Preparation of amine-functionalized mesoporous magnetic colloidal nanocrystal clusters for glucoamylase immobilization

Accepted Manuscript Preparation of amine-functionalized mesoporous magnetic colloidal nanocrystal clusters for glucoamylase immobilization Jianzhi Wang, Guanghui Zhao, Yanfeng Li, Xiaomeng Peng, Xinyu Wang PII: DOI: Reference:

S1385-8947(14)01059-6 http://dx.doi.org/10.1016/j.cej.2014.08.007 CEJ 12512

To appear in:

Chemical Engineering Journal

Received Date: Revised Date: Accepted Date:

26 May 2014 29 July 2014 1 August 2014

Please cite this article as: J. Wang, G. Zhao, Y. Li, X. Peng, X. Wang, Preparation of amine-functionalized mesoporous magnetic colloidal nanocrystal clusters for glucoamylase immobilization, Chemical Engineering Journal (2014), doi: http://dx.doi.org/10.1016/j.cej.2014.08.007

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1

Preparation of amine-functionalized mesoporous magnetic colloidal

2

nanocrystal clusters for glucoamylase immobilization ∗

3

Jianzhi Wang, Guanghui Zhao, Yanfeng Li , Xiaomeng Peng and Xinyu Wang

4

State Key Laboratory of Applied Organic Chemistry, Institute of Biochemical Engineering &

5

Environmental Technology, College of Chemistry and Chemical Engineering, Lanzhou University,

6

Lanzhou 730000 (China),

7

Abstract

8

A facile one-pot synthesis of size-tunable mesoporous carboxyl-functionalized

9

magnetic colloidal nanocrystal clusters (MCNCs) with high magnetization (82.0

10

emu/g), large surface area (95 m2/g), and excellent colloidal stability has been

11

developed. The mesostructured MCNCs were synthesized by a solvothermal approach

12

with iron (III) chloride hexahydrate as a precursor, ethylene glycol as a reducing agent,

13

ammonium acetate as a porogen, and ethylenediaminetetraacetic acid disodium salt

14

(EDTA-2Na) as a surface-modification agent. Glucoamylase was immobilized onto

15

the mesoporous MCNCs via the different routes. These immobilized glucoamylase

16

exhibited excellent thermal stability and reusability in comparison with the free

17

enzyme. The residual activity of immobilized enzyme remained above 65% after 6 h,

18

while free glucoamylase was only left over 45% of the initial activity. And the

19

residual activity of the immobilized enzyme was about 60 % of the initial activity

20

after the 10th reuse.

∗

To whom correspondence should be addressed. Tel.: 86-931-8912528; Fax: 86-931-8912113. E-mail address: [email protected]. 1

21

Keywords: Mesoporosity; Magnetic colloidal nanocrystal clusters; Immobilisation;

22

Glucoamylase; Biocatalysis; Enzyme Activity.

23

1. Introduction

24

Magnetic colloidal nanocrystal clusters (MCNCs) have attracted significant

25

interest because of the unique superparamagnetic properties, high magnetization, and

26

high water dispersibility [1], which has been widely used in diverse areas of

27

bioseparation [2,3], MRI contrast agents [4, 5], heterogeneous catalysts [6, 7] and

28

drug delivery [8, 9]. For bioapplication applications, magnetism makes possible

29

heterogeneous catalysis by which fast separation of biocatalysts is made feasible, and

30

the process can be carried out continuously [10-12]. In principle, the ideal MCNCs for

31

bioapplication should possess suitable surface area, narrow size distribution, strong

32

magnetic response, abundant functional groups and excellent biocompatibility.

33

Therefore, the synthesis of monodisperse MCNCs with a hydrophilic functional

34

surface, especially for the template-free synthesis of functional mesoporous MCNCs,

35

is still an essential yet challenging step as they have much potential in biological and

36

medical fields, such as the immobilization of proteins, peptides, and enzymes [13].

37

Over the past two decades, there have been various techniques for the preparation

38

of these MCNCs, such as coprecipitation and microemulsion methods [14, 15].

