ORIGINAL PAPER

Thermal denaturation of recombinant human lysozymefrom rice: effect of pH and comparison with human milk lysozyme

Eduardo Castillo • Indira Franco • Marıa D. Perez •

Miguel Calvo • Lourdes Sanchez

Received: 28 June 2011 / Revised: 21 September 2011 / Accepted: 12 October 2011 / Published online: 29 October 2011

� Springer-Verlag 2011

Abstract Thermal denaturation of recombinant human

lysozyme from rice has been studied by differential scan-

ning calorimetry at acidic (4.2), neutral (7.2) and basic

(9.2) pH levels at various heating rates, and it has been

compared with thermal denaturation of human lysozyme

isolated from milk at the same pH levels at a heating rate of

10 �C/min. Data obtained from heat-induced unfolding and

subsequent refolding after heating indicate that thermal

denaturation of both lysozymes undergoes a two-state

process. The maximum temperature of the endothermic

peaks and the enthalpy change of denaturation indicate that

recombinant and milk lysozymes possess similar thermo-

stability, which is higher at acidic than at neutral pH. On

the other hand, both proteins are more thermolabile at basic

pH. Lysozyme from human milk shows a higher tendency

to aggregate than recombinant human lysozyme from rice

during the thermal denaturation process.

Keywords Human recombinant lysozyme � Human milk

lysozyme � Differential scanning calorimetry �Thermodynamical parameters

Introduction

Lysozymes are hydrolytic enzymes that cleave the b-(1,4)

glycosidic bond between N-acetylmuramic acid and

N-acetylglucosamine in peptidoglycan, the major bacterial

cell wall polymer. Lysozymes are divided into six families

according to genetical, immunological and structural sim-

ilarities [1, 2]. Most lysozymes are implicated in defensive

bactericidal systems in a wide range of taxonomically

diverse organisms including, among others, plants, inver-

tebrate and vertebrate animals [2]. One of these lysozyme

families is the c-type group, to which hen egg-white

lysozyme (HEWL) and human lysozyme (hLz) belong.

HEWL makes up to 3.4% of the total egg-white protein

content, whereas hLz is present in different human secre-

tions, such as tears, saliva and milk. Particularly, human

milk is rich in lysozyme, although this concentration has

great individual and temporal variations. The concentration

of lysozyme in colostrum is about 100 mg/L, then

decreases to 20–40 mg/L and in the second half of the first

year is increased, especially in the weaning stage, when it

reaches concentrations of 800–1,000 mg/L [3]. This con-

centration is about 3,000 times higher than that of cow’s

milk, which ranges between 0.05 and 0.21 mg/mL [4, 5]. In

addition, the enzymatic activity of hLz is 10 times greater

than that of bovine lysozyme [6], and it has been reported

that both proteins are antigenically different [7], which

suggests the existence of structural differences between

them.

