Nanoporous Phyllosilicate Assemblies for Enzyme Immobilization

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Nanoporous Phyllosilicate Assemblies for Enzyme Immobilization Shuang Mei, Jiafu Shi, Shaohua Zhang, Yue Wang, Yizhou Wu, Zhongyi Jiang, and Hong Wu ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00642 • Publication Date (Web): 16 Jan 2019 Downloaded from http://pubs.acs.org on January 19, 2019

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ACS Applied Bio Materials

1

Nanoporous Phyllosilicate Assemblies for Enzyme

2

Immobilization

3

Shuang Mei,a,c Jiafu Shi,b,c,d* Shaohua Zhang,a,c Yue Wang,a Yizhou Wu,a,c Zhongyi

4

Jianga,b,c Hong Wu,a,c,e*

5

a. Key Laboratory for Green Chemical Technology of Ministry of Education, School

6

of Chemical Engineering and Technology, Tianjin University, Tianjin 300072,

7

China

8 9 10 11 12 13 14 15

b. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P. R. China c. Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, China d. School of Environment Science and Engineering, Tianjin University, Tianjin 300072, China e. Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin 300072, China

16

Corresponding authors:

17

Jiafu Shi, Email: [email protected]; Hong Wu, Email: [email protected]

18

ACS Paragon Plus Environment

ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

19

ABSTRACT:

20

Physical/chemical adsorption is well known as a facile and effective method for

21

enzyme immobilization, while ideal adsorbents with high structural stability, high

22

loading capacity and low leaching ratio are still under exploration. In this study,

23

nanoporous assemblies of two-dimensional (2D) copper phyllosilicate (L-CuSiO3) are

24

prepared as an adsorbent to immobilize horseradish peroxidase (HRP) for

25

phenol-containing wastewater treatment. Specifically, the robust chemical bonds of

26

Si-O-Si and Si-O-Cu in L-CuSiO3 ensure its superior structural stability; the

27

well-developed porous structure endow L-CuSiO3 assemblies with a high specific

28

surface area of 611.7 cm3 g-1, which enables a fast and high enzyme loading of 140

29

mg g-1 within 4 h; and the well distributed Cu (Ⅱ) ions ensure the stable attachment of

30

enzyme through Cu (Ⅱ)-arginine (in HRP) coordination with a leaching ratio less than

31

10%. Meanwhile, the scaling assembly of L-CuSiO3 renders the resultant biocatalysts

32

(HRP-loaded L-CuSiO3 assemblies) ease-of-recycling performance. Given the above

33

features, the HRP-loaded L-CuSiO3 assemblies exhibit better stability and two-fold

34

higher activity by contrast with HRP adsorbed on conventional mesoporous SiO2 and

35

SiO2 nanoparticles, and it also acted as an efficient bioreactor in the application of

36

catalytical removal of phenol pollutants from wastewater. Our L-CuSiO3 assemblies

37

show great potential in immobilization of enzymes for industrial biocatalysis.

38

KEYWORDS: Copper Phyllosilicate (L-CuSiO3); Nanoporous Assembly; Enzyme

39

Immobilization; Horseradish Peroxidase; Phenol Removal

40


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41

INTRODUCTION

42

As a highly efficient catalyst working under ambient conditions, enzymes well

43

meet the requirement for “green chemistry”.1 However, water-soluble enzymes in

44

their native forms are not amenable for practical applications due to the low

45

operational stability and poor reusability.2-3 Physical/chemical adsorption of enzymes

46

onto nano/micro-materials is a simple but efficient way to prevent the enzyme from

47

dissolving in water, thus leading to enhanced stability and recyclability.4 An “ideal”

48

adsorbent for enzyme immobilization is expected to have the following features

49

including i) high structural stability in acidic/alkaline conditions, ii) high surface area

50

and porous structure for high-capacity and fast loading of enzymes, and iii)

51

appropriate interactions between enzyme and adsorbents for non-leaching of enzymes

52

and better stabilization of enzymes.3, 5-6

53

Since the first case of enzyme immobilization by adsorption appeared in 1960s, 7

54

numerous efforts have been devoted to developing high-performance adsorbents for

55

enzyme immobilization. Accordingly, the adsorbents that reported currently varied

56

from organic polymers to inorganic materials, such as chitosan8, alginate9, clay10,

57

silica11, metal-organic frameworks12-13, covalent-organic frameworks14, and so on.

