Covalent Immobilization of Cellulase Using Magnetic Poly(ionic liquid

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Covalent immobilization of cellulase using magnetic poly(ionic liquid) support; improvement of the enzyme activity and stability Seyed Hassan Hosseini, Seyedeh Ameneh Hosseini, Nasrin Zohreh, Mahshid Yaghoubi, and Ali Pourjavadi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03922 • Publication Date (Web): 11 Jan 2018 Downloaded from http://pubs.acs.org on January 11, 2018

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Journal of Agricultural and Food Chemistry

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Covalent Immobilization of Cellulase Using Magnetic Poly(ionic

2

liquid) Support; Improvement of the Enzyme Activity and Stability

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Seyed Hassan Hosseini†,*, Seyedeh Ameneh Hosseini†, Nasrin Zohreh‡, Mahshid Yaghoubi§ and

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Ali Pourjavadi§

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†

Department of Chemical Engineering, University of Science and Technology of Mazandaran, Behshahr, Iran ‡

11 12

§

Department of Chemistry, Faculty of Science, University of Qom, Qom, Iran

Polymer Research Laboratory, Department of Chemistry, Sharif University of Technology, Tehran, Iran

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Corresponding Author: Dr. Seyed Hassan Hosseini, [email protected]; Phone/fax: +982166165301

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21 1 ACS Paragon Plus Environment


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ABSTRACT: A magnetic nanocomposite was prepared by entrapment of Fe3O4 nanoparticles

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into the cross-linked ionic liquid/epoxy type polymer. The resulting support was used for

24

covalent immobilization of cellulase through the reaction with epoxy groups. The ionic surface

25

of the support improved the adsorption of enzyme and a large amount of enzyme (106.1 mg/g)

26

was loaded onto the support surface. The effect of the presence of ionic monomer and covalent

27

binding of enzyme was also investigated. The structure of support was characterized by various

28

instruments such as FT-IR, TGA, VSM, XRD, TEM, SEM and DLS. The activity and stability of

29

immobilized cellulase were investigated in the prepared support. The results showed that the

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ionic surface and covalent binding of enzyme onto the support improved the activity, thermal

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stability and reusability of cellulase compared to free cellulase.

32 33 34 35

KEYWORDS: Cellulase; Covalent Immobilization; Magnetic Support; Ionic Liquid

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2 ACS Paragon Plus Environment

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1. INTRODUCTION

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The production of ethanol, as an alternative green motor fuel from lignocellulosic biomass is

45

highly interesting since it can be produced without using food resources and it has also high

46

octane number which reduces greenhouse gas emissions

47

cellulosic materials includes enzymatic hydrolysis of cellulose and hemicellulose which leads to

48

the production of fermentable reducing sugars and then the resulting sugars fermented to produce

49

ethanol 5-7. Unfortunately, the current technologies for hydrolysis of lignocellulosic materials are

50

relatively expensive and the process has no economic advantage due to cellulase enzyme, which

51

is necessary for hydrolysis, is very sensitive to changes in environmental conditions

52

widespread practical applications of cellulase are limited because of its hydrophilic nature and

53

low stability in various ranges of pH and temperature

54

cellulase, its stability and reusability should be enhanced. A practical way to the improvement of

55

enzyme stability and reusability is immobilization of enzymes onto the solid supports

56

Among the various supports for immobilization of enzymes

57

interesting because their magnetic properties allow us to separate them by using an external

58

magnet

59

where the separation of large amounts of immobilized enzyme does not need a troublous

60

filtration or centrifugation

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immobilization; physical adsorption

62

enzyme onto the solid support provides an easy way for immobilization which rarely changes the

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structure of enzyme and therefore decreases the activity loss 16, 47. However, the physical linkage

64

is not firm and the enzyme will easily detached from the support surface, so in this case the

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reusability of supported enzyme is poor

28-35

1-4

. The production of bioethanol from

10

8-9

. The

. Therefore, for a practical use of

20-27

11-19

.

, magnetic supports are more

. This advantage of magnetic supports is highly desirable in large-scale applications

32,

36-37

. There are two conventional methods for enzyme

38-41

and covalent binding

47-48

42-46

. The physical adsorption of

. In the second method, enzyme is attached to the



66

surface of support by covalent bond. The covalent binding of enzyme onto the support surface

67

increases the stability and reusability of the enzyme, however; it may reduce the activity 27, 32, 34,

68

49

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suitable for industrial applications than physical immobilization, since the expensive cellulase

70

must be reused several times 50-51.

