these additives do
not sufficiently prevent irreversible protein aggregation
or some of them are no longer stable at high tempera-
tures. We have reported that adding water-miscible
aprotic ionic liquids into an aqueous solution of proteins
can effectively hinder the formation of protein aggrega-
tion at high temperatures, and keep high remaining ac-
tivities of proteins [12]. At present, ionic liquids are at
high costs, and their use is limited, since ionic liquids are
organic salts. On the other hand, immobilization of pro-
teins on a support noncovalently and covalently has ex-
tensively been studied [13,14]. Enzymes immobilized
within carbon paste electrodes exhibit the improvement
of thermal stability [15]. Enzymes enhance thermal sta-
bility by adsorbing them onto C60 fullerenes [16]. In or-
der to develop the practical process, the factors such as
the cost of the process, the need for a specific support
material, ensuring that the substrates are not sterically
hindered from diffusing to active site of the immobilized
enzyme where they react at a suitable rate, and so on
must be taken into account [17].
The development of technologies for recycling wastes
is one of the most important challenges to establish re-
H. Noritomi et al. / J. Biomedical Science and Engineering 4 (2011) 692-698 693
cycling society. Wastes are carbonized to be applied to
soil modifiers and humidity materials, and, moreover,
activated carbons are produced from raw materials con-
taining rich carbon [18-22]. In the present work, the
finely grinded biomass charcoal powder (BCP) was pre-
pared from plant biomass wastes such as dumped adzuki
bean, bamboo, and wood by pyrolysis without combus-
tion under nitrogen atmosphere and comminution with a
jet mill. The characteristics of the production process of
BCP in the present work are as follows: First, as plant
biomass wastes are not burned in the production process
of BCP, carbon dioxide emissions are reduced, and the
atom economy of carbon is high. Second, as the produc-
tion process of BCP is carried out at low temperatures
compared to the conventional production process of
charcoal, the energy cost is held down. Thus, BCP is
obtained by environmentally benign process, is at low
costs, and has no toxicity. We have focused on the re-
maining activity of proteins after heat treatment in order
to address a question of whether or not adsorbing pro-
teins on BCP affects the thermal stability of proteins in
aqueous solutions. As a model protein, chicken egg-
white lysozyme has been employed, since it is well in-
vestigated regarding its structure, properties, functions,
and thermal stability [23-25].
2. EXPERIMENTAL
2.1. Materials
Lysozyme from chicken egg while (EC 3.2.1.17, 46400
units/mg solid, MW = 14,300, pI = 11.1) and Micrococ-
cus lysodeikticus (ATCC No. 4698) were purchased
from Sigma-Aldrich Co. (St. Louis, USA).
2.2. Preparation of Biomass Charcoal Powder
The process of preparing biomass charcoal powder (BCP)
from adzuki bean is shown in Figure 1. Under nitrogen
atmosphere, adzuki bean was dried at 180˚C for 2 hr,
was pyrolyzed at 450˚C for 2 hr, was carbonized at
350˚C for 3 hr, and was cooled at 100˚C for 1 hr by py-
rolyzer (EE21 Pyrolyzer, EEN Co. Ltd., Japan). Biomass
charcoal powder (BCP) was obtained by grinding the
resultant biomass charcoal (BC) with jet mill (100AS,
Fuji Sangyo Co. Ltd., Japan). BCP of bamboo or wood
was prepared by the same method.
2.3. Preparation of Lysozyme Adsorbed on
Biomass Charcoal Powder
In order to adsorb lysozyme on BCP of adzuki bean,
0.01 M phosphate buffer solution at pH 7 containing 500
μM lysozyme and 3 g/L BCP of adzuki bean was incu-
bated at 25˚C and 120 rpm for 24 hr. After adsorption,
lysozyme adsorbed on BCP was recovered by filtrating
the mixture with a membrane filter. The amount of ly-
sozyme adsorbed on BCP was calculated by subtracting
the amount of lysozyme included in the supernatant liq-
uid after adsorption from the amount of lysozyme in its
aqueous solution before adsorption. The amount of ly-
sozyme was measured by UV absorption at 280 nm.
2.4. Heat Treatment of Lysozyme Adsorbed on
Biomass Charcoal Powder
A requisite amount of lysozyme adsorbed on BCP was
dispersed in 0.01 M phosphate buffer solution at pH 7.0,
and then the mixture was incubated in thermostated sili-
cone oil bath at 90˚C for 30 min.
