Influence of sodium alginate pretreated by ultrasound on papain properties: Activity, structure, conformation and molecular weight and distribution

Accepted Manuscript Influence of Sodium Alginate Pretreated by Ultrasound on Papain Properties: Activity, Structure, Conformation and Molecular Weight and Distribution Liping Feng, Yanping Cao, Duoxia Xu, Sasa You, Fu Han PII: DOI: Reference:

S1350-4177(16)30081-5 http://dx.doi.org/10.1016/j.ultsonch.2016.03.015 ULTSON 3157

To appear in:

Ultrasonics Sonochemistry

Received Date: Revised Date: Accepted Date:

20 December 2015 14 March 2016 15 March 2016

Please cite this article as: L. Feng, Y. Cao, D. Xu, S. You, F. Han, Influence of Sodium Alginate Pretreated by Ultrasound on Papain Properties: Activity, Structure, Conformation and Molecular Weight and Distribution, Ultrasonics Sonochemistry (2016), doi: http://dx.doi.org/10.1016/j.ultsonch.2016.03.015

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1

Influence of Sodium Alginate Pretreated by Ultrasound on Papain Properties:

2

Activity, Structure, Conformation and Molecular Weight and Distribution Liping Fenga,b,c, YanpingCaoa,b,c,*, Duoxia Xua,b,c, Sasa Youa,b,c, Fu Hana,b,c

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a

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Nutrition & Human Health (BTBU), Beijing Engineering and Technology Research Center of

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Food Additives, Beijing Technology & Business University, Beijing, 100048, China

7

b

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Beijing Key Laboratory of Flavor Chemistry, Beijing Technology & Business University, Beijing,

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100048, China

School of Food & Chemical Engineering, Beijing Advanced Innovation Center for Food

Beijing Higher Institution Engineering Research Center of Food Additives and Ingredients,

10

c

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Beijing, 100048, China

Beijing Laboratory for Food Quality and Safety, Beijing Technology & Business University,

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*Corresponding author.

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Tel.: + 86-10-68985645

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Fax: + 86-10-68985645

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Address: No.11, Fucheng Road, Beijing 100048, China

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E-mail: [email protected]

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Abstract: The aim of the study was to investigate the impact of sodium alginate

19

(ALG) pretreated by ultrasound on the enzyme activity, structure, conformation and

20

molecular weight and distribution of papain. ALG solutions were pretreated with

21

ultrasound at varying power (0.05, 0.15, 0.25, 0.35, 0.45 W/cm2), 135 kHz, 50 °C for

22

20 min. The maximum relative activity of papain increased by 10.53% when mixed

23

with ALG pretreated by ultrasound at 0.25 W/cm2, compared with the untreated ALG.

24

The influence of ultrasound pretreated ALG on the conformation and secondary

25

structure of papain were assessed by fluorescence spectroscopy and circular dichroism

26

spectroscopy. The fluorescence spectra revealed that ultrasound pretreated ALG

27

increased the number of tryptophan on papain surface, especially at 0.25 W/cm2. It

28

indicated that ultrasound pretreatment induced molecular unfolding, causing the

29

exposure of more hydrophobic groups and regions from inside to the outside of the

30

papain molecules. Furthermore, ultrasound pretreated ALG resulted in minor changes

31

in the secondary structure of the papain. The content of α-helix was slightly increased

32

after ultrasound pretreatment and no significant change was observed at different

33

ultrasound powers. ALG pretreated by ultrasound enhanced the stability of the

34

secondary structure of papain, especially at 0.25 W/cm2. The free sulfhydryl (SH)

35

content of papain was slightly increased and then decreased with the increase of

36

ultrasonic power. The maximum content of free SH was observed at 0.25 W/cm2,

37

under which the content of the free SH increased by 6.36% compared with the

38

untreated ALG. Dynamic light scattering showed that the effect of ultrasound

39

treatment was mainly the homogenization of the ALG particles in the mixed 2

40

dispersion. The gel permeation chromatography coupled with the multi-angle laser

41

light scattering photometer analysis showed that the molecular weight (Mw) of

42

papain/ALG was decreased and then increased with the ultrasonic pretreatment.