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The epoxy functionalized supports are ideal materials for covalent immobilization of enzymes 52-

72

54

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groups on the surface of enzymes and form a strong linkage with minimum changes in the

74

structure of enzymes

75

happens, the enzyme should initially adsorbed by the surface, then the ring opening reaction

76

occurs and a covalent bond is established

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hydrophobic surface, the immobilization of enzyme onto these supports has been carried out at

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higher temperature or lower pH values57-58. On the other hand, multi-point covalent binding of

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enzyme onto these kinds of supports reduces the activity of enzymes 55, 59.

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In this work, we prepared a magnetic nanocomposite based on entrapment of Fe3O4 nanoparticles

81

into an epoxy modified ionic liquid type polymer. The cellulase enzyme was then immobilized

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onto the support by covalent binding. The ionic liquid part of polymer chains improved the

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adsorption of enzyme. The immobilization of enzyme is carried out at mild condition to prevent

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denaturation of the enzyme. The activity and stability of immobilized cellulase are then

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investigated and compared to a non-ionic support.

. Despite the loss of enzyme activity resulted from covalent binding, this method is still more

. Epoxy groups are stable at neutral pH but they easily react with amine, hydroxyl and thiol

55-56

. However, before any reaction between enzyme and epoxy groups

56

. Since most of commercial epoxy supports have

86 87

2. MATERIALS AND METHODS


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2.1. Materials and instruments. Ferric chloride hexahydrate (FeCl3.6H2O), ferrous chloride

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tetrahydrate

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(trimethoxysilyl)propylmethacrylate (MPS), and methylenebisacrylamide (MBA) were obtained

91

from

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(methacryloylamino)propyl

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Azobisisobutyronitrile (AIBN) purchased for Sigma-Aldrich. GMA was distilled before use and

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stored in refrigerator and AIBN was recrystallized from ethanol. Cellulase from Aspergillus,

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carboxymethyl cellulose sodium salt (CMC, Mw=90,000), D-glucose, dinitrosalicylic acid

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(DNS), potassium sodium tartrate, coomassie blue G-250 and bovine serum albumin (BSA) were

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obtained from Sigma-Aldrich. The de-ionized water was used in this study and filtered using a

98

U.S. Filter purification system.

99

FT-IR spectra of samples were taken using an ABB Bomem MB-100 FT-IR spectrophotometer.

100

Thermogravimetric analysis (TGA) was acquired under a nitrogen atmosphere with a TGA Q 50

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thermo-gravimetric analyzer. Morphology of catalyst was observed with a scanning electron

102

microscope (SEM) instrument (Philips, XL30). Transmission electron microscopy (TEM)

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images were taken with a TOPCON-002B electron microscope. The magnetic property of

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catalyst was measured by a vibrating sample magnetometer (VSM, Meghnatis Daghigh Kavir

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Co., Kashan, Iran). The activity was measured using LAMBDA 25 UV/Vis Spectrophotometers

106

(PerkinElmer, USA) by measuring the amount of released glucose equivalents during the

107

hydrolysis of CMC solution based on analysis using the DNS assay.

Merck

(FeCl2.4H2O),

without

ammonia

further

(25%),

purification.

trimethylammonium

tetraethyl

Glycidyl chloride

orthosilicate

methacrylate (MAPTAC,

(TEOS),

(GMA),

75%)

and

3-

32,2’-

108 109

2.2. Synthesis of magnetic supports. The Fe3O4 magnetic nanoparticles were prepared based on

110

co-precipitation method according to our previously reported method


60

. Silica coated magnetic


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nanoparticles were prepared by hydrolysis of TEOS in alkaline solution. Fe3O4 nanoparticles (2.0

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g) were ultrasonically dispersed in 300 mL of ethanol/water (4:1) mixture and pH of solution

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was adjusted to 10 using ammonia solution. Then, TEOS (15 mL) was added to solution and the

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mixture was stirred using a mechanical stirrer under nitrogen atmosphere at 50 °C. The mixture

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was stirred for 7 h, and then the silica coated nanoparticles (MNPs) were magnetically separated

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and washed with deionized water (3×100 mL) and ethanol (2×50 mL). The final dark brown

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product was dried at 50 °C under vacuum for 24 h.

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Vinyl coating of magnetic nanoparticle was done with MPS. MNP (1.0 g) was ultrasonically

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dispersed in 50 mL ethanol and then 2 mL of ammonia solution was added to the flask. An

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excess amount (10 mmol) of MPS was then dropwise added and the mixture was stirred at 60 ˚C

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for 48 h. The MPS coated magnetic nanoparticles (MNP@MPS) were magnetically separated

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and washed several times with methanol (3×50 mL) and dried under vacuum at 50 ˚C.