Lysozyme plays a role in the development of the gas-

trointestinal microbiota in the newborn. It has been found

that the predominant anaerobic flora in infants fed breast

milk is composed of Bifidobacterium bifidum, contrary to

that observed to those who are fed formula, in which the

major microbiota is composed of coliform bacteria [8]. One

of the reasons for which the intestinal flora of infants fed

breast milk is rich in Bifidobacterium, bacteria essential for

health and well-being of the child, may be due in part to the

high content of hLz. Lysozyme plays a dual role: by

hydrolyzing the peptidoglycan, it kills the harmful bacteria

E. Castillo � I. Franco � M. D. Perez � M. Calvo �L. Sanchez (&)

Tecnologıa de los Alimentos, Facultad de Veterinaria,

Universidad de Zaragoza, Miguel Servet 177,

50013 Zaragoza, Spain

e-mail: lousanchez@unizar.es

123

Eur Food Res Technol (2011) 233:1067–1073

DOI 10.1007/s00217-011-1612-8

and, in addition, it induces the liberation of N-acetylglu-

cosamine, which is an important bifidogenic factor. Thus,

despite the high content of lysozyme in breast milk, the

number of bifidobacteria is increased in the intestinal tract,

suggesting that these bacteria are resistant to the antibac-

terial activity of this enzyme [9]. It has also been docu-

mented that breastfed babies suffer fewer infections, and

these are shorter than those of infants fed with infant for-

mula [10]. In Japan, studies were conducted supplementing

infant formula with HEWL, and it was found that the

intestinal microbiota of infants fed with that milk was

richer in bifidobacteria than that of those fed with com-

mercially available infant formulae [11]. These results

suggest that supplementation of infant formula with lyso-

zyme would be very convenient. However, the addition of

egg protein that is the main source of lysozyme for com-

mercial use in infant food may cause allergic reactions, so

it would be desirable to supplement infant formula with

hLz [12]. Furthermore, the structure of hen egg lysozyme,

as well as its enzymatic and antibacterial activities, is

different to those of human milk.

Human lysozyme has been isolated from milk, pancreatic

juice [13], neutrophils [14] and urine of hemodialysis

patients [15]. Since human milk is the main source of this

protein, it is obviously impossible to obtain enough hLz to be

added to commercial products. However, hLz has been

expressed successfully in transgenic cattle [16] and in Pichia

pastoris [17]. The expression of recombinant proteins in rice

is an alternative to produce large quantities of proteins with

low cost. The use of plants for large-scale expression of

human proteins offers some advantages over microbial or

mammalian cell culture systems, because transgenic plants

do not carry or propagate human diseases or mammalian

viral vectors [18]. In addition, the expression of recombinant

proteins in edible plant tissues could reduce protein purifi-

cation requirements for some applications [19]. Rice is a

particular well-suited crop for this purpose, since their seeds

allow for long-term storage and hold GRAS status. There-

fore, rice-based foods are regarded as hypoallergenic, and

rhLz obtained from transgenic rice could be used in a par-

tially purified form as a baby formula ingredient [20].

The use of rhLz as a food additive or ingredient requires

the knowledge of its thermal stability; therefore, the pur-

pose of this study was the determination of the thermal

stability of rhLz by differential scanning calorimetry

(DSC), a technique that provides useful information con-

cerning factors that affect protein stability [21]. The ther-

moresistance of rhLz was determined at three different pH

levels: acidic, neutral and basic, since no thermal stability

studies of rhLz has been carried out at pH above the acidic

range. In addition, the thermoresistance of rhLz has been

compared with that of lysozyme from human milk by the

same technique.

Materials and methods

Proteins and human milk

Recombinant hLz isolated from rice was kindly provided

by Ventria Bioscience (Sacramento, CA) as a white powder

of a high purity above 98% as determined by native PAGE.

Human milk samples were generously donated by

healthy mothers by mediation of Dr. Luis Ros from the

Hospital Miguel Servet of Zaragoza (Spain).

Isolation of lysozyme from human milk

Milk was skimmed by centrifugation at 2,5009g at 4 �C for

30 min. Caseins were separated by acid precipitation adding

0.1 M HCl to reach a pH of 4.5 and subsequent centrifuga-

tion at 2,5009g at room temperature for 30 min. The clear

whey obtained was dialyzed against 50 mM sodium acetate

buffer, pH 4.5 overnight. Afterward, to avoid undesired

interactions between hLz and other whey proteins, 100 lL

of Triton X-100 was added to 250 mL of the dialyzed whey,

and then, it was applied on a SP-Sepharose column

(/1.5 cm 9 8 cm) previously equilibrated with 50 mM

sodium acetate buffer, pH 4.5 containing 50 mM NaCl. The

column was washed with the same buffer, and the bound

proteins were sequentially eluted with 50 mM sodium ace-

tate buffer, pH 4.5 and a NaCl stepwise gradient (0.2, 0.5 and

1 M). Fractions were collected and analyzed by SDS–

PAGE, and those with the highest content of hLz were

concentrated by ultrafiltration and applied to a Sephadex

G-50 column (/1 cm 9 80 cm), which was eluted using

1 M NaCl and 10 mM potassium phosphate buffer, pH 7.4.

The fractions collected were analyzed by SDS–PAGE, and

those containing pure hLz were dialyzed against distilled

water and lyophilized.