43

Results demonstrated that the activity of immobilized papain improved by ultrasonic

44

pretreatment was mainly caused by the variation of the conformation of papain and

45

the effect of interactions between papain and ALG. This study is important to explain

46

the intermolecular interactions of biopolymers and the mechanism of enzyme

47

immobilization treated by ultrasound in improving the enzymatic activity. As

48

expected, ALG pretreated by appropriate ultrasound is promising as a bioactive

49

compound carrier in the field of immobilized enzyme.

50

Key words: Ultrasound pretreated sodium alginate; Papain; Activity; Conformation;

51

Intermolecular interactions

3

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1. Introduction

53

Ultrasound as a novel technology has attracted a wide range of interest from

54

fundamental academic research to many different industrial applications in recent

55

years. It has been proved to be an effective technique for the synthesis of

56

nanomaterials as well as for the deposition and insertion of nano-particles on/into

57

mesoporous ceramic, polymer supports, fabrics, and glass [1, 2]. It can be divided into

58

two intensity ranges. Low-intensity (high-frequency, 100 kHz - 1 MHz, power < 1

59

W/cm2) ultrasound is most commonly applied as an analytical technique to provide

60

information on the physicochemical properties of food such as firmness, ripeness,

61

sugar content and acidity, etc. High-intensity (low-frequency, 16-100 kHz, power in

62

the range 10-1000 W/cm2) ultrasound is suitable for the applications including

63

homogenization, emulsification and extraction [3-6].

64

The effect of ultrasound on liquid system is mainly attributed to the cavitation

65

bubbles generating intense shear stress [7]. The bubbles are rapidly formed and

66

violently collapsed, leading to extreme temperature (5000 K) and pressure (120 MPa)

67

which can produce very high shear energy waves and turbulence in the cavitation

68

zone [8]. The energy released from collapsing cavitation bubbles can be transferred to

69

the sonication matter through the appropriate choice of sonication medium and

70

particle concentration [9]. Under such conditions, molecules trapped in the bubble

71

(water vapor, gases and vaporized solutes) can be brought to an excited-state and

72

dissociate [10]. The above mentioned factors of temperature, pressure and turbulence

73

combined together to affect the ultrasound treated system. Moreover, hydroxyl free 4

74

radicals could be generated by the rupture of molecules bond in aqueous solution,

75

leading to the activation effect. There are some literatures focusing on studying

76

ultrasound as an enzymatic pretreatment to reduce particle size or accelerate the

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reaction rate [11]. Bashari et al. found that ultrasound could improve the catalytic

78

activity of immobilized dextranase [12]. Ultrasound is also known to perturb weak

79

interactions and induce conformational changes in protein structures [13].

80

Papain is a kind of cysteine protease obtained from carica papaya. It is a compact

81

globular protein (molecular mass = 21 kDa) containing 212 amino acid residues with

82

three disulfide bonds. The active site of papain consists of Cys25 - His159 - Asn175

83

[14]. Sodium alginate (ALG) is one of the widely used polysaccharides as an enzyme

84

carrier and composed of β-D-mannuronate residues and α-L-guluronate residues. It

85

has been demonstrated that alginate is a promising bioactive compound carrier [15].

86

Recently, ALG immobilized papain has received a great deal of attention. However,

87

the grid structure of polymer carriers may hinder the macromolecules’ diffusion and

88

restrain the release of substrate such as casein. It was reported that the activity of

89

papain immobilized by ALG-chitosan was enhanced successfully by the ultrasound

90

treatment and the optimal frequency was 135 kHz [16]. The activity of immobilized

91

papain improved by ultrasonic treatment was probably caused by the increase of the

92

diffusion properties of the casein at different frequencies and powers, respectively

93

[17]. Meanwhile, the results could also be due to the variation of the secondary and

94

tertiary structures of papain or the effect of interactions between papain and

95

polysaccharides [18]. To the best of our knowledge, there are few published paper 5

96

related to clearly demonstrate the latter reason. Additionally, few studies focused on

97

investigating the enzyme-polysaccharide liquid system pretreated by ultrasound,

98

especially the polysaccharide was pretreated by ultrasound.

