Enzyme Nanocarriers

C H A P T E R

6 Enzyme Nanocarriers Fei Peng*,†, Hang Yin*,†, Shi-Lin Cao*, Wen-Yong Lou*,† *

Laboratory of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, Guangzhou, China †Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, South China University of Technology, Guangzhou, China

6.1 INTRODUCTION Biocatalysts play an important role in the chemical industry, and an enzyme that is one part of the biocatalyst has served as a critical tool in the field of chemical synthesis [1]. Especially in the past two decades, this enzyme has been widely exploited in the fine and bulk chemical, food, pharmaceutical, cosmetic, textile, pulp, and paper industries [2]. Furthermore, enzymatic processes that are more environmentally friendly, most cost-effective and, ultimately, more sustainable are carried out under mild conditions in water with high rates and selectivities [3]. Given the advances in enzymatic techniques, enzymatic application in green and sustainable chemical manufacturing has been taken more seriously. Despite all these advantages, the industrial application of enzymes is still limited by their instability in harsh operational conditions, low shelf-life, and difficulty in recycling [4]. To overcome the inevitable drawback of natural enzymes, employing enzymes immobilized on solid supports is a straightforward method for enhancing the practical performance of enzymes. The immobilization of enzymes can offer more effective control of the catalysis process, and maintain high enzyme stability in operational conditions. Currently, some traditional materials such as sol-gel matrices, hydrogels, organic microparticles, and mesoporous silica are utilized to immobilize enzymes. Except for the profits brought by these solid supports, it should be noted that enzymes can suffer leaching, enzyme denaturation, and sometimes, reduction of enzyme loading. Therefore, to further enhance the efficiency of enzyme immobilization, it is necessary to forge some novel materials that occupy massive surface area, tailored pore size, and tunable shape structure to build a proper chemical and physical environment for enzymes.

Advances in Enzyme Technology https://doi.org/10.1016/B978-0-444-64114-4.00006-6

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# 2019 Elsevier B.V. All rights reserved.

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Lately, three types of nanomaterials have emerged as promising biocatalytic support platforms, including inorganic nanoflowers, metal-organic frameworks, and magnetic nanoparticles. They have also spurred a significant amount of research. So far, hundreds of papers and articles on the immobilization of enzymes in different materials have been published. For example, Lee et al. reported a glutaraldehyde treating method to synthesize nanoflowers to immobilize lipase, which exhibits improved reusability [5]. Ze et al. designed and built a kind of PA-Td-Cu MOF nanoenzyme that not only possessed high catalytic activity compared with horseradish peroxidase, but was also capable of exuding fluorescence to indicate the concentration of H2O2 [6]. Cao et al. reported that glucose oxidase was immobilized on a new core-shell magnetic ZIF-8/cellulose nanocomposite, and the nanocatalyst offered excellent protein loading and enhanced relative activity. In brief, these three sterling candidates for enzyme immobilization are highly desirable for practical applications. In this chapter, we focus on the preparation processes for the preceding enzyme carriers, and discuss the use of these carriers for efficient enzyme immobilization. An extensive list of enzyme immobilization processes is provided, and details about the structures and catalytic performance of these immobilized enzymes are discussed to provide guidance on designing better nanobiocatalysts. Finally, we hope this chapter will trigger relevant researchers’ motivation to further explore enzyme immobilization.

6.2 METAL-ORGANIC FRAMEWORKS More than two decades have passed since Yaghi first wrote the term “metal-organic framework” in his paper [7]; and the exploitation of metal-organic frameworks (MOFs) has substantially widened our horizon due to its admirable characteristics on enzyme immobilization. MOFs, which are exactly porous crystalline materials, consist of metal ions or clusters coordinated to organic linkers, and feature well-defined pore structures, large surface areas, and structural flexibility [8]. These elements make host MOFs filled with more regions to trap guest molecules, enabling them to be the potential immobilization matrices for biomolecules, especially certain enzymes [9]. Notably, flexible rational design on the architecture and functionalization of organic structure or the change of metal nodes has enabled the massive synthesis of MOFs. However, the industry application of MOFs on enzyme catalysis is still in its infancy, albeit tailored to >20,000 types of MOF. To circumvent this, researchers need to dial back on synthesizing MOFs whose properties are never fully explored, and focus on refining those that have proven stability or activity [10]. Given that MOFs are composed of metal nodes and organic ligands, bonding and non-bonding interactions between MOFs supports and enzymes are possible [11]. Typically, MOF-enzyme composites can be categorized into four main types in terms of their synthetic approach: surface attachment, covalent linkage, pore entrapment, and co-precipitation [12]. While the first three methods involve a similar immobilization process to the one reported for other porous or dense solid-state support materials, co-precipitation also known as in situ synthesis is specific to MOF materials due to the possible use of gentle synthesis conditions that prevent enzyme degradation [9]. Herein, numerous compelling areas of work have been reported on MOF-enzyme composites [13],

