Increase in thermal stability of proteins adsorbed on biomass charcoal powder prepared from plant biomass wastes

doi:10.4236/jbise.2011.411086

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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: [email protected]

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.

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kt (1)

where t, 450

, 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

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 (%)

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

H. Noritomi et al. / J. Biomedical Science and Engineering 4 (2011) 692-698

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

BCPofwood

BCPofbamboo

BC Pofad zu k ibe an

Remain ingactivity(%)

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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