99

Therefore, the purpose of this study was to investigate properties of papain

100

mixed with ALG pretreated by ultrasound in liquid system rather than entrapped in

101

the ALG gel. Comparison of papain features in ALG liquid system based on

102

ultrasound eliminated the influence of casein diffusion. Hosseini et al. reported that

103

ultrasonication promoted ALG-ALG interactions and decreased the interaction

104

strength between β-lactoglobulin and ALG [19]. It was stated that ultrasound

105

pretreatment is an efficient method in rapeseed proteolysis to produce peptides

106

through its impact on the molecular conformation [20]. In our current study, it was

107

hypothesized that ultrasonic pretreatment of ALG would affect the interactions

108

between papain and ALG, molecular conformation of papain and further impact the

109

immobilized enzyme activity. Based on our group previous study [16], the ultrasound

110

pretreated condition were carried out at different powers (0.05, 0.15, 0.25, 0.35, 0.45

111

W/cm2), 135 kHz, 50 °C for 20 min.

112

To investigate the impact of ALG pretreated by different ultrasonic powers on

113

enzymology characteristics of papain by using the dynamic light scattering (DLS),

114

fluorescence

115

chromatography coupled with a multi-angle laser light scattering photometer analysis

116

(GPC-MALLS). The aim is to evaluate enzyme activity, structure, conformation and

117

molecular weight and distribution of papain impacted by ultrasound pretreated ALG

spectroscopy,

circular

dichroism

6

(CD)

and

gel

permeation

118

which is to explain the mechanism of ultrasound-accelerated enzymolysis of papain

119

immobilization in order to extend the application of ultrasound in the process of

120

immobilized enzyme.

121

2. Materials and methods

122

2.1. Materials

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Sodium alginate (ALG, Mw =1.93×105 g/mol, M/G=1.51, 200 ± 20 mPa.s

124

viscosity) and 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) were purchased from

125

Aladdin Reagent Company (Shanghai, China). Papain (21 kDa, 2.77×105 U.g-1) was

126

purchased from Sigma Chemicals. Casein was purchased from Beijing Ao Bo Xing

127

Bio-Tech. Co., Ltd. (Beijing, China). Folin phenol reagent and L-Glutathione were

128

purchased from Sigma Chemicals. All other chemicals and solvents used were of

129

analytical grade.

130

2.2. Ultrasound equipment

131

An assembled ultrasonic bath system equipment with two sets of JXD-02

132

multi-frequencies processing system and the low temperature circulating water tank

133

was employed (JXD-02, Beijing Jinxing Ultrasonic Equipment Technology Co., Ltd.,

134

China).The experimental ultrasound apparatus used in this work has been described in

135

detail in our previous work [17]. Ultrasonic intensity was measured by calorimetry

136

using a thermo couple (model: TASI 601, TASI Ltd., Suzhou, China) and expressed in

137

W/cm2. The ultrasound generators probe could deliver a maximum power of 0.45

138

W/cm2 and a maximum frequency of 135 kHz. The length, width and depth of the

139

ultrasonic bath were 20, 20, and 15 cm, respectively. 7

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2.3. Preparation of samples and ultrasound pretreatment

141

ALG solutions (0.95 wt%) were prepared in 0.1 M phosphate buffer solution at

142

pH 7.0. The solutions were stirred with heating at 50°C to ensure complete dispersion

143

and hydration, and then cooled to the room temperature. Papain solution (0.48 wt%)

144

was prepared in 0.1 M phosphate buffer solution at pH 7.0. ALG solutions prepared

145

with ultrasound treatment were carried out at different powers (0.05, 0.15, 0.25, 0.35,

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0.45 W/cm2), 135 kHz, 50 °C for 20 min based on our group previous study [16].

147

After ultrasonic treatment, the papain diluted appropriate times was mixed with the

148

ALG solution in a volume ratio of 1:1 and then the mixtures were analyzed according

149

to the experiment requirement. In this study, papain was used without ultrasound

150

treatment which is focus on the effect of ultrasound pretreatment of ALG on the

151

properties of papain.

152

2.4. Enzyme activity measurement

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The papain was mixed with the above ultrasound pretreated ALG (2.3) in a

154

volume ratio of 1:1. After that, the concentration of papain was 0.024 mg/mL. The

155

activity of papain was determined according to the method of Folin-phenol described

156

by Li [21] and calculated by the standard curve of tyrosine solution obtained by

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Ultraviolet-visible spectrophotometer (UV-1240, Shimadzu, Co., Ltd., Tokyo, Japan).

158

In the process of enzyme activity measurement, the casein was used as substrate.