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encapsulation strategies for enzymes in MOF micropores [14], and MOFs with multiple types of active sites as multifunctional catalysts for synergistic catalysis or tandem reactions [15]. For further details of recent advances in enzyme catalysis within MOFs, it is essential to distinguish the synthesizing methods of MOFs. The approaches can be classified mainly into four types: solvent-thermal synthesis, microwave synthesis, sonochemical synthesis, and mechanochemical synthesis. Different methods offer different yields and particle sizes, and undergo different reaction times using the same reaction materials. Afterward, we will discuss these methods, as well as relevant examples of manufacturing MOFs. Solvent-thermal synthesis, which is the most typical method, involves both metal ions and organic ligands heated in a solvent within sealed vessels. Yaghi et al. successfully designed a classic IRMOF series with different pore sizes by heating an N, N0 -diethylformamide (DEF) solvent mixture of Zn(NO3)24H2O and 12 kinds of links in a closed vessel, respectively [16]. Here, DEF works as an activating agent for removing unreacted metal ions or radicals that attached on the surface of the MOF or in the pore of the material. It can be costly or cause difficulty in clearing inevitably. Alternatively, high-speed synthesis with microwaves has been employed broadly in recent years [17]. Compared with solvent-thermal synthesis, microwave heating would dramatically cut reaction times, increase product yields, and enhance product purities [18]. Ni and coworkers first describe this rapid method for the assembly of three known MOFs, IRMOF-1, IRMOF-2, IRMOF-3 [19]. Another method, sonochemical synthesis, has recently been exploited to synthesize small MOF particles. This facile, physical synthetic method provided with acoustic cavitation and nebulization, leads to the creation of extreme conditions associated with high temperature and pressure. Son et al. first prepared a high-quality MOF-5 of 5–25 μm in size using a sonochemical method in considerably decreased synthesis time (30 min), compared with traditional solvent-thermal synthesis (24 h) [20]. While all of the methods described herein rely on solvents, mechanochemical synthesis can be carried out under mechanical force, that is, solvent-free conditions. For this technique, MOFs is obtained by grinding both organic linkers and metal ions in a ball mill without extra heating. Peng et al. introduce an N-coordination to modify UiO-66(Zr) with dopamine (DA) for enhancing its acetaldehyde and chlorobenzene/H2O adsorption, using the mechanochemical synthesis strategy [21]. Additionally, postsynthetic modification (PSM), known as a postpartum recovery method to enhance the stability of MOF material, permits the installation of various chemical functionalities into the skeleton of MOFs after their formation. Cohen and co-workers have reported some of their works and published relevant reviews on this topic [22, 23]. Simultaneously, the activation of MOFs is also crucial for PSM to prepare topologically identical MOFs. Information about well-activated MOFs can also be taken from the primary literature [24]. In brief, the evolution of MOF synthesis routes would pave the way for practical applications of MOF materials. But we cannot escape from the truth that available methods emphasizing the prospective industrial utilization are almost all based on laboratory scale experiments. Hence, advanced protocols are still required for industrial applications. Compared with previously porous materials used to immobilize enzymes [25], MOFs are emerging candidates that pursue high stability, selectivity, and efficiency of catalysts. Typically, MOF-enzyme composites are divided into three strategies: surface adsorption, covalent linkage, and encapsulation within pores of MOF (Fig. 6.1). Researchers are supposed to select

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FIG. 6.1 The model cascade enzyme system of glucose oxidase (GOx) and horseradish peroxidase (HRP).