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The magnetic supports were prepared according to distillation-precipitation-polymerization

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method. In a 500 mL single-necked flask, 0.50 g MNP@MPS was ultrasonically dispersed in

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200

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(methacryloylamino)propyl trimethylammonium chloride (MAPTAC), 0.5 g MBA and 100 mg

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AIBN were added to flask. The flask was completely deoxygenated by bubbling purified argon

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for 30 min and then equipped with fractionating column, Liebig condenser, and a receiver. The

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flask was stirred and polymerization initiated by increasing the temperature from ambient

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temperature to the boiling state. The process was stopped after about 130 mL of methanol was

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distilled from the reaction mixture within 5 h. The obtained magnetic ionic liquid support were

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magnetically separated and washed two times with water (2×50 mL) and three times with

mL

dry

methanol.

Then,

1.0

g

glycidyl

methacrylate


(GMA),

1.0

g

3-

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methanol (3×50 mL) to obtain the magnetic coated poly(ionic liquid-co-GMA) (Noted as

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MNP@P(IL/GMA).

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For investigation of effect of ionic surface two other supports were prepared as follow;

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In a same procedure as mentioned above one support was prepared in which polymer around the

137

MNPs were composed of 2.0 g GMA (without use of MAPTAC monomer). This support was

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noted as MNP@P(GMA).

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Another support was prepared based on same procedure and the polymer around the MNPs were

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composed of 2.0 g MAPTAC (without use of GMA monomer). This support was noted as

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MNP@P(IL).

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2.3. Cellulase immobilization. Cellulase was immobilized onto the magnetic supports based on

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following procedure; 500 mg of each magnetic support (MNP@P(GMA), MNP@P(IL/GMA)

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and MNP@P(IL)) was added to 20 mL of phosphate buffer (pH=7, 10 mM) and the mixture was

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ultrasonically dispersed for 5 min. Then 10 mL cellulase solution with different concentrations

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(1-30 mg/mL) was added to the mixture and the mixture was shacked for 7 h at 25 ˚C. After

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completion of immobilization, sample loaded enzyme was magnetically recovered and washed

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with water (2×10 mL) and the supernatants were collected for protein analysis. The epoxy

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containing samples were washed with NaCl solution (0.5 M) and incubated at 25 ˚C for 30 min,

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to remove physically adsorbed enzyme from the support. Each loading experiment was

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triplicated. The enzyme content was determined by the Bradford method using bovine serum

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albumin as a standard protein

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immobilization (Yield %) were calculated using the following equation:

61

. The enzyme loading capacity (ELC) and yield of

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ELC =

Yield (%) =

− ∑

− !"!# × 100

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Where MIE is the initial enzyme mass, MRE is the residual enzyme mass in the supernatant

157

solutions and Msup is the support mass.

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2.4. Measurement of cellulase activity. The activity of free and immobilized cellulase was

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assayed according to the standard procedure of IUPAC. 0.5 mL of cellulase loaded sample with

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specific concentration was added to a solution of CMC (1% in 10 mM buffer). The reaction was

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carried out at defined temperature for 1 h and the amount of produced glucose was examined as

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enzyme activity using DNS reagent and measurement of absorbance at 540 nm. The international

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unit of cellulase activity is defined as the amount of cellulase that hydrolyzes CMC and produces

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1 µmol glucose per minute. The relative activity was calculated based on highest activity of free

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cellulase. For pH (pH=3-7 using 10 mM buffer) and temperature (30-70 ˚C) optimization, the

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reaction for each sample was performed with equal amount of loaded cellulase. All activity

168

experiments were triplicated to obtain the average for more accurate results.

169 170

2.5. Calculation of kinetic parameters. The Michalis-Menten kinetic constants Km and Vmax for

171

cellulase were calculated from Lineweaver-Burk plot. Glucose production after 30 min was

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determined for reactions using 1.0, 2.0, 3.0, 5.0, and 10.0 g/L of CMC for free and immobilized

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cellulase samples.

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2.6. Reusability and leaching test. For reusability assay, appropriate amount of each cellulase

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loaded sample containing 10 mg of cellulase was weighted and added to 50 mL solution of 1%

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CMC at acetate buffer (pH=5, using 10 mM buffer). The reaction was carried out at 60 ˚C for 12

178

h and after that, the cellulase loaded sample was magnetically separated and added to a fresh

179

CMC solution. The experiment was repeated for 6 times under same condition and each

180

experiment was triplicated to obtain the more accurate results.