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2.5. Intrinsic fluorescence analysis

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The mixtures of untreated and ultrasound pretreated ALG and papain were

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diluted five times and then were measured at room temperature (25 ± 1°C) with 8

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fluorescence spectrophotometer (RF 5301, Shimadzu, Co., Ltd., Tokyo, Japan) at 280

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nm (excitation wavelength, slit = 5 nm), 290-500 nm (emission wavelength, slit = 5

164

nm) and 1200 nm/min of scanning speed.

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2.6. Circular dichroism analysis

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Circular dichroism (CD) spectra were recorded with a spectropolarimeter (optical

167

physics applications, British; Chirascan), using aquartz cuvette of 1 mm optical path

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length at room temperature (25 ± 1°C). CD spectra were scanned in the far UV range

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(190-260 nm) with three replicates at 0.1 nm as bandwidth. The papain was mixed

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with the above ultrasound pretreated ALG in a volume ratio of 1:1. After that, the

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concentration of papain for CD analysis was 0.12 mg/mL. The secondary structures of

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the papain were analyzed by using a Chirascan software. These data were expressed

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as ellipticity, θ (mdeg). All spectra were corrected by subtracting the baseline. Finally,

174

spectra were deconvoluted using the deconvolution software CDNN2.1 to obtain

175

information about the secondary structures of papain [22].

176

2.7. Determination of free SH content

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The free sulfhydryl (SH) content of the papain was determined using Ellman’s

178

reagent DTNB according to the method described by Shimada and Cheftel [23,24]

179

and Lagrain et al. [25], with some modification. The assay relies on the reaction of

180

thiols

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5-thio-2-nitrobenzoic acid. The papain was added to the native and ultrasonic

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pretreated ALG at a concentration of 2.4 mg/mL. The mixtures were incubated for 30

183

min at 25 °C in a shaking water bath. 600 µL of the mixtures were added to 2.4 ml of

with

the

chromogenic

DTNB

9

to

form

the

yellow

dianion

of

184

0.1 M phosphate buffer, pH 7 followed by rapid addition of 30 µL of 0.4% Ellman’s

185

reagent (4 mg DTNB/mL phosphate buffer). Then the solution was rapidly mixed and

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allowed to stand at 20 °C for 15 min, the absorbance was read at 412 nm. The

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phosphate buffer was used instead of papain solutions as a reagent blank. A molar

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extinction coefficient of 1.36×10 4 M-1.cm-1 was used for calculating the content of SH

189

in papain [26].

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2.8. Dynamic light scattering measurements (DLS)

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The preparation of mixture of ALG and papain was the same with the above 2.5.

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Samples were diluted to minimize multiple scattering effect. Particle size distributions

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of the mixtures were measured using a Zetasizer Nano-ZS90 (Malvern Instruments,

194

Worcestershire, UK) at a fixed detector angle of 90°. Results were described as

195

number particle size distribution.

196

2.9. Gel permeation chromatography coupled with a multi-angle laser light scattering

197

photometer analysis

198

Molecular weight and polydispersity of polymers were measured by gel

199

permeation chromatography coupled with the multi-angle laser light scattering

200

(GPC-MALLS, Wyatt Technology Co, USA) equipped with two Viscotek A6000M

201

columns. The GPC-MALLS system consists of a Waters 2690D separations module, a

202

Waters 2414 refractive index detector (RI) and a Wyatt DAWN EOS MALLS detector.

203

Papain solution (0.48 wt%) was prepared in 0.1 M phosphate buffer and mixed with

204

ALG pretreated by different ultrasonic powers in a volume ratio of 1:1 until complete

205

dissolution. The flow rate was set to 0.5 mL/min phosphate buffer. All samples (300 10

206

µL) were filtered through 0.22 µm nylon filters before being injected into the GPC

207

column. Molecular mass distributions of the papain-ALG were determined through

208

the designated software.

209

2.10. Statistical analysis

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All experiments described above were made in triplicate foreach sample. Data

211

were subjected to analysis of variance (ANOVA) using the software package SPSS

212

19.0 for Windows (SPSS Inc., Chicago, IL). Unless otherwise noted in the text, a P <

213

0.05 level was used where values were considered as being significantly different.

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3. Results and discussion

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3.1. Effect of ultrasound pretreated ALG on the enzyme activity of papain

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The effect of ultrasound pretreated ALG with different powers on the relative

217

activity of papain was shown in Fig. 1. The activity of papain in untreated (without

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ultrasound treatment) ALG was 2.57×10 5 U.g-1, which was described as the control.