39

However, the relatively poor size uniformity and monodisperse of the nanoparticles

40

obtained strongly affect their magnetic properties. Exciting progress has been made in

41

synthesizing iron oxide magnetic nanocrystals with controlled size and shape by

42

high-temperature solution-phase reaction of Fe(acac)3 [16, 17]. These small magnetic

2

43

nanocrystals have specific surface area and uniformly size, but the weak magnetic

44

response and high reaction temperature limits their technical use. Among the various

45

synthetic methodologies, Li group [18] first described the one-pot solvothermal

46

synthesis

47

cluster-structure magnetic nanomaterials. This facile method evokes much interest,

48

and is subjected to extensive studies for the preparation of MCNCs that satisfy all

49

major requirements in biotechnology. Yin et al. [19] synthesized highly

50

water-dispersed MCNCs using poly (acrylic acid) to the surface of MCNCs as a

51

stabilizer. Cheng et al. [20] fabricated sodium citrate stabilized MCNCs to improve

52

biocompatibility, and the tuning effect of sodium citrate on the magnetite nanocrystal

53

clusters was well illustrated. Li and coworkers [21] also reported the synthesis of

54

amine-functionalized MCNCs using 1, 6-hexanediamine as precipitation agent and

55

amine-functional agent. Unfortunately, saturation magnetization is improved by the

56

sacrifice of the large surface area so as to restrict their application [22]. Therefore, it

57

is a new challenge to fabrication of functional mesoporous MCNCs with high

58

magnetization and large surface area that satisfy all major requirements in

59

biotechnology.

method

that

has

attracted

researchers’

attention

for

preparing

60

Herein, we report, for the first time, the synthesis of the mesoporous

61

carboxyl-functionalized MCNCs with tunable size and high magnetization. As

62

depicted in Figure 1, the nanocrystals were assembled into interior porous clusters by

63

using iron (III) chloride hexahydrate as precursor, ethylene glycol as reducing agent,

64

ammonium acetate as a porogen and EDTA-2Na as a surface modification agent.

3

65

Moreover, the particle sizes can be simply controlled by varying the relative

66

concentrations of EDTA-2Na. To estimate the applicability of the obtained MCNCs in

67

biotechnology, the mesoporous carboxyl-functionalized MCNCs were used to

68

immobilize

69

mesoporous MCNCs were used to immobilize glucoamylases by electrostatic

70

adsorption. The properties of the immobilized glucoamylases also were studied

71

systematically.

72

2. Materials and Methods

73

2.1 Enzymes and Reagents

glucoamylases

by

covalent

bonding,

and

amine-functionalized

74

Glucoamylase (exo-1, 4-a-D-glucosidase, EC 3.2.1.3 from Aspergillus niger 10U

75

mg-1) was purchased from Yixing Enzyme Preparation Company (China); Bovine

76

serum

77

hydrochloride

78

tris(2-aminoethyl)amine (TAEA) were purchased from Sigma Chemical Co.; Other

79

chemicals and reagents were analytical grade, obtained from Tianjing Chemical

80

Reagent Company (China).

81

2.2 Preparation of mesoporous carboxyl-functionalized MCNCs.

albumin

(BSA),

(EDC·HCl,

N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide

99%),

N-hydroxysuccinimide

(NHS,

97%),

and

82

The mesoporous carboxyl-functionalized MCNCs were prepared through a

83

modified solvothermal reaction [23-25]. Typically, anhydrous NH4OAc (2.0 g),

84

FeCl3·6H2O (0.8 g) and EDTA-2Na (with various weight: 0, 0.1, 0.3, 0.5, and 1.0 g)

85

were dissolved in ethylene glycol (40 ml) under vigorously stirring to give a

86

homogeneous yellow solution. The solution was sealed in a Teflon-lined

4

87

stainless-steel autoclave heated at 200 °C for 12 h, and the mixture was then cooled to

88

ambient temperature. The resulting black magnetite particles were washed with

89

deionized water and ethanol, and dried at 60 °C before characterization and

90

application.