58

Amongst, silica-based materials were quite attractive as a potentially “ideal”

59

adsorbent due to their features of enzyme compatibility, easy preparation/engineering,

60

high stability against acid/alkaline, etc.15-17 In this regard, structural regulation and

61

surface modification of silica-based adsorbents were implemented to acquire high

62

enzyme adsorption rate/capacity, low enzyme leaching and desirable enzyme

63

activity/stability. Structure regulation has been applied to downsize silica-based

64

adsorbents into nanoscale and/or engineer them with numerous mesopores to increase

65

their specific surface area,18-20 where more adsorption sites were generated for



66

enzyme immobilization. Accordingly, the enzyme loading capacity could reach as

67

high as 122~450 mg g-1 for a broad range of nanostructured silica, including

68

mesocellular foam21, SBA-1522, etc. Surface modification mostly aimed to inhibit the

69

enzyme leaching, where the surface of silica-based materials was functionalized with

70

organic ligands such as polyethylenimine, octadecyltrimethoxysilane, carboxylic acid,

71

and so on.11, 23 With the help of these ligands, additional electrostatic interactions or

72

covalent bonds between enzymes and silica-based materials were incorporated, which

73

would help to prevent the enzymes from detaching even after tens of recycling

74

times.24-25 Nonetheless, the structural regulation and surface modification were

75

commonly a two- or more-step processes, which were time-consuming and laborious.

76

Besides, the covalent bonds between reactive organic ligands and enzymes may cause

77

the conformation changes and then deactivation of enzymes.

78

Recently, coordination of enzyme with transition metal ions has been developed as

79

a green and emerging method for enzyme immobilization.26 To be more specific,

80

metal-enzyme coordination, which owned bond energy of higher than physical

81

interactions (20-25 KJ mol-1)27 but weaker than covalent bonds (i.e. > 300 KJ mol-1

82

for C-N bonds of methylamine)28, could help to regulate the enzyme-sorbents

83

interactions, achieving the stabilization of enzymes on the sorbents without

84

remarkably altering their conformation. In this regard, silica-based nanostructured

85

materials decorated with metal ion may merge the merits of both high surface area of

86

nanostructured silica and appropriate interaction of metal ions toward enzymes.

87

However, the post decoration of metal ion on silica materials were quite difficult as

88

the rather weak interactions between both compounds.29 Besides, similar to organic

89

ligand modified silica-based nanostructured materials, the structure regulation and

90

surface decoration were two separated processes.24 It was then expected that


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91

metal-silica nanostructures prepared through simple yet effective methods would be

92

highly desired.

93

Herein, porous assemblies of copper phyllosilicate (L-CuSiO3) were prepared

94

through a modified ammonia evaporation hydrothermal method, and used as

95

adsorbents for efficient enzyme immobilization. The assembling units of the porous

96

assemblies were composed of two-dimensional (2D) L-CuSiO3 with blending silica

97

tetrahedron and copper-oxide octahedron.30-31 In the assemblies, L-CuSiO3 with large

98

external accessible surface and porous structure could help to achieve a high enzyme

99

loading capacity and fast loading rate. The enzymes could be adsorbed on the

100

L-CuSiO3 surface through amino-Cu (II) coordination in a controlled manner. The

101

morphology and chemical composition of L-CuSiO3 assemblies can be controlled

102

through altering the preparation conditions. Horseradish peroxidase (HRP) that could

103

catalyze many reactions in the presence of hydrogen peroxide for the removal of

104

phenols and its derivatives in wastewater32-34 was applied to evaluate the adsorption

105

behavior of HRP and further elucidate the adsorption mechanism on the L-CuSiO3

106

assemblies. Systematic comparison of enzyme adsorption and catalytic performance

107

between L-CuSiO3 assemblies and its well-explored counterparts, such as mesoporous

108

SiO2, SiO2 nanoparticles, and so on, were conducted to highlight the superiority of our

109

L-CuSiO3 assemblies. Further applications of HRP-loaded L-CuSiO3 assemblies in

110

phenol removal were performed to exert their potential in industrial biocatalysis.

111

EXPERIMENTAL SECTION

112

Materials. Horseradish peroxidase (HRP, EC 1.11.1.7, 300 U mg-1) was obtained

113

from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China). The silica sol and

114

Cu(NO3)2·3H2O were purchased from Sigma-Aldrich (USA). SiO2 (No: S5505-100G,

115

0.2-0.3 μm avg. part. size (aggregate), 2.3 lb/cu.ft (bulk density)), 2,2′-azinobis



116

(3-ethylbenzthiazoline-6-sulfonate) (ABTS) and 3,3',5,5'-tetramethylbenzidine (TMB)

117

were bought from Sigma-Aldrich (USA). SBA-15 (No: XFF01, pore diameter: 6-10

118

nm, SSA: 550-600 m2 g-1) were bought from Jiangsu XFNANO Materials Technology

119

Co., Ltd. (Jiangsu, China). Coomassie brilliant Blue G-250 was obtained from

120

Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). All other chemicals

121

of analytical grade were used without further purification.