219

The relative activity of papain mixed with ultrasound pretreated ALG was increased

220

with the increase of ultrasound power until it reached 0.25 W/cm2. When the

221

ultrasound power exceeded 0.25 W/cm2, the relative activity was decreased. The

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maximum relative activity of papain was observed when ALG was pretreated at 0.25

223

W/cm2, 135kHz，50 °C for 20 min, under which the relative activity increased by

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10.53% compared with the control. It might be explained by that the shearing force,

225

shock waves and free radicals induced by ultrasound might crush the ALG-ALG

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cross-linkage and change the interactions between ALG and papain. It resulted in the

227

enlargement of specific surface areas of papain and thus increased the effective 11

228

contact areas between papain and substrate. It was also possible because that the

229

hydroxyl radicals produced by ultrasound could react with the intermediate molecules

230

produced by the ALG which further changed the physical and chemical properties

231

of ALG and then increased the activity of papain. Also, it was reported that ultrasonic

232

treatment could accelerate the graft reaction between peanut protein isolate and

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polysaccharides [27]. The radiation force induced by the oscillation of the stable

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cavitation bubbles might alter the configuration of ALG and the interactions between

235

ALG and papain，thereby indirectly improving the activity of the papain.

236

3.2. The fluorescence analysis

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It is known that the intrinsic fluorescence of enzyme is mainly attributed to the

238

amino acid groups of tryptophan. Therefore, the effect of ultrasound pretreated ALG

239

on the molecular conformation of papain was investigated by tryptophan fluorescence

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spectrum. As can be seen in Fig. 2, the fluorescence intensities of the papain mixed

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with ALG by different treatment conditions were in the order of 0.25 W/cm2 > 0.15

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W/cm2 > 0.35 W/cm2 > untreated > papain alone > 0.45 W/cm2 > 0.05 W/cm2. It

243

demonstrated that the medium power of ultrasonic pretreatment of ALG increased the

244

number of tryptophan on papain surface, especially at 0.25 W/cm2. While, the power

245

of 0.45 W/cm2 and 0.05 W/cm2 pretreated ALG decreased the number of tryptophan

246

on papain surface. It has been reported that ultrasound induced the molecular

247

unfolding of protein, changed hydrophobic interactions of protein molecules and

248

caused more groups and regions to expose from inside to the outside of the molecules

249

[28]. Similarly, ultrasound pretreated ALG by the medium intensity of power induced 12

250

the molecular unfolding of papain molecules, causing more hydrophobic groups and

251

regions exposure from inside of the molecules to the outside. This finding could also

252

be explained by the fact that ultrasound pretreated ALG might change the

253

hydrophobic interactions between ALG and papain. The increase of hydrophobicity

254

indicated that the unfolded hydrophobic groups induced by ultrasound might

255

rearrange more intensively to reach the minimum energy state.

256

3.3. Secondary structure analysis

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CD spectra was employed in this study to provide insights into the secondary

258

structure of the papain, papain/ALG and papain/ALG pretreated by different

259

ultrasonic powers. It has been reported that the CD spectra in the far-UV wavelength

260

range from 190 to 250 nm are perceived as very useful measurement for

261

conformational change of proteins and obtained mainly due to electronic transitions

262

between molecular orbital in ground and excited states [29]. The CD spectra of the

263

native papain revealed a positive peak of the molar ellipticity at 192 nm (Fig. 3). The

264

two negative peaks at 208 and 222 nm displayed by the native papain were not found

265

after mixed with untreated and ultrasound pretreated ALG. The presence of a peak at

266

above 200 nm of different wavelength confirmed that the native papain, papain/ALG

267

and papain/ALG pretreated by ultrasound exhibited a change of β-strand content. It

268

was interesting to find that the shape and the amplitude were affected after the ALG

269

pretreated by different ultrasonic powers. The content of α-helix, β-sheet and random

270

coil structures showed slightly changes in the percentages of the secondary structure

271

elements (Table. 1). The content of α-helix, β-turn and random coil in papain 13

272

increased according to the different ultrasound treatment, compared with the untreated

273

sample. It suggested that the increase of α-helix, β-turn and random coil structures

274

caused by ultrasound was mainly converted from decreasing the content of β-sheet.