91

2.3 Amine functionalization of mesoporous MCNCs.

92

The carboxyl-functionalized mesoporous MCNCs were first activated with

93

EDC/NHS mixture [26]. 0.2 g of mesoporous MCNCs in 20 mL of phosphate buffer

94

solution (50 mM, pH 7.0) were sonicated for 1 h to obtain a homogeneous dispersion.

95

This dispersion was mixed with 10 mL of 400 mM EDC and 100 mM NHS in

96

phosphate buffer solution, and shook gently for 1 h at room temperature. The obtained

97

products were denoted as Fe3O4-EDC. Subsequently, 1 ml of ethylene diamine (EDA)

98

or TAEA was slowly added to yield a final concentration of 500 mM. The reaction

99

mixture was incubated overnight while stirring. Finally, the amine-functionalized

100

mesoporous MCNCs was taken out, washed several times with de-ionized water and

101

then dried in atmosphere. Following this process, the Fe3O4-EDA or Fe3O4-TAEA

102

was obtained.

103

2.4 Immobilization of glucoamylase on the amine-functionalized mesoporous MCNCs.

104

Schematic representatives for the preparation of supports and the enzyme

105

immobilization are shown in Fig. 1. Glucoamylase was immobilized onto the

106

Fe3O4-EDA or Fe3O4-TAEA by electrostatic adsorption. Necessary qualities of

107

supports were put into glucoamylase buffer solution (pH 4.0, 50 mM acetate buffer),

108

and then the reaction was taken out at 30 °C in a shaking-table with rotational speed

5

109

as 120 rpm for 6 h. After immobilization was completed, the immobilized

110

glucoamylase was obtained by magnetic separation, and washed with acetate buffer

111

(0.1 M, pH = 4.0) two times to remove the unreacted glucoamylase. The resultant

112

immobilized glucoamylase was kept at 4 °C prior to use. The amount of immobilized

113

protein on the support was determined by measuring the initial and final

114

concentrations of protein in the reaction medium by Bradford’s method [27]. BSA

115

was used as standard to construct a calibration curve. The immobilization capacity of

116

the protein on the support was defined as the amount of protein (mg) per gram of the

117

support. For comparison, the immobilization ability of pure (3-aminopropyl)

118

triethoxysilane (APTES)-modified Fe3O4 (Fe3O4-NH2) was also studied. Details of the

119

preparation were thoroughly described in supplementary material.

120

2.5 Immobilization of glucoamylase on the mesoporous carboxyl-functionalized

121

MCNCs.

122

Glucoamylase was immobilized onto the mesoporous MCNCs activated with

123

EDC/NHS (Fe3O4-EDC) by covalent bonding. The immobilization process was

124

carried out at 30 °C in a shaking air bath for 6 h. After this, the immobilized

125

glucoamylase was recovered by magnetic separation, and thoroughly rinsed with

126

acetate buffer solution (50 mM, pH 4.0) two times to remove unbound glucoamylase.

127

The washed solution was collected to assay the amount of residual enzyme. The

128

resulting immobilized glucoamylase was stored at 4 °C prior to use. The amount of

129

immobilized enzyme on the Fe3O4-EDC nanoparticles was determined as described

130

above.

6

131

2.6 Enzyme Activity Assay

132

The enzyme activity was determined by using soluble starch as a substrate [28].

133

In the standard conditions, the substrate composed of 0.5 mL of 10 wt.% soluble

134

starch gelatinized in water and 2.5 mL acetate buffer solution (50 mM, pH 5.5). The

135

reaction was started by addition of 0.5 mL free glucoamylase (0.236 mg mL-1) or 0.1

136

g of immobilized glucoamylase. The mixture was incubated at 30 °C under reciprocal

137

agitation at 120 strokes per minute. After 15 min of reaction, agitation was stopped,

138

and then the reaction was terminated by adding 5 mL of NaOH solution (0.1 M). The

139

glucose content was determined using the 3, 5-dinitrosalicylic acid (DNS) method

140

[29]. The amount of glucose was obtained from the calibration curve and used in the

141

calculation of enzyme activity. One unit of glucoamylase activity (U) was defined as

142

the amount of glucoamylase that produced 1.0 mmol of glucose from dissolubility of

143

starch per minute in the assay condition. The activity recovery (%) was the ratio

144

between the activity of bound glucoamylase and the total activity of glucoamylase

145

added in the initial immobilization solution. All experiments of activity measurement

146

were carried out at least three times, and the experimental error was less than 3.0 %.