one suitable method to immobilize enzymes, pursuing an ideal catalyst that enjoys high selectivity, structure, productivity, substrate specificity, and space-time yield. It lies on the character of the enzyme and MOF frameworks, which probably involves the robust peptide bonds, high surface area of MOFs, the pore size of MOFs, and so on. Here, we intend to simply exhibit how MOF materials work as supports for enzyme immobilization. Enzyme immobilization is facilitated through the formation of bonding and non-bonding interactions between enzymes and solid supports [12]. For the most widely used method between enzymes and solid supports, surface adsorption means that enzymes are anchored by weak interactions, such as van der Waals forces, hydrogen bonding, and ionic binding. The unity of diverse forces usually facilitates the stability of MOF catalysts. Owing to the nature of the adsorption method, we do not have to take into account the pore size or the existence of unique functional groups. Nevertheless, enzymes attached on the surface of MOFs mostly have an unsatisfied stability. Compared with surface adsorption, covalent binding of enzymes can render a strong bond formed by amino groups and carboxylate groups originated from enzymes and MOFs. In general, DCC (N,N0 -dicyclohexylcarbodiimide), an enzyme coupling agent, was needed for the activation of carboxylate groups of MOFs to enhance enzyme immobilization [26]. But, this costly and time-consuming method also confuses researchers due to its complicated protocol. Except for the two methods mentioned herein, one pot synthesis strategy has recently stimulated considerable interest, because it allows enzymes to immobilize into the MOF over the synthesis of the MOF. It can hinder enzyme leaching and enlarge the selection of proteins and MOFs on one hand, and on the other hand, it creates a new problem, in that organic solvents used in the synthesis of MOFs may denature the enzymes. To inhibit enzyme denaturation, tailored MOF pores can encapsulate enzymes via providing local buffering or optimizing of the enzyme’s microenvironment, and in particular, the mesoporous MOF can load enzymes in a high volume under a well-dispersed state [27]. Furthermore, it can reduce leaching of enzymes and prevent aggregation of enzymes. For this

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encapsulation route, we should also consider the challenge of decomposition that MOFs may undergo at elevated temperatures [8]. Thus, making stable MOFs is crucial to the success of MOF-enzyme composites. In addition to optimized routes of MOF-enzyme composites, modelling studies can facilitate the explicitness of enzyme diffusion mechanisms into MOF cavities [28]. Overall, each MOF-enzyme composite route has its advantages and drawbacks, the property of both MOFs and enzymes would determine the choice of the appropriate method. In recent decades, the versatility of MOFs has been explosively demonstrated by researchers on catalytic applications. Due to the richness of strategies for MOF materials and MOF-enzyme bioreactors, an extensive species of enzyme has been reported to be immobilized with MOFs. Herein, we will discuss the controllable design process and enzyme properties in detail. Three representative enzymes are selected to show the advantages of enzyme immobilization in MOFs, especially from the perspective of catalytic activity, reusability, and stability. We hope our fellow chemists can improve the application of MOFs on industrial catalysis on the basis of their predecessors’ work. Lipase-catalyzed transesterification has been considered as one of the most promising techniques for producing biodiesel, which will be subject to environmental and economic sustainability [29]. Compared with free lipase, immobilized lipase often has good enzymatic performance in relation to activity, stability, and reusability. Given that, the porcine pancreatic lipase (PPL) can exhibit essential biocatalysis in the chemical and pharmaceutical industries [30]. Liu et al. reported that microporous MOF materials can adsorb PPL without any chemical modification to synthesis warfarin, which is a common anticoagulant in the clinic [31]. The four microporous MOFs involved were UiO-66(Zr), UiO-66-NH2(Zr), MIL-53(Al), and carbonized MIL-53(Al); and one mesoporous silica SBA-15 was synthesized according to published procedures via solvent-thermal synthesis. The adsorption of PPL was implemented by mixing a MOFs and PPL solution in a solution of methanol and DMSO, along with using a vortex. Subsequently, this PPL@MOF bioreactor was used for catalysis of 4-hydroxycoumarin (A) and benzylideneacetone (B) to synthesize warfarin in turn. The catalytic activity and loading capacity of different MOF bioreactors was further probed to demonstrate the feasibility of microporous MOF material for enzyme adsorption. Cao et al. [32] were the first to report the use of HKUST-1 as supports for Bacillus subtilis lipase (BSL2) immobilization by surface attachment. HKUST-1, also known as tunable Cu-BTC mesoporous MOFs, are fabricated through their previous method, which is a template-free strategy under solvothermal conditions [33]. BSL2-surfactant complexes were first prepared by mixing BSL2 solution and surfactant in a homogenizer. Then, BSL2surfactant complexes diffused in isooctane can be immobilized in HKUST-1 materials via stirring. The esterification reaction between lauric acid and benzyl alcohol was notably facilitated by the catalysis of BSL2@HKUST-1. The result indicated that immobilized BSL2 had significantly enhanced pH and temperature stability and enzymatic activity when compared with free enzymes. Moreover, the initial reaction rate of immobilized BSL2 is around 17 times that of free form. Notably, the BSL2@HKUST-1 could be operated for 10 cycles of successive reuse retaining 90.7% of the initial enzymatic activity and 99.6% of its original conversion. Compared with one enzyme catalysis reaction, a multi-enzymatic reaction permits the combination of cascade reactions simultaneously, or in a stepwise pattern, without