275

Such an increase of the α-helix fractions in papain due to the ultrasonic pretreatment

276

of ALG could be explained by the fact that the hydrophobic interactions between

277

ALG and papain were changed by ultrasound. The small difference between different

278

ultrasonic powers could be attributed to the fact that ALG pretreated by ultrasound

279

treatment enhanced the stability of the secondary structure of papain. The structure of

280

the secondary structure of papain was the most stable when mixed with ALG

281

pretreated by 0.25 W/cm2 as expected.

282

Since the secondary structure of papain depends on both the local sequence of

283

amino acids and the interactions with environmental biopolymers. Results certified

284

that ultrasound pretreated biopolymer changed the interactions between ALG and

285

papain, leading to the minor changes in the secondary structure of papain. This

286

behavior could be correlated with literature which found that ultrasonic treatment (20

287

kHz, 450 W) resulted in an increase in the α-helix component and a decrease in the

288

β-sheet component of whey protein concentrate [30]. Furthermore, an increase

289

of α-helix in the papain was induced by different ultrasonic time were reported which

290

was consistent with the present results [18]. With the increase of the intensity of

291

ultrasonic treatment, hydrogen bonding and vander Waals interactions in polypeptide

292

chains could be ultimately damaged, resulting in the modification of protein

293

secondary structures [31]. Therefore, it suggested that the hydrogen bonding and 14

294

vander Waals interactions in papain molecules were slightly affected by the

295

ultrasound pretreated ALG.

296

3.4. Determination of free SH groups

297

Fig. 4 showed that ALG led to the decrease in the SH content of papain while

298

papain mixed with ALG pretreated by ultrasound showed the increase in the SH

299

content. It was observed that the SH content of papain mixed with ALG pretreated by

300

ultrasound at 0.25 W/cm2 was increased to 173.10 ± 0.46 µmol/g compared with

301

the untreated ALG/papain mixture showing 162.75 ± 0.49 µmol/g . The content of

302

free SH decreased slightly with the increase of ultrasonic power ranging from 0.25

303

W/cm2 to 0.45 W/cm2. The increased content of free SH might be attributed to the

304

broken of intermolecular disulfide bonds in papain. Hu et al. found similar result that

305

free SH content of SPI increased with ultrasonic intensity (from 200 W to 600 W)

306

[26]. In addition, Fernandez-Diaz et al. pointed out that electric pulse processing led

307

to partial unfolding of the ovalbumin protein, thus exposing SH groups to the surface

308

[32]. The ultrasonic pretreatment might have the similar effect on SH groups of

309

papain. The buried SH groups of papain were exposed due to the changes of the

310

hydrophobic environment after ultrasonic pretreatment. It was agreed with the above

311

result of fluorescence spectrum analysis.

312

3.5. Particle size distribution

313

The impact of ultrasonic pretreatment of ALG on the particle size distribution of

314

papain/ALG was shown in Fig. 5. The particle size distribution was obtained based on

315

the intensity of scattered light, which could be converted to volume or number 15

316

distribution. The dependencies of the number distribution, volume distribution and

317

intensity distribution on the particle diameter were d, d 3, and d6, respectively.

318

Therefore, the peaks tended to be skewed towards larger particle size when analyzing

319

the volume and intensity distribution. For the bipolymers, the result was described as

320

particle number size distribution. Fig. 5 illustrated the number size distributions of the

321

native papain, papain/ALG and papain/ALG pretreated by different ultrasonic powers,

322

respectively. It showed that ultrasonic pretreatment resulted in a pronounced left shift

323

of the particle size distribution of papain/ALG to lower diameters. It indicated that

324

ultrasonic treatment decreased the particle size of papain/ALG. The result of untreated

325

papain/ALG showed a unimodal distribution of particles with a major peak at 550 nm.

326

However, the value of this peak decreased and another peak at 8 nm appeared when

327

ALG pretreated by ultrasound at 0.25 W/cm2.

328

This finding was consistent with previous studies showing that particle size of

329

β-lactoglobulin and ALG was decreased after ultrasonic treatment [19]. Similarly, it

330

was also reported that ultrasound could result in the reduction of soy protein isolate

331

particle size [20, 33]. The changes of particle size distribution of papain/ALG

332

solutions after ultrasonic pretreatment might be attributed to the shearing force,

333

micro-streaming and turbulent force of the sonication. The covalent bonds between

334

molecules are likely to be disrupted. It was assumed that the shearing effect resulted

335

from the cavitation bubble collapses was mainly responsible for the changes in the

336

structure of biopolymers such as the breakage of the chemical bonds within the

337

macromolecule [34]. Indeed, ALG pretreated by ultrasound could probably undergo 16

338

some sonochemical reactions including conjugation, oxidation, C-D, C-heteroatom,

339

and C-C bond formations.