147

2.7 Characterization

148

The morphologies of the samples were further investigated by transmission

149

electron microscopy (TEM, FEI Tecnai G20) and scanning electron microscopy (SEM,

150

JSM-6701F, JEOL, Japan); FT-IR spectra were recorded by a Fourier-transform

151

infrared spectrophotometer (American Nicolet Corp. Model 170-SX) using the KBr

152

pellet technique; The crystal structure of the magnetic nanoparticles were examined

7

153

by the X-ray diffraction (XRD, Rigaku D/MAX-2400 X-ray diffractometer with

154

Ni-filtered Cu Kα radiation). The magnetization curves of the magnetic nanoparticles

155

were measured with a vibrating sample magnetometer (LAKESHORE-7304, USA) at

156

room temperature. The Brunauer-Emmett-Teller (BET) specific surface areas were

157

measured with a Micromeritics ASAP 2010M instrument. Thermogravimetric analysis

158

of the magnetic nanoparticles was observed by a TG-DSC apparatus (NETZSCH STA

159

449C) by heating the samples from room temperature to 800 °C under N2 atmosphere

160

at a heating rate of 20.0 K min-1.

161

3. Results and Discussion

162

3.1 Characterization of mesoporous MCNCs.

163

For investigate the effect of EDTA-2Na on the morphology of magnetite

164

nanocrystal clusters, a series of experiments with different amounts of EDTA-2Na (0,

165

0.1, 0.3, 0.5, and 1.0 g) were carried out. Fig. 2 shows the morphology variation of

166

products with different amounts of EDTA-2Na added. As a control, Fig. 2a shows

167

uniform MCNCs formed without the addition of EDTA-2Na, which have a hollow

168

magnetite nanocrystal clusters with a diameter of 225-250 nm instead of mesoporous

169

magnetite nanocrystal clusters. However, when the initial EDTA-2Na concentration is

170

increased from 0.1 to 1.0 g, the mesoporous magnetite nanocrystal clusters

171

spontaneously aggregated by the primary Fe3O4 nanocrystals are uniform both in size

172

and in shape. When the feeding amount of EDTA-2Na is 0.3, the mesoporous

173

magnetite nanocrystal clusters became morphologically, and the mesoporous structure

174

becomes more distinct (Fig. 2c). Meanwhile, when the feeding amount of EDTA-2Na

8

175

is further increased to 1.0 g, the compact structure of the MCNCs become

176

morphologically rough, irregular, and structurally loose (Fig. 2e). The diameter of the

177

mesoporous MCNCs varies from 325, 365, 265, to 225 nm with an increase of

178

EDTA-2Na content from 0.1 to 1.0 g, as shown in the SEM (Fig. 3), thus indicating

179

EDTA-2Na plays an important role in the formation of pure mesoporous magnetite

180

nanocrystal clusters. The decrease of the grain size could be attributed to the

181

adsorption of the EDTA-2Na on the nanocrystals which blocked the growth of the

182

magnetic nanocrystals, which was consistent with the previous report [20].

183

In order to understand the influence of EDTA-2Na on the subunits of MCNCs,

184

X-ray diffraction was used to study the variation of the subunits of the MCNCs. The

185

powder X-ray diffraction (XRD) patterns for all of the MCNCs are compiled in Fig.

186

4A. The characteristic diffraction peaks of all of the MCNCs can be well indexed to

187

the (220), (311), (400), (422), (511), and (440) planes according to JCPDS 19-629.

