Accepted Manuscript

Advances on methods and easy separated support materials for enzymes

immobilization

Dong-Mei Liu, Juan Chen, Yan-Ping Shi

PII: S0165-9936(18)30036-0
DOI: 10.1016/j.trac.2018.03.011
Reference: TRAC 15120

To appear in: Trends in Analytical Chemistry

Received Date: 1 February 2018

Revised Date: 13 March 2018
Accepted Date: 13 March 2018

Please cite this article as: D.-M. Liu, J. Chen, Y.-P. Shi, Advances on methods and easy separated
support materials for enzymes immobilization, Trends in Analytical Chemistry (2018), doi: 10.1016/
j.trac.2018.03.011.

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 ACCEPTED MANUSCRIPT

1 Advances on methods and easy separated support materials for enzymes

2 immobilization

4 Dong-Mei Liu1, 2, Juan Chen1∗, Yan-Ping Shi1∗

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5

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6 CAS Key Laboratory of Chemistry of Northwestern Plant Resources and Key

7 Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical

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8 Physics, Chinese Academy of Sciences, Lanzhou 730000, People’s Republic of China

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2
9 University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing
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10 100049, P. R. China

11
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12 Abstract
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13 Compared with free enzymes, immobilized enzymes are more robust and resistant to
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14 environmental changes. In addition, with enhanced stability, immobilized enzymes can

15 be separated from the reaction mixture and used for repeated cycles. These advantages
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16 prompt their applications in various fields. This review outlines the existing methods
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17 and easy separated support materials for enzymes immobilization. After a brief
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18 introduction on the immobilized enzyme, the immobilization methods of adsorption,

19 entrapment, covalent attachment and cross-linking are discussed. The emphasis is given

20 on the easy separated support materials of magnetic nanoparticles (MNPs), membranes

21 and capillary columns. An outlook on the immobilized enzyme is given at last.

∗
Correspondence: Juan Chen, and Yan-Ping Shi; Tel.: 86-931-4968208; fax: 86-931-4968094; E-mail:
chenjuan@licp.cas.cn (J. Chen), shiyp@licp.cas.cn (Y. -P. Shi).
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22

23 Keywords: Enzyme immobilization; Immobilization methods; Easy separated support

24 materials; MNPs; Membranes; Capillary columns

25

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26 Abbreviations

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27 Magnetic nanoparticles (MNPs)

28 Nanoparticles (NPs)

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29 Metal-organic frameworks (MOFs)

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30 Polyvinylpyrrolidone (PVP)
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31 l-Ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC)

32 N-hydroxysuccinimide (NHS)
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33 Cross-linked enzyme aggregates (CLEAs)

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34 Horseradish peroxidase (HRP)

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35 3-Aminopropyltriethoxysilane (APTES)

36 Polyethyleneimine (PEI)
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37 Cetyltrimethyl ammonium bromide (CTAB)

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38 1,2-Bis(trimethoxysilyl)ethane (BTME)
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39 Magnetically-separable mesoporous silica (Mag-MSU-F)

40 3-Chloropropyltriethoxysilan (CPTES)

41 Dimethyloctadecyl [3-(trimethoxysilyl)propyl] ammonium chloride (DMOAP)

42 N-(2-aminoethyl)-3-aminopropyltrimethoxy-silane (EDS)

43 Polypropylene (PP)
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44 Hollow fiber membrane (HFM)

45 Poly (vinylidene fluoride) (PVDF)

46 Open tubular column immobilized enzyme microreactor (OTC-IMER)

47 Monolithic column immobilized enzyme microreactor (MC-IMER)

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48 Gold nanoparticles (GNPs)

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49

50 1. Introduction

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51 Enzymes, widely exist in plants, animals and microorganisms, are proteins that catalyze

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52 the biochemical and chemical reactions. They are catalysts with the advantages of high
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53 catalytic activity, selectivity, specificity and biodegradability. In addition, they can

54 operate in mild pH, temperature and pressure [1, 2]. However, enzymes cannot be
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55 recovered and reused after the first run, which will be no longer economic [3]. This
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56 drawback can be overcome by the immobilization techniques. Immobilized enzyme was

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57 first reported in 1916. In this work, the authors observed that the physically adsorbed

58 invertase on charcoal retained its catalytic activity perfectly [4]. The immobilization
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59 allows the easy recovery of enzyme, rapid termination of enzyme assay and repeated
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60 enzyme assay, which will reduce the assay cost. In addition, after immobilization, the
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61 storage stability, pH and thermal resistances are usually improved. Based on these

62 advantages, immobilized enzymes have been widely applied in various fields like

63 pharmaceutical industry, food industry, wastewater treatment and textile industry and so

64 on.

65 Hitherto, immobilization techniques have been developed maturely. And physical

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66 adsorption [5], covalent attachment [6], crosslinking [7] and entrapment [8] are the

67 commonly used ones. Besides immobilization techniques, support materials are also of

68 vital importance for enzymes immobilization. Inert polymers and inorganic materials

69 are usually used as carrier matrices for enzymes immobilization [3]. However, it is

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70 tedious and laborious to separate and recover immobilized enzymes after the

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71 immobilization and enzymatic reaction processes. Therefore, separation of immobilized

72 enzymes is a key problem in real application. The easy separated support materials of

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73 magnetic nanoparticles (MNPs), membranes and capillary columns can be separated

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74 without centrifugation, thereby simplifying the separation and recovery processes.
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75 This review focusses on the existing methods and easy separated support materials

76 for enzymes immobilization. The physical methods of adsorption and entrapment, and
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77 chemical methods of covalent attachment and cross-linking are evaluated. And

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78 compared with previous reviews [3, 9], for the first time, the easy separated support
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79 materials are discussed to solve the separation problems of immobilized enzymes. In

80 addition, future perspectives of immobilized enzymes are presented as well.

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81
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82 2. Immobilization methods
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83 According to the interaction modes between enzymes and support carriers,

84 enzyme-immobilization methods can be classified into physical methods and chemical

85 methods. Adsorption and entrapment could be assigned to physical methods, in which

86 there are no covalent interactions between enzymes and support carriers, while covalent

87 attachment and cross-linking belong to chemical methods. In some cases, multiple

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88 enzyme-support interactions are exploited for enzymes immobilization. The

89 enzyme-immobilization methods stated above are shown as Fig. 1.