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The activities of free and immobilized cellulase on samples after storage in acetate buffer (50

182

mM, pH 5.0) at 4 °C were investigated for 25 days.

183

To examine the heterogeneity of bonded cellulase same experiment was operated. After 5 h,

184

cellulase loaded sample was separated from the solution and rest of solution allowed to continue

185

the reaction for another 7 h without the cellulase loaded sample. The, glucose production after

186

this time was measured using DNS reagent.

187 188

3. RESULTS AND DISCUSSION

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Scheme 1 shows the synthetic route for preparation of magnetic support. The first step is

190

preparation of magnetic nanoparticles with vinyl groups on the surface as a core of our magnetic

191

support. Silica shell around the Fe3O4 increases the stability of magnetic core against heat and

192

acidic medium. Functionalization of MNPs with vinyl groups facilitates the polymerization

193

around the MNPs as well as covalent attachment of polymer chains onto the MNPs. Epoxy

194

magnetic support was prepared through the distillation-precipitation-polymerization method

195

without using any surfactant with MNP@MPS as the core, GMA and MAPTAC as monomers,

196

MBA as cross-linker, and AIBN as initiator in methanol. After the initiation of the

197

polymerization, cross-linked polymer chains (which are insoluble) continuously precipitates



198

from the solution and grabs the MNPs to form a core-shell structure. The resulting polymer

199

shells around the MNPs are in multi-layer form and contain numerous epoxy and ionic groups.

200

The presence of ionic groups in the polymer chains can increase the enzyme adsorption from the

201

solution and after that the amine groups of enzyme react with epoxy groups to form a strong

202

covalent bond. To investigate the effect of ionic groups on the surface of support in

203

immobilization of enzyme, two other samples were prepared, one with GMA monomer alone

204

(noted as MNP@P(GMA)) and another one with MAPTAC alone (noted as MNP@P(IL)). The

205

immobilization of cellulase onto MNP@P(GMA) and MNP@P(IL/GMA) includes two steps:

206

first, physical adsorption of cellulase and second covalent attachment of adsorbed enzyme. Since

207

cellulase has hydrophilic nature, its adsorption onto the surface of MNP@P(GMA) is slower

208

than MNP@P(IL/GMA) which is due to the presence of ionic groups onto the surface of

209

MNP@P(IL/GMA). The immobilization of cellulase onto the MNP@P(IL) only occurs by

210

physical adsorption.

211 212

Scheme 1

213 214

Table 1 shows the composition of each sample. The amount of ionic monomer MAPTAC was

215

calculated by titration of chloride ion onto the support surface using standard Mohr’s method.

216

The available epoxide groups on the surface of supports were also determined by pyridine-HCl

217

method 27. The total amounts of MNPs core are calculated based on TGA curves of each sample.

218

The results showed that the molar ratio of GMA:MAPTAC in MNP@P(IL/GMA) is 1.82.

219 220

Table 1


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221 222

Figure 1 shows the FT-IR spectra of bare Fe3O4, Fe3O4@SiO2, MNP@MPS,

223

MNP@P(GMA), MNP@P(IL/GMA) and MNP@P(IL). The FT-IR spectrum of bare Fe3O4

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shows the stretching vibration of Fe-O band at 586 cm-1 and this band is observed at spectrum of

225

all samples confirming the presence of magnetic core in all samples. The presence of silica shell

226

around the Fe3O4 is confirmed by the appearance of strong stretching vibration of Si-O at 1085

227

cm-1. The successful modification of MPS is proved by observed peaks at 2929, 1707, and 1404

228

cm-1 in the FT-IR spectrum of MNP@MPS which are attributed to C-H, esteric C=O, and C=C

229

bonds, respectively. The FT-IR spectrum of MNP@P(GMA) shows a strong vibration bond at

230

1725 cm-1 associated to esteric C=O of poly(GMA). In this spectrum, the observed peaks at 1631

231

and 1524 cm-1 are attributed to amidic C=O and N-H of MBA, respectively. The peak of epoxy

232

groups are completely covered by strong peak of F-O. The same peaks are observed in the FT-IR

233

spectrum of MNP@P(IL/GMA) except the peak of amide groups became stronger. The spectrum

234

of MNP@P(IL) also shows the characteristic peaks of polymer shell. The FT-IR spectrum of

235

cellulase immobilized supports (not presented) did not showed the characteristic peaks of

236

cellulase due to the strong peaks of supports covered weak peaks of enzyme (considering that the

237

amount of cellulase is much lower than support). All of these results confirm the successful

238

synthesis of magnetic supports.