188

Among them, the primary magnetite nanocrystals from the MCNCs give rise to the

189

strongest and sharpest X-ray diffraction peaks, indicative of the formation of

190

large-size, highly crystalline Fe3O4 [22]. To further investigate the effects of

191

EDTA-2Na on their magnetic properties, all of the MCNCs were investigated using a

192

vibrating sample magnetometer (VSM) at room temperature. As shown in Fig. 4B, the

193

bare MCNCs displayed an extremely high saturation magnetization of up to 93.7

194

emu/g, the mesoporous MCNCs synthesized with EDTA-2Na of 0.1, 0.3, 0.5 and 1.0

195

g have saturation magnetization values of 84.1, 65.6, 54.7 and 36.4 emu/g,

196

respectively. This trend may be attributed to the EDTA-2Na largely restrict particle

9

197

growth, and thus the primary magnetite nanocrystals are so small grain sizes that they

198

elicit a considerably decreased magnetization in MCNCs with an increase of the

199

EDTA-2Na feeding amount [30]. These results indicated that the nanocrystal clusters

200

exhibited strong magnetic responsiveness, which suggests that this level of saturation

201

magnetization is sufficient for applications in enzyme immobilization. Additionally,

202

thermogravimetric analysis (TGA) profiles confirmed that EDTA-2Na was anchored

203

within the MCNCs, so that weight loss increased as a function of the feeding

204

EDTA-2Na (Fig. S1). This result is accordance with the results of saturation

205

magnetization. Compared to the other magnetic mesoporous spheres prepared in

206

former literatures [31, 32], the magnetic mesoporous spheres we prepared here

207

showed narrow size distributions, excellent mesoporous structures, rich functional

208

group and high saturation magnetization.

209

In addition, the porous nature of MCNCs was also confirmed by measurement of

210

nitrogen

adsorption-desorption

measurements.

211

adsorption-desorption isotherms of MCNCs obtained with different EDTA-2Na weigh.

212

All the samples are considered as the type-IV isotherm, which is characteristic of

213

mesoporosity. The Brunauer-Emmett-Teller (BET) model was used to estimate the

214

mesoporous parameters for the various EDTA-2Na-stabilized MCNCs, as shown in

215

Table 1. Upon increasing the EDTA-2Na content from 0.1 to 1.0 g, the surface area of

216

the MCNCs, increased from 33 to 95 m2/g. This increasing surface area could be

217

attributed to the decreasing crystal size triggered by EDTA-2Na. The bare MCNCs, as

218

expected, give the lowest surface area due to the structure of hollow chambers and

10

Fig.

S2

shows

the

N2

219

mesoporous walls assembled by dense packing of the primary nanoparticles. The

220

higher BET surface area and larger pore volume strongly support the fact that the

221

product has a mesoporous structure, which is in accordance with the observations

222

from SEM and TEM. However, as the concentration of EDTA-2Na increased in the

223

polyol reaction, the pore size of the resulting CMNCs gradually decreased from about

224

20.2 nm to 7.7 nm. Accordingly, high surface area and proper pore size are very

225

important for potential application in catalysis, drug delivery, enzyme immobilization,

226

and so on. The optimum pore size for immobilization of the enzyme was similar to

227

that for glucoamylase. When the feeding amount of EDTA-2Na is 0.3, the BET

228

surface area and total pore size are calculated to be 40 m2/g and 8.8 nm, respectively.

229

The pore size of MCNCs is a good match for the dimensions of glucoamylase

230

molecules (8.5 nm) [33]. This type of porosity would provide an efficient transport

231

pathway for reactants to the interior of the MCNCs, which is beneficial for catalytic

232

properties. Base on the above two facts (large surface area and suitable pore size),

233

thus we hypothesize that carboxyl-functionalized mesoporous MCNCs (0.3 g of

234

EDTA-2Na) are available for enzyme immobilization.

235

To evaluate the capability of the mesoporous MCNCs for enzyme immobilization,

236

the carboxyl-functionalized mesoporous MCNCs (0.3 g of EDTA-2Na) were

237

functionalized via two strategies, and it is visually summarized schematically in Fig. 1.