122

Characterizations. SEM images of the L-CuSiO3 assemblies were recorded by an

123

environmental scanning electron microscope (Nanosem-430, FEI). The elemental

124

analysis was measured by energy dispersive spectroscope (PHI-1600 ESCA,

125

Perkin-Elmer). TEM observation of the L-CuSiO3 assemblies was performed on a

126

transmission electron microscopy instrument (JEM-2100F, JEOL). Fourier transform

127

infrared spectra (FTIR) of the samples were measured by a FTIR spectrometer

128

(Nicolet-560, Nicolet). Textural properties of the samples were determined at 77K

129

using a nitrogen adsorption method on Autosorb-IQ-MP (nova2000e, American

130

Quanta chrome), data analysis was performed by NovaWin software. The samples

131

were degassed at 90 °C for 12 h at vacuum before adsorption experiment. The

132

contents of the L-CuSiO3 assemblies were detected through XPS (X-ray photoelectron

133

spectroscopy, PHA 5000C ESCA). The Confocal laser scanning microscope (CLSM)

134

images were taken with a LSM 710 Confocal Microscope and the HRP was stained

135

with FITC. Ultraviolet spectrophotometer (UV-3010, Hitachi) was used to analyze the

136

concentration of the enzyme and the reaction products.

137

Preparation of L-CuSiO3 assemblies. L-CuSiO3 assemblies of different Cu mass

138

percent (wt%) were prepared through a modified ammonia evaporation hydrothermal

139

(AEH) method.35 Briefly, a certain amount of Cu(NO3)2·3H2O (5, 10, 20, 40 wt% by

140

Cu wt% in the final assemblies) were mixed with 25 wt% ammonia aqueous solution


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141

under stirring for 10 min. Then, the silica sol was added into Cu-ammonia mixture

142

and stirred for 6 h. The pH of the suspension was about 12. A water bath was

143

pre-heated to 353 K for ammonia evaporation. After the deposition of copper species

144

on the silica sol, pH value of the suspension was decreased. The evaporation process

145

was terminated when the pH value was decreased to 6~7. The mixture was then sealed

146

in an autoclave, heated to 463~482 K and maintained for 12 h. After cooling down to

147

room temperature, the resultant solid was filtrated and washed several times with

148

deionized (DI) water. The final sample were obtained after dying at 60 °C overnight.

149

Determination of Enzyme Loading. The immobilization of HRP was performed

150

as follows: a certain amount of adsorbents were dispersed in a centrifuge tube with 9

151

mL of phosphate buffer solution (PBS, 50 mM, pH 7.0), then different amount of HRP

152

dissolved in DI water (10 mg mL-1) was added. The mixture was kept in a shaking

153

bed at 4 °C for a certain time. A centrifugation process (5000 rpm, 3 min) followed by

154

buffer washing course were performed to remove free HRP. The immobilized enzyme

155

was stored in aqueous solution at 4 °C for further enzyme assays.

156

The enzyme loading capacity was measured by Bradford method.36 The

157

concentration

of

enzyme

in

the

solution

was

158

spectrophotometry. Specifically, 1 mL of enzyme solution was added into 5 mL of

159

Coomassie brilliant Blue G-250 for 3 min in dark to measure the absorbance at 595

160

nm. The loading capacity was calculated with the following equation: Loading Capacity (mg g -1 ) =

161

measured

C0V0 - C1V1 m

with

UV-vis

(1)

162

where C0 and V0 were the initial enzyme concentration (mg mL-1) and volume (mL);

163

C1 and V1 are the finial enzyme concentration (mg mL-1) and volume (mL) of

164

immobilized supernatant; m was the weight (g) of adsorbents used for immobilization.

165

Investigation of Enzyme Leaching. The leakage of enzyme on different



Page 8 of 31

166

adsorbents along with incubation time was measured by the enzyme concentration of

167

supernatant. A certain volume of HRP-loaded assemblies was added into 10 mL of

168

PBS 7.0. Then, the suspensions were kept at 4 °C under shaking. A certain volume of

169

mixture was fetched every 2 h, and centrifuged to collect the supernatant for enzyme

170

concentration measurement using Bradford method. The enzyme leaching ratio was

171

defined as: Leaching Rate (%) =

172

CV 100% m

(2)

173

where Ct was the concentration (mg mL-1) of HRP at a certain time; V was the volume

174

of total suspension (mL); m0 was the weight (mg) of enzyme added.

175

Assay of HRP Activity. The catalytic activity of HRP was measured according to

176

the previous literature.37 ABTS/TMB are commonly used as simulative material for

177

contaminants. The oxidation of ABTS or TMB was catalyzed by HRP in the presence

178

of hydrogen peroxide (H2O2), and the reaction product was measured with UV/vis

179

spectrophotometry at 420 nm (ɛ420nm = 3.6×104 M-1 cm-1) for ABTS or 450 nm (ɛ450nm

180

= 5.9×104 M-1 cm-1) for TMB.37-38 Specifically, 18 μmol of ABTS or TMB and 9 μmol

181

of H2O2 in PBS 7.0 (total volume was 9 mL) was prepared as the reaction solution.