90 2.1 Physical methods

91 2.1.1 Adsorption

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92 Adsorption is a simple and convenient method for enzymes immobilization. It

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93 contains physical adsorption, ion adsorption and affinity adsorption, among which,

94 physical adsorption is the usually used one. In this immobilization mode, the commonly

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95 used supports are cation and anion exchange resins, activated charcoal, silica gel,

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96 alumina, controlled pore glass and ceramics. By H-bondings, van der Waals forces,
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97 electrostatic interactions or hydrophobic interactions, enzymes are adsorbed on the

98 surfaces of support carriers [5, 10, 11] or immobilized in the pores of mesoporous
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99 materials [12, 13]. For examples, lipases were immobilized on the resins of
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100 Phenyl-Sepharose CL-4B and Octyl-Sepharose CL-4B by hydrophobic adsorption [11],

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101 and laccases were adsorbed on the surfaces and into the pores of micro-mesoporous

102 Zr-metal organic frameworks (Zr-MOFs) [12]. The carboxylated Zr-MOFs were
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103 prepared using surfactant-templating method. At pH 3.0, positively charged laccases

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104 were adsorbed on the negatively charged Zr-MOFs. With the immobilization time of 1h,
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105 immobilized laccases showed maximum catalytic activities owing to the fact that

106 laccases were adsorbed both on the surfaces and into the pores of Zr-MOFs.

107 The adsorption of enzymes on support matrices is easy to be implemented with low

108 cost just by mixing them for a certain incubation time. In this method, no additional

109 coupling agents and modification steps are required for enzymes immobilization and the
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110 support matrices can be regenerated. Moreover, the immobilization conditions are

111 mildly disruptive to enzymes thus preserving their initial catalytic activities. However,

112 in physical adsorption, the interactions between enzymes and immobilization support

113 carriers are weak and reversible. The binding forces are susceptible to changes in pH,

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114 temperature and ionic strength, resulting in the poor stability of immobilized enzymes.

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115 In addition, the leached enzymes will contaminate the substrate solution.

116 2.1.2 Entrapment

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117 Enzymes tend to aggregate thus decreasing the catalytic activities. Entrapment is

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118 an immobilization method in which enzymes are occluded in polymeric networks with
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119 low cost. This might be a practical strategy for avoiding the aggregation of enzymes.

120 Various matrices can be used for entrapment, such as chitosan, calcium alginate,
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121 collagen, cellulose triacetate, poly acrylamide, gelatin, agar, silicon rubber, polyvinyl
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122 alcohol and polyurethane. Bilal and coworkers have reported some works for dyes
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123 degradation by entrapping enzymes within chitosan beads [14], calcium alginate beads

124 [15], or polyvinyl alcohol-alginate beads [8]. In this method, enzymes are immobilized
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125 by gel/fiber entrapping or micro-encapsulation. Enzymes are retained in the networks

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126 while substrates and products are allowed to pass through, which will decrease the
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127 leaching of enzymes, improve the stability, and allow the generation of enzymatic

128 reactions. Since there are no covalent bonds between enzymes and support matrices,

129 enzyme conformations are maintained, ensuring the high catalytic activities. However,

130 high diffusion barriers prevent the macromolecular substrates from passing through the

131 networks, which is a major demerit of this technique.

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132 Besides occlusion in polymeric networks, MOFs have been recently developed to

133 embed enzymes. In this mode, zeolitic imidazolate framework (ZIF-8) is commonly

134 used to entrap enzyme. In addition, polyvinylpyrrolidone (PVP) is usually utilized as

135 dispersant and stabilizer to protect enzyme from inactivation by organic solvent. The

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136 PVP-protected enzyme is mixed with zinc nitrate and 2-methylimidazole to synthesize

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137 the enzyme-embedded MOF [16, 17].

138 2.2 Chemical methods

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139 2.2.1 Covalent attachment

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140 Covalent attachment is a conventional method for enzymes immobilization. In this
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141 mode, covalent bonds are formed through chemical reactions between support materials

142 and enzymes’ side chain amino acids, such as lysine, cysteine, or aspartic and glutamic
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143 acid residues [18]. In addition, the functional groups of amino, carboxylic, imidazole,
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144 indolyl and phenolic hydroxyl groups are favorable for the formation of covalent bonds
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145 [19]. Covalent bonds are stable to prevent the leakage of enzymes from the support

146 matrices thus improving the stability of the immobilized enzymes. However, enzyme
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147 active sites might be inactivated due to the chemical reactions between enzyme
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148 molecules and support matrices, which will result in the decreases in catalytic activities.
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149 In this mode, dicyclohexylcarbodiimide, diazotization, bromide cyanogen and

150 glutaraldehyde coupling methods have been maturely developed.

151 Dicyclohexylcarbodiimide method is known as l-ethyl-3-(dimethyl-aminopropyl)

152 carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) method. In the

153 literature [20], EDC and NHS were used as catalysts to facilitate the amine coupling
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154 reactions, thus promoting the covalent attachment of lysozymes to carboxylated

155 polyamide layers. The amide bonds were formed owing to the reactions between

156 carboxyl groups of polyamide layers and amino groups of lysozymes. Glutaraldehyde

157 coupling method is most commonly used to immobilize enzyme on the

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158 amino-functionalized support matrix based on Schiff base reaction. In this process, one

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159 aldehyde group of glutaraldehyde molecule reacted with the amino group of enzyme

160 molecule, and the other aldehyde group reacted with the amino group of support matrix

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161 [21-23].

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162 2.2.2 Cross-linking
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163 Based on intermolecular reactions, enzymes are cross-linked to the support

164 matrices using bifunctional reagents. In this mode, with covalent bonds, enzymes are
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165 immobilized firmly to improve the reusability and stability. However, enzymes may lose
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166 their catalytic activities during the cross-linking process. Glutaraldehyde, isocyanate
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167 and N, N’-ethylene bis maleimide are the commonly used bifunctional reagents, in

168 which glutaraldehyde is most commonly used.

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169 Moreover, enzymes can be cross-linked to each other to synthesize the cross-linked
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170 enzyme aggregates (CLEAs). For the preparation of CLEAs, precipitants must be first
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171 used for enzymes aggregation, then using bifunctional reagents, the aggregated enzymes

172 are cross-linked to each other (Fig. 2). After aggregation, the enzyme active sites are

173 protected, thus preserving the catalytic activities. In the study [24], 75% ammonium

174 sulfate was used to precipitate horseradish peroxidase (HRP), and then using

175 glutaraldehyde or ethylene glycol-bis (succinic acid N-hydroxysuccinimide) as

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176 cross-linker, the HRP-CLEAs were formed. The resultant HRP-CLEAs were used to

177 degrade pollutants and retained almost 60% of their initial activities after 7 repeated

178 cycles.