238

Fig. 5 shows FTIR spectra of carboxyl-functionalized mesoporous MCNCs,

239

Fe3O4-EDA and Fe3O4-TAEA, respectively. The FTIR spectrum of the parent

240

mesoporous MCNCs (Figure 5a) exhibited the characteristic absorption peaks of

11

241

carboxyl group of EDTA-2Na at 1600 cm-1 due to the presence of the -COO-. Upon

242

Fe3O4-EDC followed by EDA and TAEA grafting, the peak at 1600 cm-1 (Figure 5b, c)

243

disappeared, and new peaks appear at 1632 cm-1 (N-H) because of the amine groups

244

present in EDA or TAEA. These results confirm the successful functionalization of

245

carboxyl-functionalized mesoporous MCNCs.

246

3.2 Immobilization of glucoamylase.

247

Three kinds of supports (Fe3O4-EDC, Fe3O4-EDA and Fe3O4-TAEA) were

248

achieved and used to enzyme immobilization with different methods. First,

249

glucoamylase was immobilized onto the mesoporous Fe3O4-EDC by covalent bond.

250

Besides, the Fe3O4-EDA and Fe3O4-TAEA with a ζ-potential of 20.6 mV and 18.3

251

(pH 4.0) were obtained (see Fig. S3), while the ζ-potential of glucoamylase was -3.2

252

mV at pH 4.0. Thus, Fe3O4-EDA or Fe3O4-TAEA could be used to adsorb

253

glucoamylase

254

APTES-modified Fe3O4 (Fe3O4-NH2) nanoparticles also were used to immobilize

255

enzyme by electrostatic adsorption. Details of the preparation were thoroughly

256

described in supplementary material.

directly by electrostatic

adsorption.

By contrast,

the

solid

257

Table 2 shows the activity of the immobilized glucoamylases under the optimum

258

reaction conditions, and all the immobilized glucoamylases were prepared under the

259

optimum conditions. In the activation process, the order of amount of -NH2 bonded

260

(test by elementary analysis) is: Fe3O4-TAEA›Fe3O4-EDA›solid Fe3O4-NH2

261

nanospheres. It can be seen that the amounts of bound protein are 29.2±0.81 mg/g on

262

the mesoporous Fe3O4-EDC, 27.6±0.96 mg/g on the solid Fe3O4-NH2, 44.0±1.05

12

263

mg/g on the mesoporous Fe3O4-EDA and 44.7±0.75 mg/g on the Fe3O4-TAEA.

264

Interestingly, compared to that on the solid Fe3O4-NH2, there is lower than that of

265

these mesoporous Fe3O4 nanoparticles. This result can be explained by the fact that

266

the remarkable larger space and the functional groups of the mesoporous MCNCs can

267

provide more potential reaction sites for the coupling of enzyme [34]. The maximum

268

amount of bound protein is higher than previous research [35]. According to the data

269

presented in Table 2, the binding capacity of glucoamylase immobilized via

270

electrostatic adsorption was found to be higher than the capacity of this enzyme

271

bound via covalent bond. Furthermore, the results confirmed that immobilization via

272

electrostatic adsorption are faster than that via covalent bond. Because all of the

273

functional groups are exposed at the mesoporous MCNCs surface and the low

274

diffusion resistance, the glucoamylase solution could rapidly diffuse to the surface of

275

the MCNCs. Anyway, all the mesoporous MCNCs have excellent immobilization

276

capacities for glucoamylase immobilization.

277

3.3 Properties of immobilized glucoamylase.

278

The change in optimum pH depends on the charge of the enzyme and the basic

279

character of the support material. The pH dependence of the free and immobilized

280

enzyme activity at pH ranging from 1.5 to 8.0 at 30 °C was investigated. As shown in

281

Fig. 6A, the free enzyme exhibited better residual activity than all immobilized

282

enzyme when the pH was exceeded 4.0. However, the glucoamylase immobilized

283

exhibited better residual activity than the free glucoamylase when the pH below 5.5.