Enzymatic activity assay

Lysozyme activity was determined by a turbidimetric

technique, measuring the decrease in absorbance at 450 nm

versus time of a Micrococcus lysodeikticus suspension

(Sigma Aldrich, Steinheim, Germany). A fresh suspension

of M. lysodeikticus (0.15 mg/mL) in 100 mM phosphate

buffer, pH 6.2, was used as substrate. For each sample,

950 lL of substrate was placed in a cuvette to which 50 lL

of sample was added. Absorbance decrease was recorded

versus time and the activity of each sample calculated from

the slope of the curve.

Differential scanning calorimetry (DSC)

Solutions of rhLz and of hLz at a concentration of 100 mg/

mL were prepared in three different buffers: 100 mM

1068 Eur Food Res Technol (2011) 233:1067–1073

123

sodium acetate buffer, pH 4.2; 15 mM potassium phosphate,

pH 7.4; and 100 mM sodium carbonate buffer, pH 9.2. For

the DSC study of hLz, 100 mM sodium carbonate buffer, pH

8.8, and 100 mM sodium phosphate buffer, pH 8.0, were also

employed. Samples and references (10 lL) were introduced

into aluminum pans (TA Instruments, New Castle, USA) and

sealed for analysis. The references consisted of pans con-

taining the corresponding buffer. DSC analysis of both

lysozymes was performed in a DuPont Thermal Analyser

(model DSC 10, Nemours, Germany), using a Thermal

Analyst 2000 System. DSC scans were programmed in the

temperature range of 35–110 �C at different heating rates: 2,

3, 4, 5, 7, 10 and 20 �C/min for rhLz and at 10 �C/min for

hLz. Lysozymes were analyzed using 3–5 replicates.

Pans containing samples of rhLz from rice treated at pH

7.2 and at a heating rate of 10 �C/min were left to cool at

4 �C during 24 h and then rescanned in the same condi-

tions of the first measurement in order to evaluate a pos-

sible renaturation of the protein after heating.

From the transition peaks obtained by DSC, several

thermodynamic parameters were obtained, such as the

temperature of maximum heat absorption (Tp), the onset

temperature (Ti) and the enthalpy change (DHap) for the

denaturation process of proteins. The last parameter (DHap)

was calculated by integrating the peak area using a straight

baseline drawn from the onset to the end of thermal tran-

sition. The value of van’t Hoff enthalpy was calculated

according to the following equation:

DHVH ¼4RT2

p

DT1=2

where Tp is the temperature of maximum heat absorption,

DT1/2 is the endothermic peak width at its half-height and R

is the universal gas constant.The activation energy (Ea) for

the denaturation process of rhLz from rice was calculated

according to Kissinger’s method [22]. Thus, the activation

energy of a denaturation process can be calculated from the

heating rate (b) and the temperature of maximum heat

absorption (Tp) using the Arrhenius equation:

LnbT2

p

!¼ �Ea

R� 1

T

where R is the universal gas constant and the temperatures

are given in Kelvin degrees.

Results

Isolation of lysozyme from human milk

Figure 1 shows the chromatogram and the enzymatic

activity as well as the corresponding SDS–PAGE of frac-

tions eluted with 50 mM sodium acetate buffer, pH 4.5

containing 0.2 and 0.5 M NaCl. The elution with 1 M NaCl

did not result in any measurable peak. As it is shown by

SDS–PAGE, besides hLz other whey proteins were present

in the two peaks obtained. The fractions with the highest

hLz content and enzymatic activity (fractions 7–10 and

31–43) were concentrated and subjected to gel filtration.

After the analysis of fractions by SDS–PAGE, it was found

that only fraction C contained pure lysozyme (Fig. 2),

because this protein interacts with other whey proteins and

cannot be easily separated from them. This fraction was

concentrated by ultrafiltration, dialyzed against distilled

water and lyophilized.

DSC of rhLz from rice to hLz from milk

Thermal stability of rhLz was studied at three different pH

levels, 4.2, 7.2 and 9.2, and at different heating rates by

means of DSC. Thermal stability of hLz was also studied at

the same pH levels as rhLz at a heating rate of 10 �C/min.

