Journal of Biomaterials and Nanobiotechnology, 2012, 3, 446-451
http://dx.doi.org/10.4236/jbnb.2012.34045 Published Online October 2012 (http://www.SciRP.org/journal/jbnb)
Immobilization of Lysozyme on Biomass Charcoal Powder
Derived from Plant Biomass Wastes
Hidetaka Noritomi1*, Reona Ishiyama1, Ryotaro Kai1, Daiki Iwai1, Masahiko Tanaka2, Satoru Kato1
1Department of Applied Chemistry, Tokyo Metropolitan University, Tokyo, Japan; 2EEN Co., Ltd., Tokyo, Japan.
Received August 16th, 2012; revised September 27th, 2012; accepted October 5th, 2012
ABSTRACT
Biomass charcoal powder (BCP) was used as a carrier matrix for immobilization of chicken egg white lysozyme. BCP
was derived from plant biomass wastes such as dumped adzuki beans by pyrolysis without combustion under nitrogen
atmosphere and grinding with a jet mill. The amount of lysozyme immobilized on BCP of adzuki beans by adsorption
was 11 μmol/g (0.16 g/g) at pH 7.0. The optimum pH values for free and immobilized lysozyme activities were 6.8 and
7.2, respectively. The optimum temperature for both free and immobilized lysozyme activities was 25˚C. The half-life
of immobilized lysozyme exhibited 1.8-fold compared to that of free lysozyme at 5˚C. Moreover, the half life of immo-
bilized lysozyme was 7 times greater than that of lysozyme at 90˚C.
Keywords: Immobilization; Biomass Charcoal Powder; Lysozyme; Activity; Stability
1. Introduction
Immobilization of enzymes onto various water-insoluble
carriers has attracted continuous attention in the fields of
biotechnology, fine chemistry, pharmaceutical, and bio-
sensor [1,2]. Some of desired carrier characteristics are
indicated as follows: 1) large surface area; 2) perme-
ability; 3) hydrophilic character; 4) insolubility; 5) che-
mical, mechanical, and thermal stability; 6) high rigidity;
7) suitable shape and particle size; 8) resistance to micro-
bial attack; 9) regenerability; 10) low cost. Various solid
materials such as natural polymers, proteins, synthetic
polymers, minerals, and fabricated materials have so far
been proposed as a carrier. It has been well known that
the performances of immobilized enzyme such as activity,
specificity, and stability largely depend on the structure
of supports and the method of immobilization. On the
other hand, the development of technologies for recy-
cling wastes is one of the most important challenges to
establish recycling society. Wastes are carbonized to be
applied to humidity material, and activated carbons [3,4].
Specifically, much attention has been paid on the char-
coal produced from plant biomass wastes by means of
pyrolysis in order to amend the soil, since it exhibits the
excellent priming effect [5]. When the charcoal prepared
from plant biomass wastes is used as a soil modifier, it
has been called biochar. However, plant biomass wastes
have not sufficiently been recycled yet, compared to other
wastes, although an enormous amount of plant biomass
wastes has been discharged in the world. Moreover, the
development in the high value-added function of char-
coal of plant biomass wastes has been desired.
Immobilization of enzymes has been performed by
means of adsorption, covalent binding, entrapment, en-
capsulation, crosslinking, and so on. Among these meth-
ods, immobilization of enzymes by adsorption has been
considered as the simplest and most economical method.
Moreover, enzymes immobilized through adsorption have
the benefit of wide applicability, since they may keep
their native structures and functions.
In the present work, the finely grinded biomass char-
coal powder (BCP) was derived from plant biomass
wastes such as dumped adzuki beans by pyrolysis with-
out combustion under nitrogen atmosphere and grinding
with a jet mill. In order to assess whether BCP is suitable
as a carrier, we have investigated the effects of pH and
temperature on the activity and stability of enzymes on
BCP. As a model protein, chicken egg-white lysozyme
has been employed, since it is well investigated regarding
its structure, properties, and functions [6].
2. Materials and Method
2.1. Materials
Lysozyme from chicken egg white (EC 3.2.1.17, 46400
units/mg solid, MW = 14300, pI = 11) and Micrococcus
*Corresponding author.

Immobilization of Lysozyme on Biomass Charcoal Powder Derived from Plant Biomass Wastes 447
lysodeikticus (ATCC No. 4698) were purchased from
Sigma-Aldrich Co. (St. Louis, USA).