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FIG. 6.2 Three immobilization strategies of MOF-enzyme composites.

segregating the intermediates [34]. Multi-enzyme embedded nanomaterials possess the advantages that the active sites of enzymes within different nanomaterials are easily brought into close proximity to enhance the catalysis efficiency. Here, we exhibit the model cascade enzyme system of glucose oxidase (GOx) and horseradish peroxidase (HRP). GOx converts glucose into gluconic acid, and generates H2O, which is the substrate for HRP to oxidize ABTS2 to form ABTS [35]. Ge et al. first reported a one-pot, ambient, and general preparation of multiple enzymeincorporated ZIF-8 using GOx and HRP as model enzymes. The MOF scaffolds are able to provide a rigid pore to embed the enzyme for preserving the enzyme’s activity, and enhancement of the enzyme’s activity. Notably, the activity of GOx and HRP@ZIF-8 was showed to be 2 times higher than that of the enzymes embedded in ZIF-8, respectively. This was attributed to the fact that the intermediate (H2O2) can be immediately employed in the next reaction without transferring from one enzymatic MOF composite to another nanocatalyst. The selectivity and storage of GOx and HRP@ZIF-8 are also evaluated to demonstrate feasibility of this promising catalyst. As shown in Fig. 6.2, to further reinforce the catalysis efficiency of GOx and HRP, Zhou’s group carried out their work, which encapsulates two enzymes into a hierarchically structured MOF. This hierarchical MOF material, PCN-888, involved three types of pores, and was used to immobilize two enzymes. Here, the largest pore was accommodated by GOx, the middle pore for HRP, and the smallest pore was reserved for the substrate diffusion pathway. The key point of precise control of enzyme distribution is a stepwise encapsulation procedure with a unique order, which is GOx first, and followed by HPR [36]. In addition, the reversed encapsulation order demonstrated the coupling of two enzymes as a control. In a word, the effect of PCN-888 indicated that the hierarchical nanoreactor had the potential to be applied in more complex systems.

6.3 ORGANIC-INORGANIC NANOFLOWERS Ge et al. first reported in 2012 [37] the preparation of organic-inorganic crystal hybrid nanoflower catalysts, in which the proteins and metal ions were used as the organic and inorganic ingredients, respectively. According to this literature, the organic-inorganic crystal hybrid nanoflowers can be formed by blending certain concentrations of CuSO4, with