Figure 3 shows the thermograms obtained for rhLz

treated at the three different pH levels at a heating rate of

10 �C/min. All measurements resulted in thermograms

showing a major endothermic peak at all pH levels and

heating rates studied, with the exception of samples treated

at pH 9.2 at heating rates of 2 and 3 �C/min, which did not

give any endothermic peak. This fact indicates that rhLz is

less stable at basic than at acidic or neutral pH.

The thermograms obtained for hLz are shown in Fig. 4.

They present also one single denaturation peak at acidic and

neutral pH levels, but it was not possible to obtain any

thermogram at pH 9.2. In addition, the study was also carried

out at pH values of 8.8 and 8.0, but in any case, a thermogram

could be obtained at a heating rate of 10 �C/min.

The thermodynamic parameters obtained at a heating

rate of 10 �C/min for the denaturation of rhLz and hLz at

the three pH levels are shown in Table 1. For both lyso-

zymes, the temperature of maximum heat absorption (Tp)

decreases as the pH rises, which points out to a greater

stability of both proteins at acidic pH. In addition, Tp

values at acidic and neutral pH levels are the same for both

lysozymes, which indicate that the thermal stability of rhLz

is comparable with that of hLz at those pH levels.

The thermogram obtained in the renaturation study of

rhLz carried out at pH 7.2 and at a heating rate of 10 �C/

min also showed one single peak, with an onset tempera-

ture (Ti) of 69.95 �C and a temperature of maximum heat

absorption of 76.63 �C, both values close to that of the first

measurement, 70.45 and 76.74 �C, respectively. However,

the enthalpy change of the second measurement was of

75.79 kJ/mL, about 70% lower than that of the value

obtained in the first run. All these data indicate that despite

aggregation that is evidenced by the DHap/DHVH ratio and

by the drop in the enthalpy change in the second

Eur Food Res Technol (2011) 233:1067–1073 1069

123

measurement, rhLz is able to refold to the native state after

heat denaturation.

Figure 5 shows that the temperature of maximum heat

absorption (Tp) of rhLz increased as the heating rate

increased. By using the equations obtained from the adjust-

ment to a linear regression plot, it was possible to calculate

the Tp values for each pH at a theoretical heating rate of 0 �C/

min. The results obtained were 76.57 �C at pH 4.2, 74.27 �C

at pH 7.2 and 64.52 �C at pH 9.2. However, no relationship

was found between the enthalpy change of the denaturation

process (DHap) and the heating rate (data not shown).

The activation energy (Ea) of the denaturation process of

rhLz was calculated according to the Kissinger’s method,

as described in the Materials and methods section (Fig. 6).

It was found that Ea was higher at neutral than at acidic or

basic pH and was slightly lower at basic than at acidic pH

(Table 1).

Discussion

Human lysozyme is a small basic globular protein belonging

to the family of the c-type lysozymes. Its tertiary structure

comprises two domains divided by a cleft where the active

site is located [23]. When globular proteins are heated, they

unfold cooperatively following a so-called two-state pro-

cess, from the native to the denatured state, with no ther-

modynamic stable species between them [24]. The overall

enzyme denaturation reaction has been described as native

protein $ denatured protein ? aggregated protein.

Denaturation of proteins, which is only the first step in

heat inactivation, is a reversible process, while aggregation

is an irreversible phenomena and leads to the inactivation

of the enzyme [25]. The unfolding is the first step of the

thermal denaturation of a protein and involves disruption of

intramolecular hydrogen bonds and is, therefore, an endo-

thermic process that can be evidenced by the endothermic

peak observed in the DSC thermograms. On the other hand,

aggregation phenomena and the breaking up of hydro-

phobic interactions take also place during denaturation

being exothermic processes and diminishing the observed

enthalpy values (DHap) [26].