340

It was interesting to find that papain/ALG pretreated at 0.25 W/cm2 had a similar

341

particle size distribution with the native papain in the part of larger size. There were

342

wide distributions of molecules after ultrasound pretreatment. These results were

343

probably attributed to some hetergeneous sonochemical interactions and structural

344

changes that occurred during the ultrasound pretreatment process. In addition, it was

345

reported that the ultrasound treatment led to a decrease of the particles size due to the

346

rupture of the previously formed aggregates [35]. It was proved that the glycosylation

347

of β-conglycinin could enhance its ability to suppress the thermal aggregation of

348

glycinin [36]. Furthermore, polysaccharide reactivity is controlled by the distribution

349

and number of functional groups attached to the polymerized sugar units that form the

350

backbone of the polysaccharide [37]. It indicated that the specific preparation

351

conditions had a great influence on the properties of ALG, which pointed out the

352

possibility to apply ultrasonic treatment conditions to control the particle size of

353

biopolymers.

354

3.6. Molecular weight and distribution

355

GPC-MALLS technique was employed to investigate whether the previously

356

described increased in hydrophobicity could alter the interactions between bipolymers,

357

leading to the formation of dimers or aggregates. Table 2 showed that the molecular

358

weight (Mw) and molecular number (Mn) of papain/ALG was decreased and then

359

increased with the increase of the ultrasonic power. The minimum value of Mw was 17

360

2.16×104 g/mol at 0.35 W/cm2. It was reported that the cavitations phenomenon could

361

be induced only when the ultrasonic power reached to a certain minimum value [38].

362

It certified that ultrasound could induce ALG degradation. At the same time, high

363

temperature and high pressure which produced by ultrasonic cavitations broke the

364

chemical bond with the change of ultrasonic power. In general, the effect of

365

ultrasound pretreatment on the activity of papain was increased with the increase of

366

the intensity in an extent. The explanation for the intensity effect lied in the

367

production of a large number of cavitation bubbles and the acoustic source to dampen

368

the efficiency of energy transmission into the reaction medium. As more and more

369

such bubbles were produced, they acted as a barrier to energy transmission into the

370

system and thus the ultrasound effect reduced [39].

371

The maximum value of polysaccharide distribution index (Mw/Mn) was 5.776 ±

372

0.072 when the papain mixed with untreated ALG. It demonstrated that fewer

373

aggregates were further formed in the papain/ALG solutions after ultrasound

374

pretreatment. It also suggested that the covalent bonds were broken between large

375

molecules or non-covalent interactions such as electrostatic interactions became

376

stronger after the ALG pretreated by ultrasound.

377

4. Conclusions

378

In our current study, we proved that ultrasound pretreated ALG could improve

379

the activity of papain. The effect of ultrasound pretreated ALG on the structure of

380

papain was dependent on the intensity of ultrasound. It was found that ultrasonic

381

treatment resulted in the partial unfolding and enhancement of intermolecular 18

382

interactions as demonstrated by the increases in free SH groups and surface

383

hydrophobicity, leading to improved activity of papain. In addition, the stability of

384

secondary structure was increased by ultrasound pretreated ALG. There was no larger

385

aggregate formation after ultrasonic treatment. Therefore, ALG pretreated by

386

appropriate ultrasound is a promising method as a bioactive compound carrier. The

387

study made an important attempt to improve enzymatic activity in the field of

388

immobilized enzyme. In addition, it could further explain the mechanism of papain

389

immobilization treated by ultrasound in improving the enzymatic activity.

390

Acknowledgements

391 392

This research was funded by the National Key Technology R&D Program of the National Natural Science Foundation of China (31371722).