179

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180 3. Easy separated support materials

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181 The separation of immobilized enzymes from the immobilization solution or

182 enzymatic reaction solution is a key problem. With the support materials of SiO2 NPs

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183 [25], mesoporous silica NPs [26], MOFs [27], etc., the immobilized enzymes are

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184 usually separated by centrifugation. However, such a process is inconvenient and
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185 tedious owing to the small size of NPs. Promisingly, the easy separated support

186 materials like MNPs, membranes and capillary columns can avert the laborious
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187 centrifugation process. This will simplify the operation procedure. The applications of
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188 easy separated support materials in enzymes immobilization are outlined in Table 1.
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189 3.1 MNPs

190 During recent decades, MNPs have gained much attentions in enzymes
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191 immobilization due to their large specific surface areas and convenient surface
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192 modification methods. The loading capacity can be greatly improved owing to these
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193 features. Most importantly, their magnetic properties can guarantee the easy separation

194 of magnetic-immobilized enzymes from reaction mixture, and therefore terminate the

195 enzymatic reactions rapidly and recover the immobilized enzymes for repeated uses.

196 Based on the excellent advantages stated above, the tedious centrifugation process will

197 be avoided, which will greatly simplify the immobilization and recovery processes.
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198 Among the various MNPs like iron oxide-based (Fe3O4 and γ-Fe2O3), pure

199 metal-based (Fe and Co), spinel-type ferromagnet-based (MgFe2O4, MnFe2O4, and

200 CoFe2O4) and alloy-based (CoPt3 and FePt) MNPs, Fe3O4 NPs are most commonly used

201 for enzymes immobilization due to their superior advantages of no toxicity and good

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202 biocompatibility [56]. Many synthetic methods have been developed for the synthesis of

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203 Fe3O4 NPs, among which chemical coprecipitation, microemulsion, thermal

204 decomposition and solvothermal synthesis are the typical ones.

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205 Fe3O4 NPs tend to aggregate because of their high surface energy caused by the

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206 large specific surface area. Moreover, Fe3O4 NPs possess high chemical activity and are
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207 easily oxidized in air, therefore resulting in loss of magnetism and dispersibility [57].

208 These factors could make the magnetic separation less effective. Hence, the naked
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209 Fe3O4 NPs cannot be used directly for enzymes immobilization. The surface
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210 modification is favorable for preventing the aggregation and oxidation of Fe3O4 NPs.
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211 Using as support matrices for enzymes immobilization, four types of modified MNPs

212 are discussed as follows.

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213 3.1.1 Silica-coated MNPs

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214 Silica coating is a typical method for the modification of Fe3O4 NPs, in which
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215 silica shells formed on the surfaces of magnetic cores. The silica shells protect the

216 magnetic cores from aggregation and oxidization thus improving the chemical stability.

217 Moreover, the silica shells can also improve the hydrophilicity and biocompatibility.

218 After modification, substantial silanol groups are introduced on the surfaces of magnetic

219 cores, which will provide the underlying basis for further modification with functional
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220 reagents for enzymes immobilization.

221 Fe3O4 NPs can be directly coated with silica shells using the well-known sol-gel

222 process [58]. In this procedure, tetraethoxysilane is hydrolyzed under alkaline condition,

223 prompting the formation of silica shells on the surfaces of magnetic cores to obtain the

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224 core-shell Fe3O4@SiO2 NPs. For further enzymes immobilization, the core-shell

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225 Fe3O4@SiO2 NPs are usually first functionalized with aminosilane coupling agents [28]

226 or epoxysilane coupling agents [32] to introduce corresponding amino groups or epoxy

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227 groups on the surfaces of support matrices. Subsequently, for amino-functionalized

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228 Fe3O4@SiO2 NPs, with the help of glutaraldehyde, enzymes are immobilized based on
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229 Schiff base reactions. While for epoxy-functionalized ones, enzymes can be directly

230 immobilized on the support matrices through the interactions between epoxy groups and
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231 enzyme amino groups. Chang et al. prepared the 3-aminopropyltriethoxysilane

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232 (APTES)-modified Fe3O4@SiO2 NPs to immobilize HRP [28]. Tural et al. modified
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233 Fe3O4@SiO2 NPs with 3-glycidyloxypropyltrimethoxysilane to introduce epoxy groups

234 on the surfaces of Fe3O4@SiO2 NPs to realize the covalent attachment of

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235 benzaldehydelyase [32]. Compared with amino-functionalized Fe3O4@SiO2 NPs, no

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236 additional coupling agents are needed for enzymes immobilization on

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237 epoxy-functionalized Fe3O4@SiO2 NPs. However, the immobilization procedure is

238 time-consuming due to the insensitive reactions between epoxy groups and amino

239 groups.

240 3.1.2 Organic polymer-modified MNPs

241 There are two modes for the modification of MNPs with organic polymers, the
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242 in-situ modification mode [59, 60] and ex-situ modification mode [33, 61]. In in-situ

243 modification mode, organic polymers are added into the precursor solution as stabilizers

244 to synthesize Fe3O4 NPs. While in ex-situ modification mode, the monomers are

245 polymerized to form polymer coatings on the surfaces of Fe3O4 NPs. The steric

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246 repulsion generated from polymer coatings will weaken the magnetic forces and van der

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247 Waals forces of Fe3O4 NPs, thus preventing their aggregation and improving their

248 dispersibility and stability.