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phosphate buffered saline containing proteins for three days, such as bovine serum albumin, laccase, lipase, carbonic anhydrase, lipase, and carbs, suggesting a common applicability of the method for various enzymes. For the mechanism of enzyme-inorganic hybrid nanoflower preparation [37, 38] (Fig. 6.3), the primary crystals consisted of protein molecules and Cu2+ was formed in the first step, mainly by the coordination facility of amide groups of the enzyme backbone. Subsequently, protein molecules were deposited to form large aggregates, and primary crystals were further generated, resulting in the separate petals based on the individual Cu2+ onto the agglomerates’ surfaces. In the final stage, the petals of a branched flower-like structure were locked by the growing copper phosphate crystals to enhance stability. Compared with common enzyme immobilization achieving a stronger stability but a lower activity than free enzyme, the application of this method can effectively improve the activity of an immobilized enzyme, which was proven for various enzymes by many authors (Table 6.1). Although enzyme-inorganic hybrid nanoflowers have a positive effect on the activity of various enzymes, different inorganic materials would cause diverse effects in the activity and the morphology of the immobilized enzyme. For example, Ge et al. [37] reported that a lipase-Cu3(PO4)23H2O hybrid nanoflower’s catalyst displayed almost the same activity, and even lower activity when compared with free lipase when using p-nitrophenyl butyrate as the substrate. However, a lipase-Zn3(PO4)2 hybrid nanoflower’s catalyst prepared by Zhang et al. [43] showed an enhancement of 147% in the activity of lipase using the same compound as the substrate in the PBS buffer. Moreover, the applications of Transmission Electron Microscope and Scanning Electron Microscope affirmed that the morphology of lipaseZn3(PO4)2 hybrid nanoflowers was different from that of lipase-Cu3(PO4)23H2O hybrid nanoflowers. The structure of lipase-Zn3(PO4)2 is a uniform ellipsoidal morphology; nevertheless, that of lipase-Cu3(PO4)23H2O is isotropic. Besides, papain-Zn3(PO4)2 hybrid nanoflowers displayed the same structure-like uniform ellipsoidal morphology [40]. The results indicate that inorganic materials can effectively impact the morphology of proteininorganic hybrid nanoflowers. Meanwhile, lipase-Zn3(PO4)2 possessed excellent storage and operational stability. After preserving at 4°C for 20 days, a lipase-Zn3(PO4)2 hybrid nanoflower catalyst still remained at 79.4% of its initial activity; nevertheless, the free lipase just reserved 24.8% of its initial activity. Furthermore, the activity of the hybrid nanoflowers only lost 5.5% of its initial activity after reusability of eight batches at 37°C of reaction temperature, and slight protein loading was lost after eight recycles. These exciting results state that designed materials can benefit from industrial enzyme catalysis.

FIG. 6.3 Forming process of protein-inorganic hybrid nanoflowers.

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TABLE 6.1 Change in Activity of Protein-Inorganic Hybrid Nanoflowers Activity of Enzyme

Entry

Enzyme

Inorganic Salt

Substrate/Solvent

Immobilized Enzyme/Free Enzyme

1

Laccase

Cu3(PO4)23H2O

Epinephrine/PBS

6.5

[37]

2

Carbonic anhydrase

Cu3(PO4)23H2O

CO2/

2.6

[37]

3

α-Chymotrypsin

Cu3(PO4)2

N-Phenyl ethyl formyl-Ltyrosine/Tris-HCl buffer

3.66

[39]

4

Papain

Zn3(PO4)2

Casein/PBS

0.81

[40]

5

Horseradish peroxidise

Cu3(PO4)23H2O

o-Phenylenediamine

6.06

[41]

6

Trypsin

Cu3(PO4)2

Nα-benzoyl-L-arginine ethyl ester/

2.73

[42]

7

Lipase

Zn3(PO4)2

p-Nitrophenyl palmitate/ PBS

1.47

[43]

8

Soybean peroxidase

Cu3(PO4)23H2O

Guaiacol/PBS

4.46

[44]

9

Lactoperoxidase

Cu3(PO4)2

Guaiacol/PBS

3.6

[45]

10

Horseradish peroxidise

FePO4

Guaiacol/PBS

7.1

[46]

References

PBS: phosphate buffer.

A multi-enzyme system can be constructed by the co-immobilization of enzymes on protein scaffolds, polymeric capsules, and so forth. In the same way, a structure-like flower hybrid complex of two enzymes and metal phosphates can be formed by a self-assembly process. Similar to the preparation of single enzyme-metal phosphates, Ge and co-workers in 2014 [47] published a hybrid nanoflower complex of two-enzyme, horseradish peroxidase (HRP) and glucose oxidase (GOx), with Cu3(PO4)2 in the three strategies: GOx@HRP in which HRP first combines with Cu3(PO4)2 forming a complex, then GOx is incorporated on the surface of HRP-Cu3(PO4)2; HRP@GOx in which GOx first combines with Cu3(PO4)2 forming a complex; then HRP is incorporated on the surface of GOx-Cu3(PO4)2; GOx-HRP in which GOx and HRP were randomly distributed in the two-enzyme-metal phosphate hybrid nanoflower. The average size of these three complexes is <10 μm. With the substrate of glucose and ABTS, the three complexes displayed enhancement in the activity compared with that of free enzymes, which is beneficial for the shorter mass transfer distance and the positive effect of copper ions. In addition, GOx@HRP showed the highest overall activity, suggesting that the distribution of enzymes would impact the overall activity of enzymes. Based on the immobilization technology for dual enzymes, a biosensor [48] comprised of microfluidic paper-based analytic devices, of which a major component is white paper, and dual enzymes