239 240

Figure 1

241 242

The thermal stability and organic contents of the supports were studied by TGA and the results

243

are shown in Figure 2a. The observed weight loss below 200 ˚C for all samples could be



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244

attributed to the evaporation of adsorbed water molecules. The TG analysis of MNP showed that

245

it was thermally stable, and there was no significant weight loss over the entire testing

246

temperature range. The MNP@MPS showed an obvious weight loss around 250 ˚C which was

247

attributed to decomposition of attached MPS on the surface of MNPs. From this weight loss the

248

loading amount of MPS was calculated 0.59 mmol g-1. The TGA curve of MNP@P(GMA)

249

showed main weight loss at 330 ˚C and the polymer content of this sample was about 43 wt%.

250

The thermal decomposition of MNP@P(IL) occurred at lower temperature and 44 wt% of

251

sample degraded at 290-450 °C. The TGA curve of MNP@P(IL/GMA) showed the thermal

252

behavior of both polymers P(IL) and P(GMA) and the degradation starts at 300 °C and weight

253

losing completed at 460 °C. The total organic content of this sample was around 49 wt%.

254

Figure 2b shows the TGA curves of cellulase immobilized magnetic supports. The weight

255

percentages of immobilized cellulase in supports were estimated from the TGA curves of

256

samples

257

MNP@P(IL/GMA) were 11.50, 3.10 and 10.20 %, respectively. From this results the amounts of

258

cellulase in MNP@P(GMA), MNP@P(IL) and MNP@P(IL/GMA) were calculated 115, 31 and

259

102 mg/g, respectively (These samples were loaded by 30 mg/mL of cellulose).

and

amounts

immobilized

cellulase in

MNP@P(GMA),

MNP@P(IL)

and

260 261

Figure 2

262 263

Figure 3 shows the XRD patterns of synthesized Fe3O4, MNP@MPS, MNP@P(GMA) and

264

MNP@P(IL/GMA). The characteristic peaks of crystalline Fe3O4 at 2θ = 30.1°, 35.4°, 43.1°,

265

53.2°, 56.9°, and 62.5° correspond to the (220), (311), (400), (422), (511), and (440),

266

respectively. The same peaks and intensities can be seen in the XRD patterns of other samples


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267

which revealed that the crystalline structure of Fe3O4 did not intact during the preparation of

268

magnetic supports.

269 270

Figure 3

271 272

The magnetic properties of samples were measured by VSM analysis at room temperature

273

(Figure 4). Naked Fe3O4 exhibited high saturation magnetization (63.2 emu/g), while coating of

274

magnetic core with silica shell reduced this value to 51.2 emu/g. The successful attachment of

275

MPS to MNPs can be confirmed from the reduction in saturation magnetization of MNP@MPS.

276

After entrapment of MNPs core in polymer chains, the saturation magnetization of

277

MNP@P(GMA), MNP@P(IL) and MNP@P(IL/GMA) dramatically reduced to 33.2, 30.6 and

278

27.9 emu/g, respectively. This reduction of magnetization is due to the presence of diamagnetic

279

materials around the magnetic Fe3O4 core. Even with the reduction of magnetization, all samples

280

can be rapidly collected using an external usual magnet.

281 282

Figure 4

283 284

The morphology of MNP@P(IL/GMA)@Cellulase was investigated by SEM and TEM

285

instruments. Figure 5a shows the TEM image of pure Fe3O4, which consist of cubic Fe3O4

286

nanoparticle with average size of 5 nm. In the TEM image of MNP@P(IL/GMA)@Cellulase, it

287

can be clearly observed that the MNPs are entrapped into the polymeric gray shell. The SEM

288

image of MNP@P(IL/GMA)@Cellulase (Figure 5c) demonstrates that the magnetic support

289

possess a rough surface due to the presence of MNPs in their structure. This special porous



290

surface of the support increases the enzyme loading capacity. The DLS analysis of Fe3O4,

291

MNP@P(IL/GMA) and MNP@P(IL/GMA)@Cellulase are shown in Figure 5d. The DLS

292

analysis showed that after coating of Fe3O4 with polymer shell the hydrodynamic diameter of

293

particles extremely increased. Moreover, cellulase immobilized support had more diameter than

294

unbonded one which confirmed the immobilization of enzyme onto the support surface. As it can

295

be seen from the DLS analysis, the size of MNP@P(IL/GMA) measured by DLS analysis (819

296

nm) is higher than the particle size obtained by TEM instrument (90 nm). This can be attributed

297

to this fact that the DLS analysis measures the hydrodynamic size of particles in the solution

298

phase in which particles are surrounded by water molecule62-63.