2.2. Preparation of Biomass Charcoal Powder
Under nitrogen atmosphere, dumped adzuki beans were
dried at 180˚C for 2 hr, were pyrolyzed at 450˚C for 2 hr,
were carbonized at 350˚C for 3 hr, and were then cooled
at 100˚C for 1 hr by pyrolyzer (EE21 Pyrolyzer, EEN Co.
Ltd., Japan). Biomass charcoal powder (BCP) was ob-
tained by grinding the resultant biomass charcoal (BC)
with jet mill (100 AS, Fuji Sangyo Co. Ltd., Japan).
2.3. Characterization of BCP
All samples were outgassed at 200˚C for 15 hr prior to
the carbon dioxide adsorption measurements. The spe-
cific surface area of BCP was calculated with use of the
Brunauer-Emmett-Teller (BET) method using a micro-
pore system (ASAP2010, Shimadzu Co. Ltd., Japan).
The ζ potentials for biomass charcoal powder were
measured by electrophoretic light scattering (ELS-Z2,
OTSUKA Electronics Co. Ltd., Japan).
The SEM micrograph was obtained using a scanning
electron microscope (JSM-7500FA, JEOL, Japan) oper-
ating at 15 kV. The sample for SEM was prepared on a
carbon tape without vapor deposition.
The surface of BCP was analyzed by x-ray photoelec-
tron spectroscopy (XPS) (Quantum-2000, ULVAC-PHI
Co. Ltd., Japan) operating at x-ray beam size of 100 μm.
2.4. Immobilization of Lysozyme onto BCP
As a typical procedure, 0.01 M phosphate buffer solution
at pH 7 containing 500 μM lysozyme and 3 g/L BCP of
adzuki beans was incubated at 25˚C and 120 rpm for 24
hr. After adsorption, the mixture was filtrated with a
membrane filter (pore size: 0.1 μm, Millipore Co. Ltd.,
USA). The amount of lysozyme adsorbed on BCP was
calculated by subtracting the amount of lysozyme in-
cluded in the supernatant liquid after adsorption from the
amount of lysozyme in its aqueous solution before ad-
sorption. The amount of lysozyme was measured at 280
nm by UV/vis spectrophotometer (UV-1800, Shimadzu
Co. Ltd., Japan).
2.5. Measurement of Activity of Free and
Immobilized Lysozyme
Lysozyme activity was determined using Micrococcus
lysodeikticus as a substrate [7]. Three hundred and fifty μL
of 0.01 M phosphate buffer solution at pH 6.2 or 7.0 of
free and immobilized lysozyme (33 μM) was added to 21
mL of 0.01 M phosphate buffer solution at pH 6.2 or 7.0
containing 200 mg/L Micrococcus lysodeikticus, and the
mixture was incubated by stirring at 25˚C. The absorbance
of the mixture was periodically measured at 450 nm by
UV/vis spectrophotometer (UV-1800, Shimadzu Co. Ltd.).
2.6. Effect of pH on Activity of Free and
Immobilized Lysozyme
The effect of pH on the activity of free and immobilized
lysozyme was assayed in the pH range from 5.0 to 9.0 at
25˚C. The buffer solutions used in the present work were
an acetate buffer solution at pH 5, phosphate buffer solu-
tions from pH 6 to less than pH 8, and borate buffer solu-
tions at pH 8 and 9. The concentration of buffer solution
was prepared at 0.01 M. Data for the activity of ly-
sozyme are the average of triple measurements. The rela-
tive activity was defined by the ratio of activity to
maximum activity.
2.7. Effect of Temperature on Activity and
Stability of Free and Immobilized Lysozyme
In order to examine the effect of temperature on the ac-
tivity of free and immobilized lysozyme, the enzyme
activity was measured in pH 6.2 at various temperatures.
In order to evaluate the storage stability of free and
immobilized lysozyme, the activity of free or immobi-
lized lysozyme was measured after free or immobilized
lysozyme was stored in 0.01 M phosphate buffer solution
at pH 6.2 and 5˚C for appropriate time. The residual ac-
tivity was determined by the ratio of activity to initial
activity.
In order to assess the thermal stability of free and im-
mobilized lysozyme at high temperatures, the activity of
free or immobilized lysozyme was measured after free or
immobilized lysozyme was stored in 0.01 M phosphate
buffer solution at pH 7.0 and 90˚C for appropriate time,
and then was cooled at 25˚C for 30 min. The residual
activity was obtained by the ratio of activity after heat
treatment to activity before heat treatment.