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249 Owing to their excellent biocompatibility and biodegradability, amino polymers

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250 like chitosan [61], polyethyleneimine (PEI) [59] and polydopamine [33] are usually
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251 used to modify Fe3O4 NPs for enzymes immobilization. Compared with silica coatings,

252 active groups can be directly introduced to the magnetic cores by organic polymers
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253 without modification of organosilanes, which will simplify the modification procedure.
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254 Chitosan-coated MNPs are usually fabricated by the in-situ modification mode [34, 35].
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255 Wu et al. reported a procedure for the in-situ synthesis of Fe3O4-chitosan NPs which

256 were further used as support matrices for lipases immobilization [35]. In this procedure,
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257 ferrous ions were added to the mixture of chitosan and sodium tripolyphosphate. After
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258 adjusting the mixture to alkaline condition, Fe(OH)2 was precipitated and oxidized to
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259 Fe3O4 NPs which were presented in the pores of chitosan gel. The obtained

260 Fe3O4-chitosan NPs showed poor dispersibility which might be due to the cross-linking

261 between chitosan molecules or the small size of the particles. Finally, based on Schiff

262 base reactions, lipases were immobilized on Fe3O4-chitosan NPs using glutaraldehyde

263 as cross-linker. In addition, ex-situ modification methods including reversed phase

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264 suspension [36], cross-linking [22] and embedding methods [23] have also been

265 reported for the preparation of Fe3O4-chitosan NPs. In our previous work, by

266 cross-linking and embedding method, Fe3O4-chitosan NPs were respectively

267 synthesized for α-glucosidase immobilization. In the cross-linking process [22],

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268 positively charged chitosan was first bound to negatively charged Fe3O4, then by

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269 cross-linking with sodium tripolyphosphate, chitosan was modified on the surface of

270 Fe3O4. The obtained Fe3O4-chitosan NPs showed excellent dispersibility, which might

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271 be attributed to the coated chitosan enhancing the repulsion of MNPs. The satisfactory

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272 dispersibility will provide more sites for enzymes immobilization. In the embedding
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273 process [23], the mixture of Fe3O4 NPs and chitosan was added to sodium hydroxide

274 solution. The Fe3O4 NPs were embedded on the chitosan membranes that formed in
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275 concentrated alkaline solution. Then using glutaraldehyde as coupling agent,

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276 α-glucosidase was immobilized (Fig. 3). Dopamine can be self-polymerized in alkaline
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277 solution to form an adherent polydopamine film on a wide variety of organic or

278 inorganic surfaces. Martín et al. prepared the core-shell Fe3O4@polydopamine MNPs to
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279 immobilize HRP [33]. Unlike Fe3O4-chitosan NPs, no further surface activation with
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280 additional coupling agent (glutaraldehyde) was needed for enzymes immobilization.
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281 HRP can be directly immobilized on the polydopamine film through Schiff base

282 reaction.

283 3.1.3 Mesoporous material-modified MNPs

284 Mesoporous materials are porous with unique features of huge surface area, high

285 pore volume, tunable pore size, desirable functionality, favorable chemical and thermal
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286 stability, and non-toxicity [62]. These merits have been considered to be favorable terms

287 for enzymes immobilization. The commonly used mesoporous materials are SBA-15

288 [63], MCM-41 [64], MCF [65] and MSU [66]. Enzymes are usually immobilized on the

289 surfaces or in the pores of mesoporous matrices by physical adsorption, covalent

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290 attachment or cross-linking. Various organosilanes are usually used to introduce amino,

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291 sulfhydryl, cyano, epoxy groups or different chain length of alkyl groups on

292 mesoporous materials [63]. These functional groups provide substantial reactive sites

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293 for enzymes immobilization, thus improving the loading capacity. In addition, the

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294 organic groups may enter into the mesoporous channel to reduce the pore size and pore
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295 volume, which will prevent the leakage of enzymes. The reviews about mesoporous

296 materials as immobilization support carriers have been comprehensively reported [62,
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297 67].
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298 During the past few years, magnetic mesoporous materials have been widely
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299 developed to immobilize enzymes due to their unique properties. More detailed

300 applications of magnetic mesoporous materials in enzymes immobilization have been

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301 summarized in Table 2. In the literature [76], Fe3O4 was first coated with a silica shell.
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302 Then using cetyltrimethyl ammonium bromide (CTAB) as template, tetraethoxysilane

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303 was hydrolyzed to form another silica coating on the surface of Fe3O4@SiO2 by sol-gel

304 process. After removing CTAB, Fe3O4@SiO2@mSiO2 was prepared. The obtained

305 Fe3O4@SiO2@mSiO2 was respectively amino-functionalized with cationic or anionic

306 surfactant and ethane-functionalized with 1,2-bis(trimethoxysilyl)ethane (BTME). The

307 non-, amino-, and ethane-functionalized Fe3O4@SiO2@mSiO2 were respectively used

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308 to immobilize cyclodextrin glucanotransferase by physical adsorption. Modified with

309 different reagents, the shell thickness, surface area, pore volume and pore size are

310 different, which will influence the loading capacity. For instance,

311 Fe3O4@SiO2@mSiO2-NH2 prepared using anionic surfactant was superior for enzyme

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312 immobilization compared to that prepared using cationic surfactant. This might be due

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313 to the higher shell thickness and surface area of Fe3O4@SiO2@mSiO2-NH2 prepared

314 using anionic surfactant. As shown in Fig. 4, the magnetically-separable mesoporous

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315 silica (Mag-MSU-F) was prepared for the immobilization of subtilisin Carlsberg [66]. In

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316 this study, subtilisin Carlsberg was first adsorbed in the pores of Mag-MSU-F, and then
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317 cross-linked with each other to obtain the subtilisin Carlsberg-CLEAs. The CLEAs were

318 limited in the pores of Mag-MSU-F, which will prevent the leakage of enzymes.
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319 3.1.4 MOF-modified MNPs

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320 In recent years, MOFs have gained considerable attentions as support carriers for
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321 enzymes immobilization due to their superior properties of tunable porosity, uniform

322 pore size, modifiable surface, ultrahigh surface area, and excellent chemical and thermal
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323 stability. These advantages will enhance the loading capacity of enzymes, improve the
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324 stability of immobilized enzymes and reduce the mass transfer resistance. MOFs are
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325 formed by self-assembly of metal ions and organic ligands. There are usually three

326 immobilization modes for enzymes immobilization on MOFs, the surface

327 immobilization, pore encapsulation and co-precipitation. In the co-precipitation method,

328 enzyme-MOF composites are synthesized in situ by mixing enzymes with metal ions

329 and organic ligands, where enzymes are embedded within MOFs. This can afford
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330 unprecedented protection of enzyme molecules from biological, thermal and chemical

331 degradation, thus improving the stability [16]. MOFs have been thoroughly reviewed as

332 the novel support materials for enzymes immobilization [79, 80].