182

Then, a certain quantity of free or HRP-loaded assemblies were added into the

183

solution. One unit (U) of HRP was defined as the quantity of HRP needed to

184

transform 1 μmol of ABTS or TMB per minute. The initial reaction rate (U mg-1 HRP)

185

was defined as, Initial Reaction Rate (U mg -1 HRP)=

186

Ct - C0 Vt m

(3)

187

where C0 and Ct were the concentration (μmol L-1) of ABTS or TMB at time 0 and t

188

respectively; V was the volume of reaction solution (l); t is the test time (min); m was

189

the weight (mg) of immobilized enzyme.


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190

Enzymatic Removal of Phenol. The degradation assay of phenol was performed at

191

room temperature with a mixture of enzyme and H2O2. Standard curve of phenol was

192

measured using the 4-aminoantipyrene (4-AAP) method.39 Phenol reacted with

193

4-AAP at pH 10.6 ± 0.2 in the presence of K3Fe(CN)6, then a salmon pink dye was

194

formed within 10 min, which could be determined at 510 nm using spectrophotometry.

195

The percentage of phenol degradation (P %) was defined as below,

Phenol Degradation Percentage (P %) 

 C0 - C1 V 100%

196

m0

(4)

197

where C0 and C1 were the initial and finial phenol concentration (mg mL-1) of the

198

supernatant, respectively; V was the volume (mL) of the reaction suspension for

199

immobilization; m was the initial weight (mg) of phenol used for degradation.

200

RESULTS AND DISCUSSION

201

Preparation and Characterizations of L-CuSiO3 Assemblies. The L-CuSiO3

202

assemblies were prepared via the ammonia evaporation hydrothermal (AEH) method

203

as illustrated in Figure S1. Accordingly, the products were named as X% L-CuSiO3,

204

where X% referred to the wt% of Cu element in the assemblies. SEM and TEM

205

images of 20% L-CuSiO3 were shown in Figure 1 and the TEM images of the other

206

L-CuSiO3 were shown in Figure S2. The assemblies were composed of numerous

207

CuSiO3 layered nanosheets (L-CuSiO3) with a width of ~3 nm. The elemental

208

mapping images (Figure 1e-f) suggested the homogeneous distribution of Cu (Ⅱ) in

209

20% L-CuSiO3. The elements of the as-prepared 5%, 10%, 20% and 40% L-CuSiO3

210

were further investigated by energy dispersive spectrometer (EDS) (Figure S3),

211

where the Si wt% to O wt% were also changed with the Cu wt%, indicating that the

212

chemical structure of the L-CuSiO3 assemblies altered with the Cu wt%. Additionally,

213

the chemical composition of L-CuSiO3 assemblies was reflected in FTIR spectra



214

(Figure S4). The peaks at around 1100 and 800 cm-1 from Si-O-Si suggested the

215

existence of silica, whereas the peaks at 760 cm-1 from the vibration of δOH suggested

216

the existence of copper-oxide octahedron.30, 35, 40

a)

b)

c) 30% 25%

Percentage

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 31

20% 15% 10% 5% 0%

10μm

50nm

e) d

d)

)

50nm

e )

50nm

1

2

3 4 5 Layer width (nm)

6

f)

50nm

217 218

Figure 1. a) SEM image, b) TEM image, c) layer width distribution, and d-f)

219

elemental mapping images of 20% L-CuSiO3.

220 221

N2 adsorption-desorption measurements were conducted to examine the textural

222

properties of 5%, 10%, 20% and 40% L-CuSiO3. All the isotherms (Figure 2a)

223

exhibited the type IV curve, indicating the existence of mesopores. The type H3

224

hysteresis loops indicated that the mesopores were derived from random accumulation

225

of L-CuSiO3 layers. The pore size distributions calculated by BJH method (Figure 2b)

226

were in line with the result of isotherm types (more details listed in Table 1). With the

227

Cu loading wt% varying from 5% to 40%, the surface areas were increased from

228

265.9 m2 g-1 to 611.7 m2 g-1, while the average pore diameters remained ~3 nm and

229

the pore volumes were mostly between 0.83-1.01 cm3 g-1. The minor changes in pore

230

diameter and pore volume might be as a result of the similar accumulation process of


Page 11 of 31

231

L-CuSiO3 based on Van deer Waals forces. Besides, with the increase of Cu wt%,

232

L-CuSiO3 in the assemblies were transformed from disc-shaped to tube-shaped

233

(Figure 1b and S1).30-31, 41 b)

a) 300 -1

N2 uptake at 77K (cm g )

5% L-CuSiO3

5% L-CuSiO3

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


10% L-CuSiO3

10% L-CuSiO3

20% L-CuSiO3

20% L-CuSiO3

40% L-CuSiO3 0.0

0.2

234

40% L-CuSiO3

0.4

P/P0

0.6

0.8

1.0

0

10

20

30

Pore diameter (nm)

40

50

235

Figure 2. a) N2 adsorption-desorption isotherms at 77 K (square for adsorption and

236

triangle for desorption), b) pore size distributions of 5%, 10%, 20% and 40%

237

L-CuSiO3.