3. Results and Discussion
3.1. Characterization of BCP
The characteristics of the production process of BCP in
the present work are indicated as follows: First, as plant
biomass wastes are not burned in the process of pyrolysis,
volatile carbon compounds such as phenols and lignin
are gradually removed out, and nonvolatile carbon com-
pounds are carbonized. Consequently, as seen in Figure
1, the morphology in BC of adzuki beans is kept, similar
to that in native adzuki beans. Carbon dioxide emissions
are reduced, and the atom economy of carbon is high,
compared with the conventional production process of
charcoal. Second, as the production process of BCP is
carried out at low temperatures compared to the conven-

Immobilization of Lysozyme on Biomass Charcoal Powder Derived from Plant Biomass Wastes 448
(c)(b)(a)
Figure 1. Photographs of BCP of adzuki beans: (a) Adzuki
beans; (b) Biomass charcoal (BC) of adzuki beans obtained
after adzuki beans were pyrolyzed; (c) Biomass charcoal
powder (BCP) of adzuki beans obtained after BC was
grinded.
tional production process of charcoal, the energy cost is
held down. Thus, BCP is obtained at low costs by envi-
ronmentally benign process, and has no toxicity.
Figure 2 shows the scanning electron micrograph of
BCP of adzuki beans. As seen in the figure, the rough
state like craters is observed on the surface of BCP of
adzuki beans. BET surface area of BCP of adzuki beans
was 204 m2/g. The order of BET surface area of BCP
was similar to that of the conventionally prepared char-
coal [8]. The mean diameter of BCP was 7.3 μm. The ζ
potential of BCP of adzuki beans was –47.6 mV at pH 7.
Figure 3 shows the ratio of elements of the surface of
BCP detected by x-ray photoelectron spectroscopy (XPS).
The main element is carbon, and oxygen and nitrogen
atoms are also located on the surface of BCP of adzuki
beans to some extent. From narrow scan spectra of XPS,
the chemical states of carbon were mainly C-C and C-H,
while as chemical states of carbon with oxygen and ni-
trogen, C-O, O-C-O, C=O, COOH, and C-N were de-
tected. These results indicate that BCP has the polar mi-
croenvironment on its surface. Accordingly, when BCP
was added into an aqueous solution, BCP exhibited good
wettability, and was steadily dispersed in an aqueous
solution.
3.2. Immobilization of Lysozyme on BCP
We have employed lysozyme as a model protein. Fig-
ure 4 shows the time course of the amount of lysozyme
adsorbed onto BCP of adzuki beans at pH 7 and 25˚C.
Amount of lysozyme adsorbed onto BCP of adzuki beans
increased with an increase in adsorption time, reached a
plateau around 24 h, and was 11 μmol/g (0.16 g/g). As
overall BCP concentration was 3 g/L in an aqueous solu-
tion, overall lysozyme concentration in the aqueous solu-
tion corresponded to 33 μM (0.48 mg/mL). It is sug-
gested that the adsorption of lysozyme on BCP is mainly
attributed to the electrostatic interaction between the ne-
gative-charged BCP and the positive-charged lysozyme,
since the isoelectric point of lysozyme is pH 11. From
the result, the adsorption of lysozyme onto BCP was car-
ried out for 24 h.
100 nm
Figure 2. SEM image of BCP of adzuki beans.
0
20
40
60
80
100
CNONaMgSiPSClKCa
Elemental ratio (atom%)
Element
Figure 3. Elemental ratios (atom%) of BCP of adzuki beans
measured by XPS.
0
2
4
6
8
10
12
14
0 10203040506
Amount adsorbed（μmol/g）
Time（hr）
0
Figure 4. Time dependence of amount of lysozyme adsorbed
on BCP of adzuki beans.
The activity of lysozyme immobilized on BCP of ad-
zuki beans was 22100 U/mg of protein, while that of free
lysozyme was 40200 U/mg of protein. Therefore, the
effectiveness factor, which was defined as the ratio of the
activity of lysozyme immobilized on BCP of adzuki
beans to that of free lysozyme, exhibited 0.55.