299 300

Figure 5

301

The maximum enzyme loading capacity on MNP@P(GMA), MNP@P(IL/GMA) and

302

MNP@P(IL) was investigated. As shown in Figure 6, MNP@P(GMA) and MNP@P(IL/GMA)

303

have higher ELC and adsorb more cellulase than MNP@P(IL). The maximum amount of loaded

304

cellulase on MNP@P(GMA), MNP@P(IL/GMA) and MNP@P(IL) was 128.9, 106.1 and 28.4

305

mg/g, respectively, which these values are in good agreement with the results of TG analysis.

306

However, by using 30 mg of cellulase for immobilization process, large amounts of enzyme

307

remained in the solution (based on the yield of immobilization). This result showed the effect of

308

epoxy groups on the surface of supports. Supports with epoxy groups covalently attach to the

309

enzyme and higher amount of cellulase can be immobilized onto the supports. Higher loading

310

amount of MNP@P(GMA) than MNP@P(IL/GMA) is attributed to this fact that

311

MNP@P(GMA) has more epoxy groups on the surface than MNP@P(IL/GMA). Comparing to


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312

other reported supports27,

35, 64-66

313

magnetic support applicable for immobilization of large amounts of enzymes.

this value of immobilized enzyme is high which make this

314

Figure 6

315 316 317

The activity of immobilized cellulase was then investigated in various pHs and temperatures.

318

Figure 7a shows the effect of pH on activity of free cellulase and immobilized cellulase on three

319

supports. The activity of free and immobilized cellulase was tested in pH range of 3-7 at 50 °C.

320

The maximum activity for free and immobilized cellulase was achieved at pH=5. However, the

321

activity of immobilized cellulase at pH=5 is less than free enzyme, but at higher pH values the

322

immobilized

323

MNP@P(IL/GMA)@Cellulase had more activity than two other cellulase immobilized supports

324

which can be attributed to the presence of ionic structure of MNP@P(IL/GMA). Another reason

325

for lower activity of MNP@P(GMA)@Cellulase than other samples, could be due to multi-point

326

covalent attachment of enzyme to MNP@P(GMA) which limited the transition of enzyme

327

conformation and reduced the enzyme activity67-68.

328

The effect of temperature on the activity of free and immobilized cellulase was then investigated

329

at different temperatures (30-70 °C) at pH=5 (Figure 7b). The result revealed that the optimum

330

temperature for free enzyme activity is 50 °C and in higher temperature the activity of free

331

enzyme dramatically decreased. Comparing to the free cellulase, the immobilized enzymes

332

showed higher activities in a wide range of temperature which proved that the cellulase

333

immobilization improved the heat resistance of cellulase. The highest activity was achieved by

334

immobilization of cellulase onto the MNP@P(IL/GMA) at 60 °C, which was even higher than

cellulase

showed

better

activity

than


free

enzyme.

Interestingly,


335

the activity of free enzyme at 50 °C. All of these results confirm that the cellulase

336

immobilization onto the MNP@P(IL/GMA) improves the enzyme activity in a wide range of pH

337

and temperature.

338 339

Figure 7

340 341

Another important aspect in practical applications of immobilized enzymes is thermal stability of

342

immobilized enzyme. The thermal stability of immobilized cellulase was compared with free

343

enzyme at pH=5 and temperature 60 °C for 12 h. The results showed that the free cellulase

344

completely losses its activity after 12 h, while the immobilized cellulase was still active. The

345

results also demonstrated that the covalently bonded cellulase (MNP@P(GMA)@cellulase and

346

MNP@P(IL/GMA)@Cellulase) had greater thermal resistance than physically immobilized

347

enzyme (MNP@P(IL)@Cellulase). Both covalently bonded cellulase had kept more than 50% of

348

its initial activities after 12 h. These higher thermal stabilities in covalent immobilized cellulase

349

arise from multi-point fixation of cellulase which limits the flexibility of enzyme and makes it

350

more stable against the change of temperature.