333 Combining the properties of MOFs and Fe3O4 NPs, MOF-modified Fe3O4 NPs

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334 have been developed [81-83], which will be splendid support matrices for enzymes

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335 immobilization. For the synthesis of Fe3O4@MOF NPs, the reaction takes place

336 between metal ions and organic ligands in the presence of carboxylated Fe3O4 NPs. The

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337 Fe3O4 NPs must be carboxylated to obtain the negatively charged surfaces, which will

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338 adsorb the positively charged metal ions and therefore ensure the growth of MOFs on
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339 the surfaces of Fe3O4 NPs. The carboxylated Fe3O4 NPs can be synthesized by in-situ or

340 ex-situ method. In the in-situ method, modifiers (polyacrylic acid, trisodium citrate
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341 dehydrate etc.) are added in the precursor solution for the synthesis of Fe3O4 NPs [82,
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342 84]. In the ex-situ method, carboxylic acid reagents are used for surface modification of
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343 Fe3O4 NPs after their synthesis [81, 83]. The synthesis process of Fe3O4@ZIF-8 is

344 shown in Fig. 5. Zheng et al. synthesized Fe3O4@ZIF-8 NPs through the reaction
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345 between zinc nitrate hexahydrate and 2-methylimidazole in the presence of Fe3O4 NPs
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346 [84]. In this procedure, Fe3O4 NPs with negative charges were prepared by the
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347 solvothermal method using trisodium citrate dehydrate as the surface modifiers. Zn2+

348 ions were bound to the carboxylic acid groups on Fe3O4 NPs to initiate the growth of

349 ZIF-8 shells on the surfaces of Fe3O4 NPs. The citric acid-treated Fe3O4 NPs were

350 mixed with Zn(NO3)2 and HCl, and then the mixture of 2-methylimidazole, PVP and

351 glucose oxidase was added to the above suspension. After stirring, the magnetic
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352 ZIF-8@glucose oxidase was prepared. MOFs can embed enzymes due to the high

353 loading capacity and strong affinity. In addition, the embedding method is facile,

354 convenient and time-saving, which will ensure the immobilized enzymes with high

355 catalytic activities [85].

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356 3.2 Membranes

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357 Enzymes are widely immobilized on membranes for the reasons that a small

358 amount of enzymes are required in the enzyme-immobilized membrane systems. In

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359 addition, the immobilized enzymes can be quickly and conveniently separated from the

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360 reaction mixture thus selectively removing products [86]. Also, the
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361 membrane-immobilized enzymes could circumvent the steric hindrance effects and

362 interfacial limitation phenomena. In the enzyme-immobilized membrane systems, the

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363 membranes aim to separate the immobilized enzymes from the reaction mixture. In this
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364 system, enzymes may be immobilized on the membrane surfaces by physical adsorption,
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365 covalent attachment or cross-linking, or inside the pores of membranes by entrapment

366 [87]. The favorable thermodynamic and kinetic process can be created through the
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367 combination of the high selectivity of enzymatic reactions with the high efficiency of
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368 membrane separation. The enzyme-immobilized membrane systems are summarized in

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369 Table 3.

370 The membrane-immobilized enzymes are usually fixed in devices to obtain the

371 bioreactors for enzyme assays, which will avoid the tedious and laborious centrifugation

372 procedure. Membranes can be classified into inorganic and organic membranes.

373 α-Alumina membrane is an inorganic membrane and the others listed in Table 3 are
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374 organic membranes. Lozano and coworkers have prepared active membranes for the

375 immobilization of Candida antarctica lipases [44]. The mixture of PEI and gelatin is

376 deposited on the membranes to introduce amino groups. Then, based on Schiff base

377 reactions, Candida antarctica lipases were immobilized on the amino-functionalized

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378 membranes using glutaraldehyde as cross-linker. In this report, membranes are

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379 amino-functionalized just by simple deposition of amino polymers on the membrane

380 surfaces, thus providing a facile method for membranes functionalization. Laccases

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381 from Trametes versicolor were immobilized on the poly (vinylidene fluoride) (PVDF)

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382 microfiltration membranes to remove phenylurea pesticides from wastewater [47]. For
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383 laccases immobilization, the PVDF microfiltration membranes were treated with

384 hydrazine hydrate to obtain the amino surfaces. Then, laccases were
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385 aldehyde-functionalized for the covalent attachment to the amino-functionalized PVDF

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386 microfiltration membranes. The authors observed that the immobilized laccases showed
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387 good stability and comparable activity compared with the free ones.

388 3.3 Capillary columns

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389 Capillary immobilized enzyme combined with capillary electrophoresis has been
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390 fully developed owing to the superior advantages of short analysis time, high efficiency
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391 separation, minimal sample consumption and flexible combination with diverse

392 detectors [48]. The enzymes immobilized in capillaries have been thoroughly

393 summarized in the review [95]. Enzymes can be immobilized on the capillaries in two

394 forms. One is on the capillary inner wall and the other is in the monolithic column. The

395 resultant reactors are correspondingly named as open tubular column immobilized
 ACCEPTED MANUSCRIPT

396 enzyme microreactor (OTC-IMER) and monolithic column immobilized enzyme

397 microreactor (MC-IMER).

398 3.3.1 OTC-IMER

399 For OTC-IMERs, enzymes are usually immobilized by electrostatic interaction [48,

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400 49] or covalent attachment [21, 50]. The capillary inner wall is negatively charged

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401 owing to the silanol groups generated after preprocessing. Then, with the help of

402 polyelectrolyte, enzymes are electrostatically adsorbed on the capillary inner wall. In

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403 addition, the silanol groups are favorable for the modification of organosilanes. Thus,

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404 the amino, sulfhydryl, cyano or epoxy groups can be easily introduced on the capillary
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405 inner wall, which will prompt the covalent attachment of enzymes.

406 Tang et al. immobilized angiotensin-converting enzyme on the capillary inner wall
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407 by ionic binding technique [48]. In this study, the negatively charged capillary inner
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408 wall was first dynamically coated with polycationic electrolyte hexadimethrine bromide,
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409 resulting in the positively charged inner wall. Then the negatively charged

410 angiotensin-converting enzyme was electrostatically adsorbed on the positively charged

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411 inner wall. Such a microreactor was used to screen enzyme inhibitors from natural
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412 extracts. As shown in Fig. 6, a neuraminidase-based IMER was prepared by covalent

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413 attachment. In this work, the pretreated capillary was first amino-functionalized with

414 APTES, following the introduction of aldehyde groups on the capillary inner wall.

415 Finally, through Schiff base reaction, neuraminidases were covalently immobilized on

416 the capillary inner wall [50]. With covalent attachment, neuraminidases were

417 immobilized firmly, thus resulting in satisfactory reusability and storage stability.
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418 Owing to their superior advantages of large specific surface area and satisfactory

419 biocompatibility, gold nanoparticles (GNPs) are usually introduced into capillaries to

420 immobilize enzymes. Zhao and coworkers [96] prepared a GNP-mediated L-glutamic

421 dehydrogenase bioreactor to screen enzyme inhibitors (Fig. 7). L-glutamic

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422 dehydrogenase molecules were first attached to GNPs to obtain the

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423 enzyme-functionalized GNPs. The negatively charged conjugates were then assembled

424 on the positively charged capillary inner wall which was treated with PEI in advance.