Immobilization of Lysozyme on Biomass Charcoal Powder Derived from Plant Biomass Wastes 449
3.3. Effect of pH on Activity of Free and
Immobilized Lysozyme
The effect of pH on the activity of the free and immobi-
lized lysozyme was assayed in the pH range from 5.0 to
9.0 at 25˚C. As shown in Figure 5, the optimum pH of
immobilized lysozyme depicted pH 7.2, while that of
free lysozyme was pH 6.8, similar to the case reported
before [7]. The enzyme molecules possess basic, neutral,
and acidic groups. Consequently, the intact enzyme con-
tains both positively or negatively charged groups. The
state of these ionic groups is influenced by the pH around
enzyme molecules. Accordingly, the interaction between
the surface of BCP and the reaction mixture needs to be
taken into consideration. This interaction may cause the
fluid environment adjacent to the surface in contact with
the enzyme to differ substantially from the bulk fluid
environment surrounding the immobilized enzyme [9].
For instance, a charged support will increase local con-
centrations of oppositely charged ions. In the present
case, negative-charged BCP may increase local concen-
trations of hydrogen ions. As a result, the relationship
between bulk solution pH and observed catalytic activity
can be shifted to high pH, compared to the pH-activity
function observed in solution, as seen in the figure.
3.4. Effect of Temperature on Activity of Free
and Immobilized Lysozyme
The effect of reaction temperature on the activity of free
and immobilized lysozyme was tested in the temperature
range from 15˚C to 55˚C at pH 6.2. As shown in Figure
6, the optimum temperature for both free and immobi-
lized lysozyme is found to be obtained at 25˚C. Accord-
ing to the Arrhenius equation, the initial rate becomes
higher at higher temperatures, while the thermal denatu-
ration of enzymes simultaneously proceeds with increas-
ing temperature. Consequently, the profile of activity
0
20
40
60
80
100
120
456789
Relative Activity (%)
pH (-)
10
Free Lysozyme
Immobilized Lysozyme
Figure 5. The effect of pH on the activity of free and immo-
bilized lysozyme at 25˚C. The relative activity at optimum
pH is taken as 100%.
0
20
40
60
80
100
120
10 20 30 40 50 60
Relative activity (%)
Temperature (℃)
Free Lysozyme
Immobilized Lysozyme
Figure 6. The effect of temperature on the activity of free
and immobilized lysozyme at pH 6.2. The activity of free
and immobilized lysozyme was measured in the tempera-
ture range from 15˚C to 55˚C. The relative activity at opti-
mum temperature is taken as 100%.
exhibits an optimum temperature. At the optimum tem-
perature or higher, the activity of immobilized lysozyme
gradually decreased with increasing reaction temperature,
whereas that of free lysozyme steeply dropped with in-
creasing temperature. The interaction between the sur-
face of BCP and the enzyme molecule may result in the
maintenance of the activity of immobilized lysozyme.
3.5. Storage Stability of Free and Immobilized
Lysozyme
Since the storage and applications of immobilized en-
zymes often need to be carried out in the artificial envi-
ronment, which is extremely different from the physio-
logical environment, it is important to investigate the
storage stability of immobilized enzymes. Figure 7 shows
time course of residual activity of free and immobilized
lysozyme through the storage at pH 6.2 and 5˚C. The
residual activity of immobilized lysozyme was superior
to that of free lysozyme. As seen in the figure, the rela-
tionship of the residual activities of free and immobi-
lized lysozyme with storage time can be correlated by
first-order kinetics. Table 1 represents rate constants and
half lives of inactivation of free and immobilized ly-
sozyme calculated from the fitting curves in Figure 7.
The half-life of immobilized lysozyme was about twice
longer than that of free lysozyme. This result indicates
that immobilizing lysozyme onto BCP enhances the
storage stability at low temperatures.
3.6. Thermal Stability of Free and
Immobilized Lysozyme
Heating time directly enhances the thermal inactivation
of proteins. Figure 8 shows time course of residual ac-
tivity of free and immobilized lysozyme through the heat
treatment at pH 7.0 and 90˚C as an accelerated test. Free
lysozyme solution immediately became turbid due to the

Immobilization of Lysozyme on Biomass Charcoal Powder Derived from Plant Biomass Wastes 450
Table 1. Rate constants and half-lives of inactivation of ly-
sozyme at 5˚C.