2.5. Measurement of Remaining Activity of
Lysozyme
Lysozyme catalyzes hydrolysis of the β-1,4 glycosidic
linkage between the N-acetylmuramic acid and N-ace-
Figure 1. Process of preparation of biomass charcoal powder derived from adzuki bean.
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H. Noritomi et al. / J. Biomedical Science and Engineering 4 (2011) 692-698
694
tylglucosamine components of peptidoglycan. This causes
breakdown and removal of peptidoglycan from the bac-
terium which results in cell bursting or lysis in natural
hypotonic solutions [13]. After the heat treatment, an
aqueous solution of lysozyme adsorbed on BCP was
cooled in thermostated water bath at 25˚C for 30 min.
After 350 μL of the cooled aqueous solution of ly-
sozyme adsorbed on BCP was added to 21 mL of 0.01 M
phosphate buffer solution at pH 7 containing 200 mg/L
Micrococcus lysodeikticus, and then the mixture was
incubated by stirring at 25˚C, the absorbance of the
mixuture was periodically measured at 450 nm by UV/
vis spectrophotometer (UV-1800, Shimadzu Co. Ltd.).
Bacterial lysis obeys a first order reaction. The lysis rate
constant (k) is calculated by

450 450
ln o
A
Akt (1)
where t, 450
o
A
, and A450 are the reaction time, the ab-
sorbance of the substrate solution at 450 nm at T = 0,
and the absorbance of the substrate solution at 450 nm at
T = t, respectively. The remaining activity (R.A.) is de-
fined as
.. 100o
RAk k (2)
where ko and k are the lysis rate constants at 25˚C of
lysozyme adsorbed on BCP before and after heat treat-
ment, respectively.
3. RESULT S AND DISCUSSION
3.1. Thermal Inactivation of Lysozyme
Modest heating causes proteins dissolved in an aqueous
solution to be denatured and inactivated by unfolding of
proteins due to the disruption of weak interactions such
as ionic bonds, hydrogen bonds, and hydrophobic inter-
actions, which are prime determinants of protein tertiary
structures as seen in Figure 2 [1,2,12,26,27]. Moreover,
the intermolecular aggregation among unfolded proteins,
the incorrect structure formation, and the chemical dete-
rioration reactions in unfolded proteins proceed. In par-
ticular, protein aggregation easily occurs upon the ex-
posure of the hydrophobic surfaces of a protein, and this
phenomenon becomes the major problem because of the
irreversible inactivation. On the other hand, when a heated
solution of denatured proteins without protein aggrega-
tion is slowly cooled back to its normal biological tem-
perature, the reverse process, which is renaturation with
restoration of protein function, often occurs. Accord-
ingly, if proteins are steadily adsorbed on BCP, and the
aggregation among unfolded proteins adsorbed on BCP
is sufficiently hindered, it is then expected that unfolded
proteins adsorbed on BCP are refolded by cooling treat-
ment, and the high remaining activity is obtained.
Lysozyme adsorbed on BCP of adzuki bean prepared
Aggregation
Unfolding
Partly Unfolded
Protein Aggregated Protein
Native Protein
Refolding
Covalent C han ges
Deamidation of Asn residues
Hydrolysis of Asp-X peptide bonds
Destruction of cystine residues
Format ion of incorrect structure
Figure 2. Schematic representation of thermal denaturation of
proteins.
in the present work had its characteristics as follows.
The mean diameter of BCP of adzuki bean was 7 μm.
Amount of lysozyme adsorbed on BCP of adzuki bean
was 11 μmol/g (0.16 g/g). As overall BCP concentration
was 3 g/L in an aqueous solution, overall lysozyme con-
centration in the aqueous solution corresponded to 33
μM (0.47 mg/mL). The effectiveness factor, which was
defined as the ratio of the lysis rate constant of lysozyme
adsorbed on BCP of adzuki bean to that of native ly-
sozyme, exhibited 0.55.
Figure 3 shows photographs of aqueous solutions
containing native lysozyme, the mixture of lysozyme
and BCP of adzuki bean, and lysozyme adsorbed on
BCP of adzuki bean before and after heat treatment was
carried out at 90˚C for 30 min as an accelerated test.
Native lysozyme solution immediately became turbid
due to the formation of protein aggregation, as soon as
heat treatment was carried out, as shown in Figure 3(d).