238

Table 1. Surface area, average pore diameter and pore volume of L-CuSiO3

239

assemblies with different Cu wt%. 5% L-CuSiO3

10% L-CuSiO3

20% L-CuSiO3

40% L-CuSiO3

Surface area (m2 g-1)

265.9

289.3

409.2

611.7

Average pore diameter by BJH method (nm)

3.392

3.046

3.387

3.398

Pore volume by BJH method (cm3 g-1)

0.855

1.089

0.831

0.850

240 241

Analysis of Enzyme Adsorption. The nanoporous L-CuSiO3 assemblies were then

242

adopted as adsorbents for enzyme immobilization, while the adsorption behaviors of

243

different samples were examined. Notably, two other conventional silica-based

244

adsorbents, i.e., SBA-15 and SiO2 nanoparticles (SEM images shown in Figure S5),



245

were also prepared to better elucidate the characters and superiority of L-CuSiO3

246

assemblies on enzyme immobilization. As shown in Figure 3a, the enzyme loading

247

capacity on L-CuSiO3 assemblies was increased with Cu wt%, in line with the results

248

of surface area. When the Cu wt% was higher than 10%, all L-CuSiO3 assemblies

249

exhibited elevated enzyme loading capacity by contrast with SBA-15 and SiO2. Since

250

10% L-CuSiO3 had lower surface area (289.3 m2 g-1) when compared with SBA-15

251

(550-600 m2 g-1), it could be conjectured that enzyme might have been adsorbed on

252

the external surface rather than in the pores of L-CuSiO3 assemblies. The specific

253

surface area of SiO2 nanoparticles was ~207.3 m2 g-1 calculated by the bulk density of

254

aggregate, which was lower that of L-CuSiO3 assemblies, thus showing a relatively

255

lower loading capacity.

256

Further depiction of the adsorption behavior of enzyme was reflected by HRP

257

adsorption isotherm on L-CuSiO3 assemblies with different Cu wt% (Figure 3b).

258

Generally, when the initial concentrations of HRP varied from 0.01 to 0.12 mg mL-1,

259

the loading capacities of all L-CuSiO3 assemblies were gradually increased. a)

b) 160

32 28

120

24

100

20

-1

Q (mg g )

-1

L-CuSiO3

Others

140 Loading capacity (mg g )

80 60 40 20

16 12

40% L-CuSiO3

8

20% L-CuSiO3

4

10% L-CuSiO3

0

5% L-CuSiO3

0.00

uS

3

iO

3

iO

3

uS

C

C

0.04

0.08 -1

0.12

%

L-

C (mg ml )

40

% 20

10

%

L-

L-

C

C

uS

iO

3

iO uS

2

O

L5%

260

Si

A-

15

0 SB

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 31

261

Figure 3. a) Loading capacity of SBA-15, SiO2 nanoparticles and L-CuSiO3

262

assemblies with different Cu wt%, b) HRP adsorption isotherm on L-CuSiO3

263

assemblies with different Cu wt% after 4 h (Already verified that all the adsorption


Page 13 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

264


equilibrium reached within 4 h). Theoretically, two possible models, Langmuir model and Freundlich model, were

265 266

frequently used to describe the enzyme adsorption onto porous matrixes.42 Langmuir model:

267

C 1 1   C Q bQm Qm

268

(5)

Freundlich model:

269

ln Q  270

1 ln C  ln(kQm ) n

(6)

271

where C and Q were the enzyme concentration and loading capacity presented in the

272

adsorption isotherm; Qm was the maximal loading of HRP; b, n and k were the

273

constants under the same test condition.

274

Herein, these two models were used to fit the experimental results of HRP

275

adsorption isotherm. As shown in Figure 4, the correlation coefficients from

276

Freundlich model were much better than that from Langmuir model, which indicated

277

that the adsorption behaviors of HRP followed pseudo-second-order kinetic model.10,

278

43

279

interactions between the adsorbents with HRP as well as the relatively high surface

280

roughness of the adsorbents.44

The reasons of well-fitting Freundlich model could be attributed to the multisite



1a)

1b)

L-model

F-model

3 0.08

2 2

R =0.376

0.04

0 -1

0.02 0.00 0.00

2

R =0.938

1

lnQ

C/Q

0.06

-2 0.02

0.04

0.06

0.08

0.10

0.12

-4.5

-1

C (mg mL )

2a)

-4.0

-3.5

-3.0

-2.5

-2.0

lnC

2b)

L-model

F-model

3.0

0.021

2.5

0.018

2.0 1.5 2

lnQ

C/Q

0.015

R =0.488

0.012

1.0 2

R =0.955

0.5

0.009

0.0 0.006 0.003 0.00

-0.5 0.02

0.04

0.06

0.08

0.10

0.12

-1

C (mg mL ) L-model

3a)

-1.0

-4.5

-4.0

3b)

0.021

-3.5

lnC

-3.0

-2.5

-2.0

-2.5

-2.0

F-model

4

0.024

3

0.018

lnQ

C/Q

0.015 2

R =0.118

0.012

2 2

R =0.878

1

0.009 0.006

0

0.003 0.000 0.00

0.02

0.04

0.06

0.08

0.10

-1

0.12

-4.5

-4.0

-1

C (mg mL )