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GOx and HRP, showed rapid and sensitive detection for glucose, with 10 min. Detection time and a 25 μM limit of glucose detection. Protein-inorganic nanoflower hybrid catalysts have difficulty separating during the liquid phase because of their nano- or micron-scale size. In addition, nanobiocatalysts, in general, do not possess strong properties in mechanical strength, which compromises stability and reusability [3, 49]. During the fermentation process of beer, the non-enzymatic oxidative decarboxylation of α-acetolactate will generate diacetyl, which can cause an unpleasant buttery taste. The translation of α-acetolactate directly to acetoin by α-acetolactate decarboxylase (ALDC) effectively overcomes this drawback. An immobilized ALDC (Ca3(PO4)2-ALDC)@ ALG) [50], based on ALDC-inorganic nanoflower catalysts and alginate gel (ALG), was formed to resolve the drawback of beer. After achieving Ca3(PO4)2-ALDC, its surface was wrapped by calcium alginate to form (Ca3(PO4)2-ALDC)@ALG, which possessed stronger mechanical strength and easy separation. The activity of (Ca3(PO4)2-ALDC)@ALG decreases slightly compared with that of Ca3(PO4)2-ALDC. After six batches of reuse, (Ca3(PO4)2ALDC)@ALG still preserves 80% of its initial activity, but Ca3(PO4)2-ALDC only remains 55% of its initial activity. The application of (Ca3(PO4)2-ALDC)@ALG in the beer fermentation revealed a lower concentration of diacetyl after fermentation for 18 days than that without ALDC (<0.1 ppm VS 0.13 ppm).

6.4 MAGNETIC NANOBIOCATALYSTS Magnetic nanoparticles (MNPs) possess the large specific surface area, favorable surface atomic coordination, and other features that make them good candidates for the carriers for the immobilization of the enzyme [51]. In addition, the nanoparticles can easily implement enzyme recovery with the addition of an external magnetic field. All of these advantages are beneficial to the application in enzyme immobilization. Preparation MNPs mainly have two categories, a wet method and a dry method [52]. Wet preparation for the synthesis of nanoparticles includes a soluble co-precipitation method, a microemulsion method, thermal decomposition, and a hydrothermal method. Ferroferric oxide nanoparticles (Fe3O4) are, in general, employed in enzyme immobilization, because of efficient biocompatibility and easy preparation. The two main approaches, as introduced here, are co-precipitation and microemulsion. In brief, ferrous iron salt and ferric iron salt become ferrous chloride and ferric chloride in the coprecipitation. The two irons are mixed in a suitable ratio in the alkai solution (commonly, ammonia water) under the protection of nitrogen. After a certain period, nanoparticles can be achieved by the filtration, washing, drying, and so on. The reaction is shown in Fig. 6.4. The factors include the ratio of Fe2+ to Fe3+, pH, and incubation time in the preparation, affect the size and magnetic properties of the final nanoparticles. Liu et al. [53] prepared MNPs with good superparamagnetism and an average particle diameter of <15 nm through coprecipitation using FeCl2 and FeCl3 as raw materials during the addition of ammonia under nitrogen protection.

FIG. 6.4 Preparation of ferroferric oxide.