It has been reported that the precipitation due to protein
aggregation is observed above 10 μM lysozyme [25]. As
lysozyme concentration in the present work was 33 μM
which was three times higher than that, the formation of
protein aggregation was enhanced. BCP of adzuki bean
spontaneously dispersed in an aqueous solution by add-
ing BCP of adzuki bean into an aqueous solution, since
BCP of adzuki bean had good wettability to water as
seen in Figure 3(b). When the mixture of lysozyme and
BCP of adzuki bean was prepared by adding BCP of
adzuki bean into lysozyme solution, and heat treatment
was immediately carried out, the precipitation consisting
of denatured proteins and BCP of adzuki bean was ob-
served due to the aggregation of free denatured proteins,
as shown in Figure 3(e). Lysozyme adsorbed on BCP of
adzuki bean easily dispersed in an aqueous solution by
adding lysozyme adsorbed on BCP of adzuki bean into
an aqueous solution, as seen in Figure 3(c). After the
heat treatment of the solution of lysozyme adsorbed on
BCP of adzuki bean, the state of dispersion of lysozyme
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H. Noritomi et al. / J. Biomedical Science and Engineering 4 (2011) 692-698 695
adsorbed on BCP of adzuki bean in the solution was
similar to that before heat treatment, and any aggrega-
tion was not observed in the solution, as shown in Fig-
ure 3(f). Figure 4 shows the remaining activities of na-
tive lysozyme, the mixture of lysozyme and BCP of ad-
zuki bean, and lysozyme adsorbed on BCP of adzuki
bean after heat treatment at 90˚C for 30 min. Native ly-
sozyme almost lost its activity after heat treatment. The
remaining activity in the mixture of lysozyme and BCP
of adzuki bean exhibited 2%. On the other hand, the re-
maining activity in lysozyme adsorbed on BCP of adzuki
bean showed 51%.
Heat treatment
90˚C, 30 min
(a) (b) (c) (d) (e) (f)
Figure 3. Photographs of lysozyme solutions before and after
heat treatment at 90˚C for 30 min: (a) an aqueous solution
containing native lysozyme before heat treatment; (b) an
aqueous solution containing native lysozyme solution and BCP
of adzuki bean before heat treatment; (c) an aqueous solution
containing lysozyme adsorbed on BCP of adzuki bean before
heat treatment; (d) an aqueous solution containing native ly-
sozyme after heat treatment; (e) an aqueous solution containing
native lysozyme solution and BCP of adzuki bean after heat
treatment; (f) an aqueous solution containing lysozyme ad-
sorbed on BCP of adzuki bean after heat treatment. Overall
concentrations of lysozyme and BCP were 33 μM and 3 g/L,
respectively.
0 204060801
L y s ads or bed on BCP
M i x t ur e of Lys and BCP
Nat ive L ys
Rema in in g activity (%)
00
Figure 4. Effect of preparation mode on remaining activity
after heat treatment at 90˚C for 30 min: Native Lys, native
lysozyme; Mixture of Lys and BCP, the mixture of native ly-
sozyme solution and BCP of adzuki bean; Lys adsorbed on
BCP, lysozyme adsorbed on BCP of adzuki bean. Overall con-
centrations of lysozyme and BCP were 33 μM and 3 g/L, re-
spectively.
3.2. Refolding of Lysozyme Adsorbed on
Biomass Charcoal Powder
Figure 5 shows the time course of remaining activity in
lysozyme adsorbed on BCP of adzuki bean at 25˚C after
the heat treatment at 90˚C for 30 min. The remaining
activity of lysozyme adsorbed on BCP of adzuki bean
exhibited 30% just after heat treatment, increased with
incubation time, and reached a plateau at 30 min, respec-
tively. Immobilization of proteins improves thermal sta-
bility of proteins by the rigidity of protein molecules due
to the interaction of protein molecules with supports [1,
2,13]. In thermal denaturation of lysozyme without pro-
tein aggregation, when the hydrophobic core of proteins
is exposed, but the disulfide bonds keep intact, denatured
proteins gradually refold to their native structures on
cooling after thermal denaturation [28-32]. In the present
system, it was suggested that adsorbing proteins on BCP
hindered aggregation of thermally denatured proteins,
caused some of proteins to be intact at high temperatures,
and enhanced the refolding of thermally denatured pro-
teins by cooling treatment, as seen in Figure 6.