4a)

-3.0

F-model

4

0.018

-3.5

lnC

4b)

L-model

0.021

3

0.015 2

lnQ

0.012

C/Q

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 31

2

R =0.010

0.009

2

R =0.774

1

0.006 0

0.003 0.000 0.00

0.02

0.04

0.06

0.08

-1

0.10

0.12

-1

-4.5

-4.0

C mg ml

-3.5

-3.0

-2.5

-2.0

lnC

281 282

Figure 4. a) Langmuir and b) Freundlich fitting curves of 1: 5% L-CuSiO3, 2: 10%

283

L-CuSiO3, 3: 20% L-CuSiO3, 4: 40% L-CuSiO3.

284 285

The N2 adsorption isotherms of 20% L-CuSiO3 before and after enzyme


Page 15 of 31

286

immobilization were further investigated and shown in Figure 5 with surface area,

287

pore diameter and pore volume data summarized in Table 2. The types of isotherms

288

and hysteresis loops were nearly unchanged after enzyme immobilization, indicating

289

minor textural transformation of the adsorbents. Expectedly, the surface area was

290

decreased from 409.2 to 300.2 m2 g-1, while the pore volume was decreased from 0.83

291

to 0.61 cm3 g-1. However, the pore size distribution nearly stayed the same as shown

292

in the inset of Figure 5, which indicated the HRP adsorption might occur between the

293

L-CuSiO3 layers instead of the pore of every L-CuSiO3 layer. Confocal laser scanning

294

microscope (CLSM) analysis was performed to show spatial distribution of the HRP

295

that stained with FITC. As shown in Figure S6, three images were taken from

296

different depth of the HRP@L-CuSiO3 assemblies, all of which showed the uniform

297

distribution of HRP. Therefore, it could be conclude that the enzyme were located

298

between the layers of L-CuSiO3 assemblies. a) 3

3

400 0

10

30

40

50

60

200 100

adsorption desorption

0 0.0

299

20

Pore diameter (nm)

0.2

0.4

500

-1

adsorption

500

300


b) 600

-1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.6

0.8

adsorption

400 300 200

0

10

20

30

40

Pore diameter (nm)

50

60

100

1.0

adsorption desorption

0 0.0

0.2

0.4

0.6

0.8

1.0

P/P0

P/P0

300

Figure 5. N2 adsorption-desorption isotherm at 77 K and pore size distribution of a)

301

20% L-CuSiO3, b) HRP-loaded 20% L-CuSiO3.

302

Table 2. BET data of 20% L-CuSiO3 before and after immobilization of HRP.

20% L-CuSiO3

HRP-loaded 20% L-CuSiO3

Surface area (m2 g-1)

409.2

300.2

Average pore diameter by BJH method (nm)

3.387

3.392



Pore volume by BJH method (cm3 g-1)

0.831

Page 16 of 31

0.613

303 304

Investigation of Enzyme Leaching Ratio. The enzyme leaching ratio of

305

HRP-loaded L-CuSiO3 assemblies, SBA-15 and SiO2 nanoparticles were further

306

investigated along with incubation time. As shown in Figure 6, the enzyme leaching

307

ratio of L-CuSiO3 assemblies were lower than 10% after the adsorption-desorption

308

equilibrium. By contrast, the enzyme leaching ratio of SBA-15 with a pore size of

309

6-10 nm reached nearly 30%. It should be noted that the size of HRP was 4.0 × 4.4 ×

310

6.8 nm,45 smaller than that of the mesopores in SBA-15, the leaching of

311

intrapore-attached enzyme was inevitable during long-time incubation. The leaching

312

ratio of SiO2 nanoparticles was about 20% during the adsorption-desorption

313

equilibrium, lower than that of SBA-15 for the intensified aggregation during the

314

leaching test, but still twice higher that of L-CuSiO3 assemblies.

315

Since the amino acid residues of the enzyme could interact with transition metal

316

ions through coordination,26 the well-distributed Cu (Ⅱ) in L-CuSiO3 assemblies

317

would then have the function of stably binding enzyme through metal-enzyme

318

coordination. It was also reported that Cu2+ exhibited strongest affinity with arginine

319

(Arg) among metal ions: Li+, Na+, K+, Mg2+, Ni2+, Ca2+, Zn2+ and Cu2+, of which the

320

Gibbs energies ΔG0 was negative (-150 ~ -1500 KJ mol-1).46 Therefore, the

321

coordination of alkaline amino acids (such as lysine, arginine) to Cu (Ⅱ) would then

322

contribute to the low leaching ratio as demonstrated in Figure 7. To further verify the

323

interactions of adsorbents and enzyme, XPS spectra of 20% L-CuSiO3 assemblies

324

before and after enzyme immobilization were shown in Figure S7. A typical peak of