Samples Rate constant (day–1) Half life (day)
Free lysozyme 0.0625 14.3
Immobilized lysozyme 0.0485 26.2
1
10
100
0246810
Residual activity (%)
Time (day)
12
Free Lysozyme
Immobilized Lysozyme
Figure 7. Time course of residual activity of free and immo-
bilized lysozyme at pH 6.2 and 5˚C.
formation in the aggregation of thermally-denatured pro-
teins, as soon as heat treatment was carried out. On the
other hand, the white protein aggregation and the cohe-
sion among carriers immobilizing lysozyme were not
observed in the solution dispersing immobilized ly-
sozyme during the heat treatment. When proteins dis-
solved in an aqueous solution are placed at high tem-
peratures, most of proteins are immediately unfolded due
to the disruption of weak interactions including ionic
effects, hydrogen bonds, and hydrophobic interactions,
which are prime determinants of protein tertiary struc-
tures [10,11]. In addition, the intermolecular aggregation
among unfolded proteins and the chemical deterioration
reactions in unfolded proteins proceed. In particular,
protein aggregation easily occurs upon the exposure of
the hydrophobic surfaces of a protein, and this pheno-
menon becomes the major problem because of the fast
irreversible inactivation. In the present work, immobi-
lizing lysozyme onto BCP could inhibit the formation of
protein aggregation. The residual activity of free lyso-
zyme was almost lost for 30 min, while that of immo-
bilized lysozyme exhibited about 50%. As seen in the
figure, the relationship of the residual activities of free
and immobilized lysozyme with heat treatment time can
be correlated by first-order kinetics. Table 2 represents
rate constants and half lives of inactivation of free and
immobilized lysozyme calculated from the fitting curves
in Figure 8. The half-life of immobilized lysozyme ex-
hibited 7-fold, compared to that of free lysozyme. The
robust thermal stability of immobilized lysozyme may be
Table 2. Rate constants and half-lives of inactivation of ly-
sozyme at 90˚C.
Samples Rate constant (min–1) Half life (min)
Free lysozyme 0.168 4
Immobilized lysozyme0.027 28
0.1
1
10
100
0 204060801
Residual activity (%)
Time (min)
00
Free Lysozyme
Immobilized Lysozyme
Figure 8. Time course of residual activity of free and immo-
bilized lysozyme through the heat treatment at pH 7.0 and
90˚C.
attributable to the suitable interaction of lysozyme with
the surface of BCP.
Carriers immobilizing lysozyme do not attach each
other through the heat treatment, indicating that the ag-
gregation among thermally-denatured proteins immobi-
lized on each carrier does not arise. On the other hand, if
the aggregation of thermally-denatured proteins immobi-
lized on the same carrier is formed, it is probably that
more amount of lysozyme immobilized on BCP pro-
motes the formation of protein aggregation, resulting on
less residual activity. In order to elucidate the relation
between the amount of lysozyme immobilized on BCP
and the residual activity, the residual activity of immobi-
lized lysozyme having a different immobilized amount
was assayed after the heat treatment at 90˚C for 30 min.
As seen in Figure 9, the residual activity shows around
50% regardless of the amount of lysozyme immobilized.
This result indicates that the irreversible aggregation
among thermally-denatured proteins immobilized on the
same carrier hardly occurs under the present experimen-
tal condition.
4. Conclusion
BCP had polar functional groups such as carbonyl and
carboxyl groups, and exhibited negative ζ potential at
pH 7.0. Lysozyme was sufficiently immobilized on BCP
of adzuki beans by adsorption. The optimum pH of im-
mobilized lysozyme was higher than that of free ly-
sozyme. The half-life of immobilized lysozyme exhibited
1.8-fold compared to that of free lysozyme at 5˚C, while
the half life of immobilized lysozyme was 7 times greater
than that of lysozyme at 90˚C. The increase in stability

Immobilization of Lysozyme on Biomass Charcoal Powder Derived from Plant Biomass Wastes 451
0
20
40
60
80
100
024681012
Residual activity (%)
Amount of lysozyme immobilized on BCP (μmol/g)
14
Figure 9. The effect of the amount of lysozyme immobilized
on the residual activity of immobilized lysozyme after the
heat treatment at 90˚C for 30 min.
shown by BCP-immobilized lysozyme would be encou-
raging for its choice in industrial application such as the
sterilizing treatment.
5. Acknowledgements
This work was supported by a Grant-in-Aid for Scientific
Research (C) from Japan Society for the Promotion of
Science (No. 24561013) and a Grant-in-Aid for Scien-
tific Research from Japan Science and Technology
Agency (No. AS2111014D).
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