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425 Lin et al. [97] fabricated a dual-enzyme co-immobilized capillary microreactor by

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426 immobilizing adenosine deaminase and xanthine oxidase on the capillary inner wall
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427 according to the method stated above. The enzyme loading capacity is much greater

428 owing to the vast surface-to-mass ratio of GNPs. In addition, thiol groups of enzyme
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429 molecules can bind to GNPs strongly, which contributes to the high stability of the
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430 conjugates. However, the conjugates are assembled on the capillary inner wall by
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431 electrostatic interaction. The GNP-mediated bioreactor might show unsatisfactory

432 stability during the rinsing process.

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433 Since there is no systematic work for studying the effects of different
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434 immobilization methods on the activity and stability of IMERs, it is unable to compare
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435 the performances of IMERs fabricated with different methods. Promisingly, the features

436 of adsorption and covalent attachment have been thoroughly discussed in the

437 immobilization methods section. The adsorption method is easy to carry out with low

438 cost and there is no modification of enzyme molecules, thus preserving the catalytic

439 activities. However, the binding forces are too weak to obtain satisfactory stability. In
 ACCEPTED MANUSCRIPT

440 contrast, enzymes immobilized by covalent attachment possess good stability but the

441 active sites might be inactivated due to the chemical reactions. Given all that, we

442 suppose that the IMER fabricated with adsorption method possesses good catalytic

443 activity but poor stability while that fabricated with covalent attachment possesses good

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444 stability but slightly low catalytic activity.

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445 3.3.2 MC-IMER

446 For MC-IMERs, enzymes can be immobilized on the porous polymer monolithic

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447 column after its preparation [51-53] or on the monolithic sol-gel column during its

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448 preparation [54, 55].
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449 Trypsin-immobilized porous polymer monolithic column was prepared by Peterson

450 et al. [53]. The capillary inner wall was first vinylized with 3-(trimethoxysilyl)propyl
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451 methacrylate. After that, the polymerization mixture was polymerized to form the
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452 porous polymer monolithic column. Then, 2-vinyl-4,4-dimethylazlactone was

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453 introduced on the monolithic column to enable the subsequent immobilization of

454 trypsin.
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455 The sol-gel method has been noted as the monolithic packing method and therefore
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456 the sol-gel encapsulation method is a typical method for enzymes immobilization. The
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457 sol-gel encapsulation method is convenient as the enzyme-immobilized monolithic

458 column can be prepared in a single step. Sakai-Kato and coworkers prepared the

459 trypsin-encapsulated monolithic column with the sol-gel method [54]. The capillary was

460 first pretreated with methacryloxypropyltrimethoxysilane in order to prevent the gel

461 from leaking out of the capillary. Then tetramethoxysilane was hydrolyzed in acidic
 ACCEPTED MANUSCRIPT

462 solution and trypsin solution was added into the hydrolyzed silane solution. The above

463 obtained mixture was introduced into the capillary inlet to prepare the

464 trypsin-encapsulated monolithic column. This work team also prepared the

465 pepsin-encapsulated monolithic column with the same procedure [55].

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466 MC-IMERs are not usually used compared with OTC-IMERs owing to their much

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467 tedious and difficult preparation procedures. For porous polymer monolithic column,

468 the polymerization mixture is critical for the formation of porous polymer monolith.

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469 Therefore, a variety of polymerization mixture must be optimized, resulting in the

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470 tedious and laborious optimization procedures. For monolithic sol-gel column, the aging
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471 process is time-consuming and enzymes might be inactivated during the aging process,

472 thus reducing the catalytic activities.

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473
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474 4. Conclusions and future perspectives

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475 This review provides an overview on the methods and easy separated support

476 materials for enzymes immobilization. The physical methods are beneficial to
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477 maintaining the catalytic activity of immobilized enzymes, while the chemical methods
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478 are favorable for keeping the stability. The combination of physical method and
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479 chemical method could be regarded as a good strategy to produce immobilized enzymes

480 possessing both high catalytic activity and good stability. The easy separated support

481 materials make the separation of the immobilized enzymes no longer tedious and

482 laborious. The magnetic-immobilized enzymes can be separated from the reaction

483 mixture under external magnetic fields. And the membrane-immobilized enzymes are
 ACCEPTED MANUSCRIPT

484 usually fixed in equipments to form the membrane bioreactors, which can be rapidly

485 separated from the reaction mixture. In addition, the capillary immobilized enzyme

486 microreactors are usually fixed in capillary electrophoresis systems for on-line analyses.

487 Immobilized enzymes have aroused great interests owing to their superior features

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488 of improved reusability, enhanced stability, extreme pH and thermal resistances,

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489 technical and economic advantages etc. However, the immobilized enzymes are

490 basically home-made and still rare in the market due to their costs and storage problems.

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491 In addition, the challenges still remain in the large-scale production of immobilized

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492 enzymes with good uniformities and activities. For these reasons, further research
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493 efforts are still needed for exploring new support matrices and developing more

494 effective immobilization methods to improve the catalytic activity, reusability and
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495 stability of immobilized enzymes. And proper characterization of the resulting

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496 immobilized enzymes is also required. In the future, immobilized enzymes will be more
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497 widely used in the biotechnological-based industrial processes.

498
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499 Acknowledgements
500 This work was funded by the National Natural Science Foundation of China (Nos.
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501 21775153 and 21575150).

502

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715 [71] Z. Ali, L. Tian, B. Zhang, N. Ali, M. Khan, Q. Zhang, Synthesis of fibrous and
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716 non-fibrous mesoporous silica magnetic yolk-shell microspheres as recyclable supports

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717 for immobilization of Candida rugosa lipase, Enzyme Microb. Technol. 103 (2017)
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718 42-52.

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720 enzyme-silica composites with high activity and enhanced stability, J. Chem. Technol.
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721 Biotechnol. 91 (2016) 1905-1913.