325

amide groups at 399.88 eV was observed in the high-resolution N 1s XPS spectra of

326

20% L-CuSiO3@HRP,47 indicating the existence of enzyme on/in the adsorbents. The


Page 17 of 31

327

peak at ~933.5 eV for Cu (II) oxide both appeared in 20% L-CuSiO3 and 20%

328

L-CuSiO3@HRP,48 indicating high structural stability of the adsorbents during

329

enzyme immobilization. Compared with 20% L-CuSiO3, 20% L-CuSiO3@HRP

330

exhibited different peaks in the range of 942-946 eV in high-resolution Cu 2p XPS

331

spectra. The spectra of 20% L-CuSiO3 showed one peak at 944.2 eV, while the spectra

332

of 20% L-CuSiO3@HRP showed two peaks at 942.6 eV and 944.6 eV, which may be

333

caused by the coordination of Cu (II) and amino acid residues of HRP.49

40

Leaching ratio (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30

SBA-15

10% L-CuSiO3

SiO2

20% L-CuSiO3

5% L-CuSiO3

40% L-CuSiO3

20

10

0

2

4

6

8

10

12

Time (h) 334 335

Figure 6. Leaching profiles of HRP loaded in/on SBA-15, SiO2 nanoparticles and

336

L-CuSiO3 assemblies with different Cu wt% (Note: The initial immobilized enzyme

337

was set as 100%).



20μm

338 339

Figure 7. Schematic diagram of enzyme immobilization.

340 341

Catalytic Activity, Recyclability and Phenol Removal Ability of HRP-loaded

342

L-CuSiO3 Assemblies. The catalytic properties of different HRP-loaded L-CuSiO3

343

assemblies were evaluated by measuring their initial reaction rates and recyclability.

344

The initial reaction rates were measured under identical conditions (with ABTS as the

345

substrate), i.e., the concentration of ABTS and H2O2 was 2 mM and 1 mM, and the

346

weight of free or immobilized HRP was the same. The optimization of immobilized

347

HRP on temperature and pH were performed and shown in Figure S8, and all the

348

catalytic activity were measured under the optimum condition as 30 °C and pH 7.0. In

349

Figure 8a, the initial reaction rates were presented as the relevant value, where the

350

reaction rate of free HRP were set as 100%. The initial reaction rates of HRP-loaded

351

L-CuSiO3 assemblies were higher than that of HRP-loaded SBA-15 and SiO2. As the

352

mass transfer is of vital importance for initial reaction rate, the well-developed pore

353

structure in the L-CuSiO3 assemblies along with the external adsorption contributed

354

much to the enhanced mass transfer, resulting in higher initial reaction rate. The lower


Page 18 of 31

Page 19 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


355

initial reaction rate for SBA-15 was caused by longer distance mass transfer through

356

narrow pore passage, while the decrease in the initial reaction rate for SiO2

357

nanoparticles might be due to the non-negligible aggregation of the nanoscale

358

adsorbent, forming clusters of about 200-400 nm. 50-53 Aggregation usually resulted in

359

reduced effective contact area of enzyme with substrate, then leading to undesired

360

decline in reaction rate.

361

The recyclability was calculated based on the changes of initial reaction rates after

362

cycled-reaction (with TMB as the substrate), where the initial reaction rate of the first

363

cycle was set as 100%. As shown in Figure 8b, the HRP-loaded L-CuSiO3 assemblies

364

could be easily separated through low-speed centrifugation (5000 rpm, 3 min) and

365

nearly no mass was lost. However, a non-negligible loss of activity after every

366

reaction cycle was observed (Figure S9). Since the molecule size of TMB and its

367

oxidation products was much smaller than the average pore size of L-CuSiO3

368

assemblies (~3.3 nm),

369

L-CuSiO3 assemblies and block them during the reaction. Especially, the zeta

370

potential of L-CuSiO3 assemblies was negative (Figure S10), and the products of

371

TMB were positive charged, the electronic interactions between the adsorbents and

372

products could cause the difficulty in the elution of products after each reaction cycle.

373

That would cause a hindered mass transfer, as well as reduced product concentration

374

in the reaction solution. Lowered initial reaction rate was thus obtained after several

375

reaction cycles. Since the primary reason for the low reusability was the strong

376

electrostatic adsorption between positively charged TMB and negatively charged

377

adsorbents, a positively charged polymer, polyetherimide (PEI), was added to shield

378

the surface positive charges. Briefly, PEI was added after the enzyme immobilization

379

and before the recycling experiment. As shown in Figure S11, over 60% of initial

38

the substrate/product could diffuse into the pores of



380

activity was remained after six reaction cycles, suggesting much enhanced reusability

381

of PEI modified L-CuSiO3 assemblies. It further indicated that the recyclability of

382

L-CuSiO3 assemblies could be improved through weakening the electrostatic

383

interactions between adsorbents and substrates/products. For a better reaction rate