284

This shift could be attributable to the amino groups on the surface of Fe3O4-EDA and

13

285

Fe3O4-TAEA, which might have been buffered and immobilized enzyme was less

286

affected by the acidity of the solution [36, 37].

287

The effect of the temperature profile on the activity of free and immobilized

288

glucoamylase was investigated at various temperatures ranging from 25-75 °C shown

289

in Fig. 6B. The immobilized enzyme showed an optimum reaction temperature

290

between 45 and 65 °C, whereas free enzyme had an optimum temperature about 40 °C.

291

Meanwhile, the residue activity of the immobilized enzyme decreased more slowly

292

than that of its free form between 50 and 75 °C. Moreover, compared to free enzyme,

293

immobilized enzyme used in this experiment retained 60% of its original activity at

294

temperature more than 70 °C, whereas the native one lost 80% of its original activity.

295

Hence, these results show that immobilized enzyme has better thermal resistance as

296

compared with the free enzyme, especially at high temperature. Such phenomenon

297

has also been observed by other researchers [34], which mean that the immobilization

298

methods preserve the enzyme activity in a wider temperature range. It might be

299

attributed to the carriers enhancing the enzyme rigidity that protected it from

300

unfolding and prevent the denaturation of enzyme at high temperature [38].

301

Due to the thermal stability of the immobilized enzyme systems is critical to its

302

practical applications, the free and immobilized gluaoamylase was stored at 65 °C and

303

the activity were also examined with different time interval. As the temperature is

304

increased, a number of bonds in the protein molecule are weakened. The first affected

305

are the longrange interactions that are necessary for the presence of three-dimensional

306

network structure of protein. As heating continues, some of the cooperative hydrogen

14

307

bonds that stabilize helical structure of protein would be broken [38]. As the helical

308

structure is broken, the immobilized enzyme also could gradually lose its activity. Fig.

309

6C shows the inactivation due to the different enzyme preparations. The result shows

310

that the preparations exhibited a similar trend: there was a significant decrease in the

311

activity of the immobilized glucoamylase and free glucoamylase over 6 h, whereas

312

the immobilized glucoamylase decreased less and more slowly than the free one. The

313

residual activity of immobilized enzyme remained above 65 % after 6 h, while free

314

glucoamylase was only left over 45% of the initial activity. The lower stability of

315

glucoamylase had also been reported by Carpio et al. [39], the immobilized enzyme

316

showed 40-50% of its initial activity after 240 min of incubation at 55 °C. Thus, these

317

results demonstrated that glucoamylase immobilized MCNCs showed significant

318

thermal stability compared to that of its free enzyme [40].

319

The reusability of immobilized enzyme is a pretty important aspect in potential

320

industrial applications. The variation in activity of the immobilized enzyme after

321

multiple reuses was showed in Fig. 7. It could be observed that the residual activity of

322

the immobilized enzyme still remained about 60 % of the initial activity after the 10th

323

reuse through the isolation of magnetic enzyme by external magnetic field. So the

324

immobilized enzyme prepared has good stability and reusability. The decrease of

325

activity was considered as the denaturation of protein and the leakage of protein from

326

the supports during the process of use [41].

327

4. Conclusions

328

In summary, we have demonstrated a flexible and simple method for the synthesis

15

329

of

monodisperse

mesoporous

330

solvothermal process. The mesoporous MCNCs simultaneously possess high

331

magnetization, large surface area, narrow size distribution, superior monodispersity,

332

and excellent colloidal stability. The surface area, the crystal size, and the saturation

333

magnetization value can be controlled by varying the concentration of EDTA-2Na. To

334

estimate the applicability of the obtained MNCs in biology-related fields,

335

glucoamylase was immobilized onto mesoporous MCNCs via the different routes.

336

Furthermore, the results confirmed that the binding capacity of glucoamylase

337

immobilized via electrostatic adsorption was higher than the capacity of this enzyme

338

bound via covalent bond. The immobilized glucoamylase showed excellent catalytic

339

activity and reusability in comparison with the free enzyme. Additionally, we expect

340

that the mesoporous MCNCs may offer new potential supports in biotechnology and

341

organocatalysis

342

microstructure.