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722 [73] X. Zang, W. Xie, Enzymatic interesterification of soybean oil and methyl stearate

723 blends using lipase immobilized on magnetic Fe3O4/SBA-15 composites as a biocatalyst,

724 J. Oleo Sci. 63 (2014) 1027-1034.

725 [74] M. Kalantari, M. Kazemeini, A. Arpanaei, Evaluation of biodiesel production using

726 lipase immobilized on magnetic silica nanocomposite particles of various structures,

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727 Biochem. Eng. J. 79 (2013) 267-273.

728 [75] F. Wang, C. Guo, L.R. Yang, C.Z. Liu, Magnetic mesoporous silica nanoparticles:

729 fabrication and their laccase immobilization performance, Bioresour. Technol. 101

730 (2010) 8931-8935.

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732 Cyclodextrin glucanotransferase immobilization onto functionalized magnetic double

733 mesoporous core-shell silica nanospheres, Electron. J. Biotechnol. 17 (2014) 55-64.

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735 magnetic mesoporous nanocomposite: A smart material for the immobilization of lipase,
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736 Polym. Compos. 37 (2016) 1152-1160.

737 [78] L. Yang, Y. Guo, W. Zhan, Y. Guo, Y. Wang, G. Lu, One-pot synthesis of
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738 aldehyde-functionalized mesoporous silica-Fe3O4 nanocomposites for immobilization of

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739 penicillin G acylase, Microporous Mesoporous Mater. 197 (2014) 1-7.

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740 [79] J. Mehta, N. Bhardwaj, S.K. Bhardwaj, K.H. Kim, A. Deep, Recent advances in

741 enzyme immobilization techniques: Metal-organic frameworks as novel substrates,

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742 Coord. Chem. Rev. 322 (2016) 30-40.

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743 [80] X. Lian, Y. Fang, E. Joseph, Q. Wang, J. Li, S. Banerjee, C. Lollar, X. Wang, H.C.
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744 Zhou, Enzyme-MOF (metal-organic framework) composites, Chem. Soc. Rev. 46 (2017)

745 3386-3401.

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747 microspheres with a designable metal-organic framework shell, J. Mater. Chem. 22

748 (2012) 9497-9500.

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749 [82] F. Pang, M. He, J. Ge, Controlled synthesis of Fe3O4/ZIF-8 nanoparticles for

750 magnetically separable nanocatalysts, Chem.-Eur. J. 21 (2015) 6879-6887.

751 [83] T. Zhang, X. Zhang, X. Yan, L. Kong, G. Zhang, H. Liu, J. Qiu, K.L. Yeung,

752 Synthesis of Fe3O4@ZIF-8 magnetic core-shell microspheres and their potential

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753 application in a capillary microreactor, Chem. Eng. J. 228 (2013) 398-404.

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754 [84] J. Zheng, C. Cheng, W.J. Fang, C. Chen, R.W. Yan, H.X. Huai, C.C. Wang,

755 Surfactant-free synthesis of a Fe3O4@ZIF-8 core-shell heterostructure for adsorption of

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756 methylene blue, CrystEngComm 16 (2014) 3960-3964.

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757 [85] C. Hou, Y. Wang, Q. Ding, L. Jiang, M. Li, W. Zhu, D. Pan, H. Zhu, M. Liu, Facile
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758 synthesis of enzyme-embedded magnetic metal-organic frameworks as a reusable

759 mimic multi-enzyme system: mimetic peroxidase properties and colorimetric sensor,
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760 Nanoscale 7 (2015) 18770-18779.

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761 [86] V. Konovalova, K. Guzikevich, A. Burban, W. Kujawski, K. Jarzynka, J. Kujawa,

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762 Enhanced starch hydrolysis using α-amylase immobilized on cellulose ultrafiltration

763 affinity membrane, Carbohydr. Polym. 152 (2016) 710-717.

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764 [87] G.M. Rios, M.P. Belleville, D. Paolucci, J. Sanchez, Progress in enzymatic
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765 membrane reactors-a review, J. Membr. Sci. 242 (2004) 189-196.

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766 [88] L. Donato, C. Algieri, A. Rizzi, L. Giorno, Kinetic study of tyrosinase immobilized

767 on polymeric membrane, J. Membr. Sci. 454 (2014) 346-350.

768 [89] X.Y. Zhu, C. Chen, P.C. Chen, Q.L. Gao, F. Fang, J. Li, X.J. Huang,

769 High-performance enzymatic membrane bioreactor based on a radial gradient of pores

770 in a PSF membrane via facile enzyme immobilization, RSC Adv. 6 (2016)
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771 30804-30812.

772 [90] K. Abrol, G.N. Qazi, A.K. Ghosh, Characterization of an anion-exchange porous

773 polypropylene hollow fiber membrane for immobilization of ABL lipase, J. Biotechnol.

774 128 (2007) 838-848.

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775 [91] H.T. Deng, Z.K. Xu, X.J. Huang, J. Wu, P. Seta, Adsorption and activity of

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776 Candida rugosa lipase on polypropylene hollow fiber membrane modified with

777 phospholipid analogous polymers, Langmuir 20 (2004) 10168-10173.

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778 [92] J.K.J. Yong, G.W. Stevens, F. Caruso, S.E. Kentish, In situ layer-by-layer

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779 assembled carbonic anhydrase-coated hollow fiber membrane contactor for rapid CO2
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780 absorption, J. Membr. Sci. 514 (2016) 556-565.

781 [93] H.T. Deng, Z.K. Xu, Z.M. Liu, J. Wu, P. Ye, Adsorption immobilization of Candida
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782 rugosa lipases on polypropylene hollow fiber microfiltration membranes modified by

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783 hydrophobic polypeptides, Enzyme Microb. Technol. 35 (2004) 437-443.

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784 [94] J.D. Kimmel, D.T. Arazawa, S.H. Ye, V. Shankarraman, W.R. Wagner, W.J.

785 Federspiel, Carbonic anhydrase immobilized on hollow fiber membranes using

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786 glutaraldehyde activated chitosan for artificial lung applications, J. Mater. Sci.: Mater.
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787 Med. 24 (2013) 2611-2621.

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788 [95] J. Iqbal, S. Iqbal, C.E. Müller, Advances in immobilized enzyme microbioreactors

789 in capillary electrophoresis, Analyst 138 (2013) 3104-3116.

790 [96] S. Zhao, X. Ji, P. Lin, Y.M. Liu, A gold nanoparticle-mediated enzyme bioreactor

791 for inhibitor screening by capillary electrophoresis, Anal. Biochem. 411 (2011) 88-93.