351 352

Figure 8

353 354

The maximum reaction rate V max and Michaelis constant K m were estimated from a

355

Lineweaver – Burk plot, using carboxymethyl cellulose as substrate. As shown in Table 2, the

356

Km and Vmax values of the immobilized cellulase onto three samples were all lower than those

357

of the free enzyme. This behavior was previously observed19, 69-73. The lower values of Km for


Page 16 of 39

Page 17 of 39


358

immobilized samples demonstrate that the immobilized cellulose exhibits greater substrate

359

affinity. The higher substrate affinity for immobilized cellulose can be attributed to increasing in

360

adsorption of CMC to the surface of magnetic supports. The result of Km values show that the

361

ionic surface of support lead to an enhancement in CMC affinity.

362

As it can be see, the Km for MNP@P(IL/GMA) is lower than MNP@P(IL) while MNP@P(IL)

363

have more ionic groups on the surface of support. An explanation for this observation can be

364

attributed to the presence of hydroxyl groups on the surface of MNP@P(IL/GMA) while there is

365

no such group on the surface of MNP@P(IL). After attachment of enzyme to the epoxy groups

366

on the surface of MNP@P(IL/GMA) a hydroxyl groups is produced. Based on the reaction

367

condition, other unreacted epoxy rings on the surface of support can also be opened by reaction

368

with water molecules during the washing process. So, the surface of MNP@P(IL/GMA) is

369

composed of hydroxyl and ionic groups. These hydroxyl groups can interact with substrates

370

through the hydrogen bonding and increase the affinity to substrate alongside with ionic groups.

371

On the other hand, decreasing in the Vmax values for immobilized samples may be caused by

372

limitation of enzyme flexibility which restrict the conformational changes of enzyme54, 58, 74. As

373

it can be seen, the pure epoxy support (MNP@P(GMA)@Cellulase) showed lower Vmax which

374

may be attributed to multi-point attachment of cellulase.

375

Table 2

376 377

Another advantage of enzyme immobilization is improvement of enzyme stability during the

378

storage. Generally, enzymes are not stable in solution and they loss their activities during storage

379

time. The effect of cellulase immobilization on storage stability was investigated at 4 °C during

380

25 days and the results are presented at Figure 9. As shown, the free cellulase lost almost all of



Page 18 of 39

381

its activity within 25 days, whereas the immobilized enzyme on MNP@P(GMA),

382

MNP@P(IL/GMA) and MNP@(IL) lost about 23, 19 and 35% of their initial activities during

383

the same storage period, respectively. The results demonstrate that upon immobilization,

384

cellulose gained more stability with respect to free cellulase.

385

Figure 9

386 387 388

The recyclability of immobilized enzyme is a critical parameter for industrial applications. The

389

recyclability of

390

MNP@P(IL/GMA) was investigated in hydrolysis of 100 mL of 1% CMC for 12 h at optimum

391

condition. The reaction was carried out and the immobilized cellulase was magnetically

392

separated and used in another run. The activity assay was calculated after each run, and the

393

change in residual activity was observed. This experiment was repeated for at least six times and

394

the results are presented in Figure 9a. The results showed that the cellulase immobilized on

395

epoxy supports (MNP@P(GMA) and MNP@P(IL/GMA)) kept more than 60 % of their

396

activities after six times of recycling while MNP@P(IL)@Cellulase lost more than 90% of its

397

activity during the six times recycling. This activity loss can be attributed to several parameters,

398

including loss of immobilized cellulase, end-product inhibition, and enzyme denaturation.

399

Probably, the activity loss for MNP@P(IL)@Cellulase caused mostly by cellulase detachment

400

which was physically adsorbed onto the support surface. To demonstrate the cellulase

401

detachment on samples, cellulase immobilized supports were separated from the reaction mixture

402

after 5 h and the rest of the solution stirred for another 7 h. The results showed that after

403

separation of MNP@P(GMA)@Cellulase and MNP@P(IL/GMA)@Cellulase (dashed lines) no

immobilized

cellulase onto

the MNP@P(GMA),


MNP@P(IL)

and

Page 19 of 39


404

significant amounts of glucose was produced. On the other hand, separation of

405

MNP@P(IL)@Cellulase from the reaction mixture did not stopped the glucose production

406

(dashed lines) which means some of cellulase molecules were detached from the surface of

407

MNP@P(IL)@Cellulase during the reaction run. These results showed that cellulase leaching

408

from MNP@P(IL)@Cellulase is higher than covalently immobilized samples.

409

Figure 10

410 411 412

In conclusion, a magnetic ionic support was prepared for covalent immobilization of cellulase.