The thermograms obtained for rhLz and for hLz showed

one single peak that indicates that the two domains of the

two types of lysozyme studied undergo simultaneous

denaturation, so the denaturation of rhLz and hLz appears

0 10 20 30 40 50 60 70A B C D E F G H 0

0,2

0,4

0,6

0,8

1

1,2

1,4

Fraction number

Ab

s (2

80 n

m)

0

0,2

0,4

0,6

0,8

1

1,2

1,4

En

zym

atic

Act

ivit

y (u

a)

0.2 M NaCl 0.5 M NaCl

Fig. 1 Chromatogram and 12.5% SDS–PAGE corresponding to the

isolation of hLz from human milk on SP-Sepharose. Filled squareabsorbance at 280 nm, filled triangle enzymatic activity. A fraction

n. 4, B fraction n. 8, C fraction n. 11, D fraction n. 32, E fraction n. 36,

F fraction n. 43, G fraction n. 46, H recombinant hLz (0.5 mg/mL)

A B C D E F G H 0

0,5

1

1,5

2

2,5

3

3,5

0 10 20 30 40

Fraction number

Ab

s (2

80 n

m)

Fig. 2 Chromatogram and

12.5% SDS–PAGE

corresponding to the gel

filtration on Sephadex G-50 of

fractions 7–10 and 31–46

obtained from the SP-Sepharose

chromatography (Fig. 1).

A fraction n. 7, B fraction n. 8,

C fraction n. 12, D fraction n.

13, E fraction n. 15, F human

whey, G fractions 7–10 and

31–46 from the SP-Sepharose

chromatography (Fig. 1),

H recombinant hLz

(0.5 mg/mL)

1070 Eur Food Res Technol (2011) 233:1067–1073

123

to be a two-state process. To ensure this fact is necessary to

confirm the refolding of the proteins after their denatur-

ation. Lysozyme from human milk was reported to refold

from denatured to native state as it has been already

reported by Dobson et al. [27]. These authors showed a

rapid refolding of hLz denatured by guanidium chloride

when compared with HEWL, which was attributed to a fast

formation of a hydrophobic core in the human protein. We

have verified the renaturation of rhLz by means of DSC, as

we have observed that the Ti and Tp values obtained for the

previous heat-denatured rhLz are close to the values of the

first calorimetric run.

In the present work, we have observed that Tp values are

similar for rhLz and hLz at acidic and neutral pH, indi-

cating a similar thermostability of both proteins at those pH

levels. In addition, Tp values decreased as the pH rose for

the two proteins, which indicate that they are more stable at

acidic than at neutral or basic pH. This behavior was

observed in previous works, being the value we have

obtained at acidic pH in accordance with those of others

[28, 29]. The thermal stability of rhLz and hLz at acidic

and neutral pH levels is also confirmed by other thermo-

dynamic parameters. The DHVH values we have obtained

for rhLz and hLz are also very similar at acidic and neutral

pH levels and decrease as the pH rises. In addition, the

DHVH values at acidic pH are in good agreement with those

reported for hLz at the same pH [28, 29]. On the other

hand, no data have been published for the thermal dena-

turation of hLz in the basic pH range. Most of the studies

made on hLz have been carried out at acidic pH, probably

because the protein turns unstable at higher pH levels

40 60 100

Temperature (ºC)

En

do

ther

mic

hea

t fl

ow

0.2 W/g

A

B

C

20 80 120

Fig. 3 DSC of recombinant rhLz from rice at a heating rate of 10 �C/

min and different pH: A pH = 4.2, B pH = 7.2, C pH = 9.2

60 100 120

Temperature (ºC)

En

do

ther

mic

hea

t fl

ow

0.2 W/g

A

B

20 40 80

Fig. 4 DSC of hLz from milk at a heating rate of 10 �C/min and

different pH levels: A pH = 4.2, B pH = 7.2

Table 1 Thermodynamic parameters of the denaturation of recombinant hLz and human milk lysozyme by DSC at a heating rate of 10 �C/min

Ti (�C) Tp (�C) DHap (kJ/mol) DHVH (kJ/mol) DHap/DHVH Ea (kJ/mol)