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Microemulsion is an isotropic, low viscosity, and transparent thermodynamically stable system composed of water, oil, and surfactant. A water-in-oil or oil-in-water microemulsion is formed by the mixing of the reactant aqueous solution, water-insoluble nonpolar substance, and surfactant as part of the dispersed phase, and the dispersing medium and emulsifier, respectively. A common microemulsion system for the preparation of MNPs is a water-in-oil system. The size distribution of the MNPs is narrow and can be controlled by the change of the latex size, which is affected by the concentrations of various contents. Loo et al. [54] used ferric chloride, ferrous chloride, and ammonia as the raw materials to successfully prepare 10 nm MNPs by the addition of a certain mass ratio of dodecyl trimethyl ammonium bromide and dodecyl ammonium bromide as surfactants. However, the MNPs are often unstable under acidic conditions, and toxic to the biocatalyst, which restricts their application in enzyme immobilization [55]. In order to solve these drawbacks, MNPs in general need further functional modification by the attachment of a more biocompatible chemical material on the surface of the particles. The carbonyl, hydroxyl, and amino groups of the material can be effectively connected with enzyme protein molecules, which enhances the stability of the enzyme and keeps its activity after immobilization on the particles. The common materials embedded on the MNPs can be divided into synthetic chemical materials and natural biological macromolecules. Two biological macromolecules of dopamine and fiber nanocrystals were introduced here in the application of MNPs on the enzyme’s immobilization. Dopamine (DA) is a neurotransmitter that can lead to senile dementia under dyssecretosis [56]. In recent years, some researchers started to use DA as a biomimetic adhesion material, resulting in a wide range of discussions and applications. The catechins of DA in the muscle fibroin are the key of adhesion. Lee et al. [57] described the formation of polydopamine (PDA) by the self-polymerization at pH 8.5 when using PA to adhere to proteins in simulation. Surprisingly, the PDA layer can be formed on the surface of different materials when they are added to the DA solution. Wang et al. [58] found that the Fe3O4 particles can be coated by PDA to form composite MNPs that can be used as the adsorbent for polyaromatic hydrocarbon in waste water. Furthermore, the PDA coated on the nanoparticles contains a number of active groups that can further bind with functional groups of inorganic and organic biological macromolecules [59, 60]. Predictably, dopamine shows great application prospects in enzyme immobilization. Zheng et al. [61] prepared MNPs based on DA and mesoporous silica supports, which can enhance the stability and activity of the enzyme compared with the enzyme immobilized on the mesoporous silica supports. Mesoporous silica supports can be employed in the immobilization of the enzyme by the adsorption, which easily causes the leakage of the enzyme, resulting in the loss of activity because of the weak acting force. Further, adhering DA on the mesoporous silica particle was applied to overcome the shortcomings in the two approaches (Fig. 6.5). In brief, the enzyme-loaded bimodal mesoporous silica was first prepared. In one approach, the particle containing enzyme was dropped in the DA solution to form enzyme-loaded bimodal mesoporous silica; in the other, after the formation of a polyelectrolytes layer on the surface of mesoporous silica, the microsphere was soaked in the DA solution to obtain enzyme-loaded, DA-capped bimodal mesoporous silica. The use of two approaches to immobilize the catalase shows significantly higher operational stability than the enzyme immobilized on the bimodal mesoporous silica. After stirring for 6 h at the phosphate buffer

6.4 MAGNETIC NANOBIOCATALYSTS

163

FIG.

6.5 The preparation of enzyme immobilized on the PDA-silica hybrid materials in the two approaches: (A) PDA-filled BMS; (B) PDA-capped BMS.

(50 mM pH 7.0), the enzyme activity decreased by <5% for the two approaches, but a >60% loss happened for the enzyme immobilized on the bimodal mesoporous silica. In addition, 78% of relative activity was achieved in the method of enzyme-loaded, DA-capped bimodal mesoporous silica. Ren et al. [62] prepared magnetic iron oxide nanoparticles adhered with DA and immobilized lipase on their surface in an easy solution (Fig. 6.6). The optimum conditions for the enzyme immobilization were that MNPs were soaked in 2.5 mg/mL of PDA solution to form the PDA-MNPs, followed by lipase immobilization at a PDA-MNPs to lipase mass ratio of 2/1. The use of this approach to prepare the immobilized lipase achieved a high specific activity recovery (73.9% of the free lipase activity), high enzyme loading (429 mg lipase/g PDA-MNPs), and better thermal and pH stabilities than its free counterpart. Furthermore, the immobilized lipase displayed good reusability and kept >70% of its initial activity after 21 recycles. The results indicate that PDA-MNPs are economical, simple, and efficient supports for enzyme immobilization. Cellulose nanocrystals (CNCs) are newly developed bio-based nanomaterials. Randy et al. [63] reported for the first time that hydrolysis of cellulose acid-catalyzed by concentrated sulfuric acid formed a suspension in which further analysis showed nanocrystals with a consistent structure of cellulose. After treated with acid, the retained rod-like cellulose with highly crystalline and pyknotic structures is CNC [64]. CNCs can be achieved from many types of cellulose, for example, bacterial cellulose, ramie, cotton, waste cotton fabrics, and microcrystalline cellulose [65]. The length of CNC depends on the raw materials and the preparation process, and can range from 20 nanometers to thousands of nanometers; nevertheless, the width of the cellulose nanomaterials is commonly less than 10 nanometers and the length [64].