19

393

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24

503

Relative enzyme activity (％)

120 110 100 90 80 70 60 0.0

0.1

0.2

0.3

0.4

0.5

2

Ultrasonic power (W/cm )

504 505

Fig. 1 Effect of ultrasonic pretreated ALG at different powers (0.05, 0.15, 0.25, 0.35,

506

0.45 W/cm2) on the relative activity of papain.

25

800 papain papain/ALG 0.05 0.15 0.25 0.35 0.45

700

Intensity

600 500 400 300 200 100 0 300

350

400

450

500

Wavelength (nm)

507 508

Fig. 2 Intrinsic tryptophan fluorescence emission spectra of the papain, papain/ALG,

509

papain/ALG pretreated by different ultrasonic powers (0.05, 0.15, 0.25, 0.35, 0.45

510

W/cm2).

26

Circular dichroism (mdeg)

190

210

220

230

240

250

260

4

4

2

2

0

0

-2

-2

-4

-4

-6

-6

-8

-8

190

511

200

200

210

220 230 Wavelength (nm)

240

250

papain papain/ALG 0.05 0.15 0.25 0.35 0.45

260

512

Fig. 3 Circular dichroism spectra of the papain, papain/ALG, papain/ALG pretreated

513

by different ultrasonic powers (0.05, 0.15, 0.25, 0.35, 0.45 W/cm2).

27

180

SH group (µmol/g papain)

175 170 165 160 155 150 145 140 papain papain/ALG 0.05

0.15

0.25

0.35

0.45

Ultrasonic power (W/cm2)

514 515

Fig. 4 The free sulfhydryl (SH) content of papain, papain/ALG, papain/ALG

516

pretreated by different ultrasonic powers (0.05, 0.15, 0.25, 0.35, 0.45 W/cm2).

28

40

papain papain/ALG 0.05 0.15 0.25 0.35 0.45

Number distribution data (%)

35 30 25 20 15 10 5 0

1

10

100

1000

10000

Size classes (nm)

517 518

Fig. 5 The molecular distribution of the papain, papain/ALG, papain/ALG pretreated

519

by different ultrasonic powers (0.05, 0.15, 0.25, 0.35, 0.45 W/cm2).

520

29

521

Table.1 Secondary structure content of the papain, papain/ALG, papain/ALG

522

pretreated by different ultrasonic powers (0.05, 0.15, 0.25, 0.35, 0.45 W/cm2). α-Helix (%)

β-sheet (%)

β-Turn (%)

Random coil (%)

papain

10.1 (-0.4)

42.9 (6.3)

18.2 (-1.2)

28.8 (-4.7)

Papain/ALG

10.5 (0.0)a

36.6 (0.0)

19.4 (0.0)

33.5 (0.0)

0.05w/cm2

11.1 (0.6)

35.2 (-1.4)

19.4 (0.0)

34.3 (0.8)

2

10.9 (0.4)

34.1 (-2.5)

20.1 (0.7)

34.9 (1.4)

0.25w/cm2

11.0 (0.5)

35.8 (-0.8)

19.5 (0.1)

33.7 (0.2)

0.35w/cm2

11.1 (0.6)

34.6 (-2.0)

19.8 (0.4)

34.4 (0.9)

0.45w/cm2

11.1 (0.6)

34.4 (-2.2)

19.7 (0.3)

34.5 (1.0)

0.15w/cm

523

a

The data in parenthesis is the difference of secondary structural element.

30

524

Table.2 Distribution and average molecular weight of the papain, papain/ALG,

525

papain/ALG pretreated by different ultrasonic powers (0.05, 0.15, 0.25, 0.35, 0.45

526

W/cm2). Mw (10 g/mol)

Mn (10 g/mol)

Mw/Mn

dn/dc

papain

0.093

0.695

1.342±0.136

0.134

papain/ALG

1.035

1.791

5.776±0.072

0.131

0.05 W/cm2

0.660

1.508

4.369±0.068

0.153

0.15 W/cm2

0.596

1.309

4.550±0.078

0.144

0.25 W/cm

2

0.432

0.808

5.344±0.115

0.153

0.35 W/cm

2

0.216

0.447

4.827±0.147

0.169

0.45 W/cm2

0.567

0.968

5.757±0.126

0.146

5

4

527 528

31

529

> To investigate the properties of papain mixed with ALG pretreated by ultrasound in liquid

530

system rather than entrapped in the ALG gel. > The particle size distribution and molecular weight

531

of biopolymers were studied. > Comprehensive studies on the effects of ultrasonic power. > ALG

532

pretreated by appropriate ultrasound is promising as a bioactive compound carrier.

533

32