384

recovery during cycling, the L-CuSiO3 assemblies are better to be used in some

385

reaction system without charged free radicals.

a)

30 Others

CuSiO3

25 Initial reaction rate /free HRP (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 31

20 15 10 5 0 SB

1 A-

5

O2 Si 5%

L-

C

uS

iO 3

10

%

L-

C

uS

iO 3

20

%

L-

C

uS

iO 3 40

%

L-

C

uS

iO 3

b)

Before reaction

During reaction

After After reaction centrifugation

386 387

Figure 8. a) Initial reaction rate of SBA-15, SiO2 nanoparticles and L-CuSiO3

388

assemblies with different Cu wt%, b) photography images of HRP-loaded 20%

389

L-CuSiO3 assemblies before reaction (H2O2 not added), during reaction, after reaction

390

and after centrifugation.


Page 21 of 31

391

As was well known, HRP was widely applied for wastewater treatment through

392

oxidation of aromatic phenols, phenolic acids, indoles and so on.33 Herein, phenol

393

aqueous solution with the concentrations ranging from 50 to 200 mg L-1 (in the range

394

of industrial wastewater) was prepared and adopted to evaluate the performance of

395

immobilized HRP for phenols removal. As shown in Figure 9, a quick equilibrium

396

was reached within 2 h under different substrate concentrations. As for HRP-loaded

397

20% L-CuSiO3, higher substrate concentration from 50 to 200 mg mL-1 caused the

398

decrease in equilibrium P% from 66 to 34%, while the actual removed phenol

399

concentration was increased from 33 to 67 mg L-1 phenol.

70 60 50

P (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


40 30

-1

50 mg L phenol -1 100 mg L phenol -1 150 mg L phenol -1 200 mg L phenol

20 10 0

400 401

0

2

4

6

8

10

Time (h) Figure 9. Phenol degradation percentage (P %) of HRP-loaded 20% L-CuSiO3.

402 403



404

CONCLUSIONS

405

In summary, nanoporous copper phyllosilicate (L-CuSiO3) assemblies were

406

reported as a high-performance adsorbents for enzyme immobilization. The L-CuSiO3

407

assemblies were prepared via ammonia evaporation hydrothermal method and

408

exhibited the chemical/structural features of high structural stability, high surface area

409

of over 600 cm3 g-1 and porous structure with a pore volume of ~1.0 cm3 g-1, and

410

evenly distributed Cu (Ⅱ). All these properties made the L-CuSiO3 assemblies nearly

411

“ideal” adsorbents for enzyme immobilization, including high and controllable

412

enzyme loading of 140 mg g-1, and low leaching of enzymes less than 10% during

413

incubation. Using the model of contaminates as substrate, the as-synthesized

414

HRP-loaded L-CuSiO3 assembles exhibited a two-fold faster initial reaction rate over

415

its counterparts, which was benefited from the external adsorption as well as

416

well-developed pore structure of L-CuSiO3. Moreover, the relatively large size of the

417

L-CuSiO3 assemblies could be easily collected through low-speed centrifugation of

418

5000 rpm, which would benefit for practical applications. And the HRP-loaded

419

L-CuSiO3 assembles also acted as an efficient bioreactor for removal of phenol

420

pollutants from wastewater.

421

ASSOCIATED CONTENT

422

Supporting information

423

Schematic diagram about the synthesis process of L-CuSiO3 assemblies; TEM images

424

of 5% L-CuSiO3, 10% L-CuSiO3 and 40% L-CuSiO3; EDS and FTIR spectra of

425

L-CuSiO3 assemblies with different Cu wt%; SEM images of SBA-15 and SiO2

426

nanoparticles; Zeta potential values of L-CuSiO3 assemblies with different Cu wt%.

427

AUTHOR INFORMATION

428

Corresponding Authors


Page 22 of 31

Page 23 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


429

*E-mail: [email protected].

430

*E-mail: [email protected].

431

Notes

432

The authors declare no competing financial interest.

433

ACKNOWLEDGEMENTS

434

We are grateful for the financial support from National Natural Science Funds of

435

China (21576189, 21621004, 21406163, 91534126), Specialized Research Fund for

436

the Doctoral Program of Higher Education (20130032110023), Open Funding Project

437

of the National Key Laboratory of Biochemical Engineering (2015KF-03) and

438

Program of Introducing Talents of Discipline to Universities (B06006).

439

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440

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Page 31 of 31

594

Graphical Abstract

HRP on CuSiO3 layers

Coordination of amino acid with Cu2+ ions 30

160

Others

CuSiO3

Initial reaction rate -1 (U mg HRP)

25

140 120

20

100

15

80 60

10

40 5

20

%

3

3

iO uS C

L%

3

3

iO uS C

L-

C 20

40

2

iO

iO uS

uS C L-

L%

5%

10

ASB

595

HRP-loaded CuSiO3 assemblies

SiO

0 15

0

Loading capacity (mg/g)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


596


Nanoporous Phyllosilicate Assemblies for Enzyme Immobilization

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