343

Acknowledgment

344

The authors thank the financial supports from the National Natural Science

345

Foundation of China (No.21374045, No.21074049), the scientific research ability

346

training of under-graduate students majoring in chemistry by the two patterns based

347

on the tutorial system and top students (J1103307), and the Opening Foundation of

348

State Key Laboratory of Applied Organic Chemistry (SKLAOC-2009-35).

349

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472

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473

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476

Table 1. Porous characteristics of the MCNCs synthesized by varying the feeding

477

amount of EDTA-2Na.

478

feeding amount

surface area

pore volum

pore size

of EDTA-2Na (g)

(m2/g)

(cm3/g)

(nm)

0

17

0.07

20.2

0.1

33

0.09

10.6

0.3

40

0.09

8.8

0.5

56

0.10

8.0

1

95

0.19

7.7

Table 2. The immobilization capacity of different supports. Supports

N

pH

(%)

Time

enzyme

Protein

Activity

(h)

concentration

bonded (mg g-1)

recovery (%)

Fe3O4-NH2

0.68

4.0

5

35

27. 57±0.96

35.8±1.9

Fe3 O4-EDC

-

4.0

6

35

29.2±0.81

46.4±1.8

Fe3O4-EDA

1.21

4.0

5

50

44.0±1.05

60.3±1.8

Fe3O4-TAEA

1.35

4.0

5

50

44.7±0.75

50.9±1.7

479 480 481 482 483 484 485 486 487 488 21

489

Captions of Figures

490

Fig. 1 Schematic representative for the preparation of the supports and enzyme

491

immobilization.

492

Fig. 2 Representative TEM images of the mesoporous MCNCs synthesized with

493

EDTA-2Na of (a) 0, (b) 0.1, (c) 0.3, (b) 0.5, and (e) 1.0 g.

494

Fig. 3 Representative SEM images of the mesoporous MCNCs synthesized with

495

EDTA-2Na of (a) 0, (b) 0.1, (c) 0.3, (b) 0.5, and (e) 1.0 g.

496

Fig. 4 (A) XRD patterns of mesoporous MCNCs obtained with EDTA-2Na of (a) 0, (b)

497

0.1, (c) 0.3, (b) 0.5, and (e) 1.0 g. (B) Magnetic hysteresis curves of mesoporous

498

MCNCs obtained with EDTA-2Na of (a) 0, (b) 0.1, (c) 0.3, (b) 0.5, and (e) 1.0 g.

499

Fig. 5 The IR spectra of (a) mesoporous MCNCs, (b) Fe3O4-EDA and (c)

500

Fe3O4-TAEA.

501

Fig. 6 pH stability (A) and thermal stability (B) of free and immobilized glucoamylase,

502

the catalytic activity of glucoamylase was determined by catalyzing the hydrolyzation

503

of the substrate at 30 °C in pH 1.5-8.0 and at 25-75 °C in pH 4.0; thermal stability (C)

504

of free and immobilized glucoamylase, tests were carried out at 65 °C.

505

Fig. 7 Reuse of glucoamylase immobilized on the supports.

506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 22

525

Fig. 1

526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551

23

552

Fig. 2

553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581

24

582

Fig. 3

583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615

25

616

Fig. 4

617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649

26

650

Fig. 5

651

652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680

27

681

Fig. 6

682 683 684 685 686 687 688

28

689

Fig. 7

690

691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714

29

715

►Mesoporous carboxyl-functionalized magnetic colloidal nanocrystal clusters

716

(MCNCs) were synthesized.

717

►The mesoporous MCNCs showed large surface area and high magnetization.

718

►The sizes of the MCNCs could be easily tuned by varying the surfactant

719

concentration.

720

►The amine-functionalized mesoporous MCNCs were utilized to immobilize

721

enzyme.

722

.

723 724 725

30