792 [97] P. Lin, S. Zhao, X. Lu, F. Ye, H. Wang, Preparation of a dual-enzyme

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793 co-immobilized capillary microreactor and simultaneous screening of multiple enzyme

794 inhibitors by capillary electrophoresis, J. Sep. Sci. 36 (2013) 2538-2543.

795

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796 Figure captions

797 Figure 1. The methods for enzyme immobilization.

798 Figure 2. Scheme of preparation of cross-linked enzyme aggregates (CLEAs).

799 Figure 3. Schematic illustration of preparation of magnetic chitosan-immobilized α-Glu

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800 [23].

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801 Figure 4. Process for immobilization of CLEAs in Mag-MSU-F.

802 Figure 5. Schematic illustration of synthesis of Fe3O4@ZIF-8.

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803 Figure 6. (a) Process for immobilizing neuraminidase on capillary inner wall and (b)

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804 schematic of on-line enzyme assay [50].
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805 Figure 7. Schematic of preparation of GNP-mediated enzyme bioreactor [96].
806
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807 Graphical abstract

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808
809

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810 Figures

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811

812 Figure 1. The methods for enzyme immobilization.

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813
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814

Figure 2. Scheme of preparation of cross-linked enzyme aggregates (CLEAs).

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816

817 Figure 3. Schematic illustration of preparation of magnetic chitosan-immobilized α-Glu

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818 [23].

819
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821 Figure 4. Process for immobilization of CLEAs in Mag-MSU-F.

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823

824 Figure 5. Schematic illustration of synthesis of Fe3O4@ZIF-8.

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825

826
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Figure 6. (a) Process for immobilizing neuraminidase on capillary inner wall and (b)
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827 schematic of on-line enzyme assay [50].
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828

829 Figure 7. Schematic of preparation of GNP-mediated enzyme bioreactor [96].

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831 Table 1. The applications of easy separated support materials in enzymes immobilization.

Easy separated
Carrier Enzyme Ref
support material
HRP, Glucose oxidase, Catalase, Pectinase,
Fe3O4@SiO2 [28-32]
Benzaldehydelyase

Fe3O4@organic polymer α-Glucosidase, HRP, Lipase, Laccase [22, 33-36]

MNP

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Fe3O4@mesoporous
Lipase, Tyrosinase, Laccase, HRP [37-40]
material

Fe3O4@MOF Glucose oxidase, Lipase [41-43]

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Inorganic membrane Lipase [44]

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Membrane
Organic membrane Lipase, HRP, Laccase [45-47]

Acetylcholinesterase, α-Glucosidase, Neuraminidase,

OTC-IMER [21, 48-50]

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Angiotensin-converting enzyme
Capillary column
MC-IMER α-Glucosidase, Trypsin, Pepsin [51-55]
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833 Table 2. The applications of magnetic mesoporous materials in enzymes immobilization.

Magnetic mesoporous Immobilization

Enzyme Template Functional reagent Ref.
carrier method

Lipase Fe3O4@mSiO2 CTAB DMOAP Adsorption [37]

Adsorption &
Laccase Fe3O4@mSiO2 EO20PO70EO20 APTES [39]
Cross-linking

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HRP Fe3O4@mSiO2 CTAB APTES Covalent attachment [40]

Adsorption &
Glucose oxidase Mag-MCF-C EO20PO70EO20 --- [65]

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Cross-linking
Adsorption &
Subtilisin carlsberg Mag-MSU-F EO20PO70EO20 --- [66]
Cross-linking

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--- Adsorption
Porcine pancreatic
Fe3O4@MCM-41 CTAB APTES Cross-linking [68]
lipase
CPTES Covalent attachment

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Nitrile hydratase Fe3O4@mSiO2 Tannic acid Glutaraldehyde Covalent attachment [69]
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Laccase Fe3O4@mSiO2 Trimethylbenzene APTES Covalent attachment [70]

Fe3O4@SiO2@hollow
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mesoporous fibrous
Candida rugosa
SiO2 CTAB APTES Covalent attachment [71]
lipase
Fe3O4@SiO2@hollow
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mSiO2
Phenylalanine
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Fe3O4@mSiO2 --- --- Entrapment [72]

ammonia lyase

Lipase Fe3O4@SBA-15 EO20PO70EO20 APTES Covalent attachment [73]

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Lipase Fe3O4@SiO2@mSiO2 CTAB EDS Covalent attachment [74]

Laccase Fe3O4@mSiO2 Jeffamine D-2000 CPTES Adsorption [75]

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Cyclodextrin ---, APTES, BTME Adsorption

Fe3O4@SiO2@mSiO2 CTAB [76]
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glucanotransferase APTES Covalent attachment

Aniline & Ammonium
Lipase Fe3O4@mSiO2 CTAB Covalent attachment [77]
persulfate
Penicillin G Trimethoxysilylpropa
Fe3O4@mSiO2 EO20PO70EO20 Covalent attachment [78]
acylase nal
834
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835 Table 3. The applications of membranes in enzymes immobilization.

Immobilization
Enzyme Membrane Functional reagent Ref.
method
Candida antarctica Gelatin & PEI
α-Alumina membrane Covalent attachment [44]
lipase B PEI
Laccase Adsorption &
PP HFM --- [45]
HRP Entrapment

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Poly(α-allylglucoside) Adsorption
Lipase PP HFM [46]
poly(γ-stearyl L-glutamate) Adsorption
PVDF microfiltration

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Laccase Hydrazine hydrate Covalent attachment [47]
membrane

Tyrosinase Polyamide membrane --- Entrapment [88]

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Hollow fiber PSF Adsorption &
Lipase --- [89]
microfiltration membrane cross-linking
Glycidylmethacrylate & Adsorption &

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ABL lipase PP HFM [90]
Diethyl amine cross-linking
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Phospholipid analogous
Candida rugosa lipase PP HFM Adsorption [91]
polymer

Carbonic anhydrase PP and PDMS HFM Polyelectrolyte Adsorption [92]

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Candida rugosa lipase PP HFM Hydrophobic polypeptide Adsorption [93]

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Carbonic anhydrase PP HFM Chitosan Covalent attachment [94]

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Highlights

Detailed methods for enzymes immobilization are discussed.

MNPs, membranes and capillary columns are presented to solve the separation

problems of immobilized enzymes.

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Applications of easy separated support matrices in enzymes immobilization are

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summarized.

Future perspectives of immobilized enzymes are presented.

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