413

The surface of magnetic support was composed of numerous epoxy and ionic groups. The ionic

414

groups improved the enzyme adsorption and then the adsorbed enzyme was covalently attached

415

to the surface by reaction with epoxy group. To investigate the effect of covalent bonding and

416

ionic surface, two other samples were prepared; one with epoxy groups and another with ionic

417

groups on the surface. The results showed that the presence of both epoxy and ionic groups on

418

the surface had a huge effect on activity, stability and reusability of immobilized cellulase. Since

419

magnetic nanoparticles were covered by epoxy polymer, a large amount of cellulase (106.1

420

mg/g) was immobilized onto the surface of support. The covalently immobilized cellulase

421

retained 60% of its initial activity after 6 cycles. All results proved that the epoxy/ionic support

422

was a good choice for covalent immobilization of cellulase and has good potential for large scale

423

production of glucose.

424 425

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426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469

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638 639 640 641 642 643 644 645


Page 24 of 39

Page 25 of 39


646 647 648 649 650 651 652 653

Figure Captions:

654

Scheme 1. Synthetic routs for preparation of cellulase immobilized magnetic supports.

655

Figure 1. FT-IR spectra of bare Fe3O4, Fe3O4@SiO2, MNP@MPS, MNP@P(GMA),

656

MNP@P(IL/GMA) and MNP@P(IL).

657

Figure 2. TGA curves of MNP, MNP@MPS, MNP@P(GMA), MNP@P(IL/GMA) and

658

MNP@P(IL) (a) and cellulase immobilized MNP@P(GMA), MNP@P(IL/GMA) and

659

MNP@P(IL) (b).

660

Figure 3. XRD patterns of Fe3O4, MNP@MPS, MNP@P(GMA) and MNP@P(IL/GMA).

661

Figure 4. The VSM analysis of bare Fe3O4, Fe3O4@SiO2, MNP@MPS, MNP@P(GMA),

662

MNP@P(IL/GMA) and MNP@P(IL) at room temperature.

663

Figure

664

MNP@P(IL/GMA)@Cellulase, DLS analysis of samples (d).

665

Figure 6. The cellulase loading capacity and efficiency for MNP@P(GMA), MNP@P(IL/GMA)

666

and MNP@P(IL).

667

Figure 7. Effect of pH (a) and temperature (b) on activity of the free and immobilized cellulase.

668

Figure 8. The thermal stability of free and immobilized cellulase.

669

Figure 9. Storage stability of the free and immobilized cellulase at 4 °C.

5.

TEM

image

of

Fe3O4

(a),

TEM

(b)


and

SEM

(c)

images

of


670

Page 26 of 39

Figure 10. Recyclibity (a) and leaching test (b) of cellulase immobilized supports.

671 672 673 674 675 676 677

Table 1. Composition of support samples Entry

Sample

MNPs (wt%)a 57 51 56

1 MNP@P(GMA) 2 MNP@P(IL/GMA) 3 MNP@P(IL) a Calculated based on TGA. b Calculated by titration with AgNO3. c Calculated by pyridine-HCl test.

Composition MAPTAC (wt%)b 18.3 35.0

678 679 680 681 682 683 684 685 686 687 26 ACS Paragon Plus Environment

GMA (wt%)c 36.4 22.8 -

Page 27 of 39


688 689 690 691 692 693 694 695

Table 2. Kinetic parameters of free and immobilized cellulase Enzyme

Km (g.L-1) 11.59

Vmax (g.L-1.s-1) ×103 17.36

Vmax/ Km (s-1) ×103 1.49

MNP@P(GMA)@Cellulase

8.93

5.48

0.61

MNP@P(IL/GMA)@Cellulase

3.49

9.67

2.77

MNP@P(IL)@Cellulase

5.13

11.6

2.26

Free Cellulase

696 697 698 699 700 701 702 703 704 705



706 707 708 709 710 711

712 713

Scheme 1.

714


Page 28 of 39

Page 29 of 39


715 716

Figure 1.

717 718



719 720

Figure 2.

721 722 723 724 725 726 727 728


Page 30 of 39

Page 31 of 39


729 730

Figure 3.

731 732 733 734 735 736



737 738

Figure 4.

739 740 741 742 743


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744 745

Figure 5.

746 747 748 749 750



751 752

Figure 6.

753 754 755 756 757 758 759 760 761 762 763


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764 765

Figure 7.

766 767 768 769 770 771 772 773 774 775 776



777 778

Figure 8.

779 780 781 782 783


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784 785

Figure 9.

786 787 788 789 790 791 792 793 794



795 796

Figure 10.

797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 38 ACS Paragon Plus Environment

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812


Graphic for Table of Content

813


Covalent Immobilization of Cellulase Using Magnetic Poly(ionic liquid

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