FIG. 6.6 The immobilization of lipase on the PDA-MNPs.

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6. ENZYME NANOCARRIERS

FIG. 6.7 The preparation of GOx-CNCs/ PEI/AuNPs.

Incani et al. [66] prepared a biosensor based on glucose oxidase from Aspergillusniger, with the CNC as support (Fig. 6.7). The CNC specific surface area was augmented, and the chemical affinity changed by adhering CNCs with cationic polyethylenimine (PEI) by electrostatic assembly to prepare a CNC/PEI nanocomposite. Subsequently, negatively charged Au were deposited on the CNCs/PEI to form enzyme carrier CNCs/PEI/AuNPs, which can be used for the immobilization of glucose oxidase after the modification with a thiol. The results demonstrated that the GOx loading decreased from 25.2 mg/g to 20.3 mg/g with the increase in the length of the thiol-linker from 11 carbons to 3 carbons, suggesting that a short length of the thiol-linker is beneficial for the amount of enzyme loaded. Although CNCs are very stable in aqueous suspension, a disadvantage is that the separation from the reaction media needs high-speed centrifugation, which has limited the applications. To enhance the easy operation, the method for the introduction of magnetic Fe3O4 nanoparticles into CNCs was investigated. Both MNPs and CNCs contain negative surface charges, resulting in unstable deposition and easy collapse. To overcome this shortcoming, chitosan was employed to combine the CNCs strongly with MNPs to form magnetic cellulose nanocrystals (MCNCs) in a simple coprecipitation-electrostatic self-assembly technique (Fig. 6.8A) [67]. Based on the enzyme carrier, further work established the highly effective precipitation-crosslinking approach used to immobilize papain (Fig. 6.8B) [68]. In brief, after addition of a precipitant, papain can automatically deposit on the MCNC surfaces in the mixture of an enzyme and the aqueous suspension of the MCNC carrier. The use of glutaraldehyde combines the enzymes to decrease the leakage from the support, forming a papain@MCNCs biocatalyst. The results indicated that the papain@MCNC biocatalyst had a high enzyme loading capacity (333 mg of protein/g MCNCs) and high enzymatic activity recovery (>80%).

6.5 CONCLUSIONS AND PERSPECTIVES The use of MOFs, protein-inorganic hybrid nanoflowers, and CNC- and PDA-based nanomaterials on the enzyme immobilization has achieved lots of exciting results in the improvement of enzyme activity and stability. The carriers can be used in not only single enzyme immobilization, but also multiple enzyme immobilization. It is noteworthy that enzyme-based biocatalytic processes are replacing traditional chemical conversion methods in laboratories and in industry because of their high efficiencies and eco-friendly properties.

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FIG. 6.8 Preparation of enzyme immobilization support (A) and the immobilization of papain (B).

However, there are still many key challenges for future research in these areas. The biocompatibility and the pore size of MOF primarily affects the catalysis activity and the loading amount of enzyme on the carrier. Exploration of these two topics can obviously accelerate the application of MOFs on enzyme immobilization at the industrial scale. Second, the interaction mechanism between the enzyme and inorganic material is still unclear. Systematic study of the interaction can be beneficial to the construction of enzyme-inorganic hybrid nanoflowers, resulting in a highly effective biocatalyst. Third, further works could focus on the control of complicated structures of PDAs and interactions between the enzyme and PDAs to enhance the immobilization effect of the enzyme. Fourth, the exploration of the interrelation between CNC morphology and the catalytic character of enzyme-CNC is beneficial to the design of the CNC carrier to achieve a highly effective nanobiocatalyst. Finally, the combination of practical experiments and molecular simulations is helpful to understand the molecular properties of the immobilized enzyme, and to easily obtain the high property of the immobilized enzyme. Nanobiocatalysts have a bright future, and we can expect to see intensive and extensive research on the topics discussed herein.

Acknowledgments We wish to thank the National Natural Science Foundation of China (No. 21878105; No. 21676104; No. 21336002), the Open Funding Project of the State Key Laboratory of Bioreactor Engineering, and the Program of State Key Laboratory of Pulp and Paper Engineering (2017ZD05) for partially funding this work.

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