3.3. Dependence of the Remaining Activity of
Lysozyme Adsorbed on Biomass Charcoal
Powder on the Temperatu re of Heat
Treatment
Figure 7 shows the relationship between the temperature
of heat treatment and the remaining activity of lysozyme
adsorbed on BCP of adzuki bean after the heat treatment
for 30 min. As seen in the figure, the dependence of the
remaining activity on the temperature exhibited the sig-
moid curve. The remaining activity of native lysozyme
dramatically decreased with an increase in temperature
in the range from 60˚C to 90˚C, and was then lost at
Figure 5. Time dependence of remaining activity of lysozyme
adsorbed on BCP of adzuki bean on cooling at 25˚C after heat
treatment at 90˚C for 30 min. After heat treatment, the aqueous
solution of lysozyme adsorbed on BCP of adzuki bean was
incubated in a water bath thermostated at 25˚C. Overall con-
centrations of lysozyme and BCP were 33 μM and 3 g/L, re-
pectively. s
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H. Noritomi et al. / J. Biomedical Science and Engineering 4 (2011) 692-698
Copyright © 2011 SciRes.
696
BCP
Heat Treatment
Protein
Adsorption
Cooling
Ref old ed Protein a ds o rbed on BCP
Protein adsorbed on BCPDen at ured Prot ein
adsorbed on BCP
Figure 6. Schematic representation of thermostabilization of proteins adsorbed on BCP.
temperatures of 90˚C or higher. On the other hand, the
remaining activity of lysozyme adsorbed on BCP of ad-
zuki bean dropped in the range from 70˚C to 98˚C, and
still exhibited 3% at 98˚C. These results indicated that
adsorbing lysozyme on BCP of adzuki bean effectively
improved the thermal stability of lysozyme at high tem-
peratures.
3.4. Relationship between the Remaining
Activity of Lysozyme Ads o rbed on Biomass
Charcoal Powder and the Kind of Biomass
Charcoal Powder Figure 7. Thermal denaturation curves of native lysozyme and
lysozyme adsorbed on BCP of adzuki bean. The aqueous solu-
tion of native lysozyme or lysozyme adsorbed on BCP of ad-
zuki bean was incubated in a silicone oil bath thermostated at
requisite temperature for 30 min. Overall concentrations of
lysozyme and BCP were 33 μM and 3 g/L, respectively.
In order to extend our study, the remaining activities of
lysozyme adsorbed on BCP prepared from different
plant biomass wastes after heat treatment at 90˚C for 30
min were investigated. Thermal stability of lysozyme
was sufficiently enhanced by adsorbing lysozyme on
BCP of bamboo or wood as well as the case of BCP of
adzuki bean, as shown in Figure 8, while several percent
of remaining activity was obtained by mixing lysozyme
and BCP of bamboo or wood. The remaining activity of
lysozyme adsorbed on BCP of adzuki bean was best
among BCP examined in the present work. Amount ad-
sorbed was almost same among three different materials,
although the mean diameter of BCP of wood was 2.5
times larger than others, as seen in Table 1. On the other
hand, as the water wettability of BCP of adzuki bean
preferred to that of bamboo or wood, the dispersibility of
BCP of adzuki bean in an aqueous solution was better
than that of bamboo or wood.
0 10203040506
BCPofwood
BCPofbamboo
BC Pofad zu k ibe an
Remain ingactivity(%)
0
Figure 8. Effect of kind of BCP on remaining activity of ly-
sozyme adsorbed on BCP after heat treatment at 90˚C for 30
min. Overall concentrations of lysozyme corresponded to 33
μM on BCP of adzuki bean, 27 μM on BCP of bamboo, and 36
μM on BCP of wood, respectively. Overall concentration of
BCP was 3 g/L.
4. CONCLUSIONS
We have demonstrated that the remaining activity of
lysozyme adsorbed on BCP of adzuki bean is sufficiently
JBiSE
H. Noritomi et al. / J. Biomedical Science and Engineering 4 (2011) 692-698 697
Ta b le 1 . Mean diameter of BCP and amount of lysozyme ad-
sorbed on BCP.
Kind of BCP Mean diameter (μm) Amount adsorbed (μmol/g)
adzuki bean 7 11
bamboo 7 9
wood 18 12
maintained after heat treatment at high temperatures,
compared to the case of native lysozyme, since adsorb-
ing proteins on BCP hinders aggregation of thermally
denatured proteins effectively, causes some of proteins
to be intact at high temperatures, and enhances the re-
folding of thermally denatured proteins by cooling treat-
ment. Regarding thermal stabilization effect of BCP on
the remaining activity of adsorbed lysozyme after heat
treatment, BCP of adzuki bean was much superior to
BCP of bamboo or wood. The results obtained at the
present work indicated that BCP had sufficient biocom-
patibility.
5. ACKNOWLEDGEMENTS
This work was supported by grants from Japan Science and Technol-
ogy Agency (AS2111014D).
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