Recombinant human lysozyme

pH = 4.2 74.19 ± 0.41 79.32 ± 0.36 254.22 ± 48.72 569.54 ± 45.99 0.45 407.13

pH = 7.2 70.45 ± 0.60 76.74 ± 0.43 256.61 ± 17.50 469.65 ± 26.92 0.55 435.7

pH = 9.2 60.46 ± 1.00 66.85 ± 0.70 169.11 ± 20.97 397.33 ± 28.11 0.43 404.3

Human milk lysozyme

pH = 4.2 75.90 ± 0.60 80.08 ± 0.32 128.69 ± 25.36 530 ± 12.04 0.18 –

pH = 7.2 69.24 ± 0.21 76.97 ± 0.16 101.35 ± 2.96 404.52 ± 53.92 0.25 –

Measurements were taken at acidic, neutral and basic pH levels; no data could be obtained for human milk lysozyme at basic pH. Onset

temperature (Ti), temperature of maximum heat absorption (Tp), enthalpy change (DHap), van’t Hoff enthalpy (DHVH) and activation energy (Ea)

for the denaturation process. The values shown are the mean ± SD of at least four repetitions

Eur Food Res Technol (2011) 233:1067–1073 1071

123

[30–32]. In fact, in our study, it was not possible to obtain

any thermogram for hLz at the three basic pH levels

studied: 9.2, 9.0 and 8.8. In addition, it was found that Tp

and DHap values were lower at basic pH for rhLz, which

confirms the thermal sensitivity of lysozyme at basic pH.

The lower DHap values obtained for hLz may be due to a

higher tendency of hLz to aggregate, since the DHVH val-

ues are very similar. These slight differences observed in

the thermodynamic values obtained for denaturation of

rhLz compared to those of hLz at basic pH may be due to

differences in the folding patterns or mechanisms at that

pH, since the behavior and the values obtained at acidic

and neutral pH levels are very similar for both proteins.

The production of recombinant proteins in bioreactors is

a rapid and often inexpensive process; however, this

technique implies a high rate of misfolded or aggregated

pH 4.2

0 5 10 15 20 2576

78

80

82

84

Heating rate (ºC/min)

Tp

(ºC

)

pH 7.2

0 5 10 15 20 2574

76

78

80

Heating rate (ºC/min)

Tp

(ºC

)

pH 9.2

0 5 10 15 20 2564

66

68

70

Heating rate (ºC/min)

Tp

(ºC

)

Fig. 5 Effect of the heating rate

on the temperature of maximum

heat absorption (Tp) for

recombinant rhLz from rice

pH 4.2

2.81 2.82 2.83 2.84 2.85 2.86-12

-11

-10

-9

-8

1/Tp (x 103)(K-1)

Ln

(β

/Tp

2 )

pH 7.2

2.83 2.84 2.85 2.86 2.87 2.88 2.89-12

-11

-10

-9

-8

1/Tp (x 103)(K-1)

Ln

(β

/Tp

2 )

pH 9.2

2.92 2.93 2.94 2.95 2.96-11

-10

-9

-8

1/Tp (x 103)(K-1)

Ln

(β

/Tp

2 )

Fig. 6 Kissinger plot for heat

denaturation of rhLz from rice.

Tp is the temperature of

maximum heat absorption, and

b is the heating rate

1072 Eur Food Res Technol (2011) 233:1067–1073

123

proteins [33]. Therefore, the expression of proteins whose

tridimensional structure is indispensable for its biological

functions, as is the case of human lysozyme, should be

conducted by other approaches. Plants are suitable for this

purpose, since their endoplasmic reticulum enables proper

folding of proteins [34]. The thermodynamic parameters

we have obtained for the thermal denaturation of rhLz from

rice evidence that the thermal stability of this protein is

similar to that of hLz from milk at both acidic and neutral

pH levels, which also points out to a correspondence in

their folding structure. On the other hand, both proteins are

more thermolabile at basic pH, though rhLz is more labile.

This finding could indicate a slight difference in the tridi-

mensional structure of the recombinant protein compared

to that of human milk that makes rhLz being less stable

upon heating at basic pH. This fact was not previously

described, since most published studies have been carried

out at acidic pH.

Acknowledgments This research has been carried out supported by

the European Social Fund and by a research fellow grant from the

Ministerio de Educacion y Ciencia of Spanish Government.

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