Review

Using Inorganic Nanomaterials to Endow Biocatalytic Systems with Unique Features Youhui Lin,1,* Zhengwei Chen,2 and Xiang Yang Liu1,2,* The rapid growth in nanotechnology and biotechnology offers a wealth of opportunities for the combination of natural enzymes with different kinds of nanomaterial. Here, we highlight recent advances in constructing nanomaterialincorporated enzymes that integrate the specific recognition and biocatalytic properties of enzymes with the attractive electronic, optical, magnetic, and catalytic properties of nanomaterials. These composite materials have some extra features that are not possessed by the enzymes themselves, such as electron transfer mediated by nanoparticles, enzyme delivery, remote activation and/or deactivation of enzymatic activity, and fabrication of catalytic entities with complementary functions. Additionally, we describe briefly the current challenges and future development of unique enzyme–nanomaterial hybrid systems. We hope that this review will help to accelerate further progress in this promising field.

Trends Enzymes and artificial enzyme-catalytic systems can be endowed with new and attractive features by using nanoscale inorganic materials. We discuss the multifunctional nanomaterial-incorporated bio- and biomimetic catalysts and their limitations. We highlight the design and development of multifunctional hybrid catalysts and discuss the challenges and future directions in this highly active field.

Biocatalysis and Nanotechnology Natural enzymes are highly effective and versatile biocatalysts that exhibit high chemo-, stereo-, and regioselectivity. In addition, their catalytic processes are conducted under mild conditions (close to atmospheric pressure, physiological pH, and temperature) [1]. Enzymes have vital roles in living organisms, mediating almost all chemical reactions involved in different cellular processes, such as signal transduction, DNA replication, immune responses, metastasis, and metabolism [2]. Aberrant enzyme activities are associated with several diseases and disorders, including HIV and cancers [3–5]. In addition to their in vivo roles, enzymes are also extensively utilized in pharmaceutical research, medicine, agrochemical production, biofuel production, environmental monitoring, the food industry, and life science studies [6,7]. Currently, the combination of nanotechnology and biotechnology in the design and fabrication of novel materials and devices has led to the establishment of new areas of nanobiotechnology [8– 13], such as the combination of nanomaterials with natural enzymes. The latest advances in nanometer-sized materials offer a novel pathway for modulating the catalytic behaviors of enzymes [13–15]. To date, gold nanoparticles (AuNPs) [2,13,15], magnetite nanoparticles (MNPs) [16,17], carbon nanotubes (CNTs) [18], graphene oxide (GO) [14,19], porous silica structures [20,21], alumina nanoparticles [22], copper phosphate [Cu3(PO4)2] [23], calcium hydrogen phosphate (CaHPO4) [24], and other types of nanomaterial [25] have been reported to modulate the structures and functions of enzymes. Moreover, in some cases, their combination with biocatalysts can also endow catalysts with superior and unique features [26–28]. Table 1 summarizes selected examples in the highly active field of nanomaterial-incorporated biocatalysis. Although many excellent review papers have been dedicated to this highly promising field [1,9–11,8,29,30], most have focused mainly on the use of different types of nanomaterial

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1 Research Institute for Biomimetics and Soft Matter, Fujian Provincial Key Laboratory for Soft Functional Materials Research, Department of Physics, Xiamen University, Xiamen 361005, China 2 Department of Physics, National University of Singapore, 2 Science Drive 3, Singapore, 117542, Singapore

*Correspondence: [email protected] (Y. Lin) and [email protected] (X.Y. Liu).

http://dx.doi.org/10.1016/j.tibtech.2015.12.015 © 2015 Elsevier Ltd. All rights reserved.

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Table 1. Examples of Tuning Biocatalysis Using Nanoscale Materials, Especially Inorganic Materials Nanomaterial

Catalysta

AuNP

Chymotrypsin [64,65], horseradish peroxidise [28], glucose oxidase [13]. Glucose oxidase [39], b-galactosidase [40], and Aeropyrum pernix glucokinase [42]

Pt or Pd nanoparticles

Aminopeptidase [43], and lipase [43,44]

CNT

Soybean peroxidise [66], chymotrypsin [67], perhydrolase S54V [18], glucose dehydrogenase or alcohol dehydrogenase [45], Taq DNA polymerase and cyclomaltodextrin glucanotransferase [50], hydrogenase [47], and horseradish peroxidise [27]

Graphene or graphene oxide

Chymotrypsin [14,19], trypsin [19], and proteinase K [19]

Porous materials

Organophosphorus hydrolase [20], butyrylcholinesterase [21], catalase [52], superoxide dismutase [52,54], and penicillinase [53]

MNPs

Lipase [17], horseradish peroxidise [16], glucose oxidase [55], horseradish peroxidise [36], lactate dehydrogenase [55], dehalogenase L-2-HADST [58], chymotrypsin [59], and b-galactosidase [59]

Alumina nanoparticles

Pepsin [22] and urease [68]

Copper phosphate or calcium hydrogen phosphate

Laccase [23], carbonic anhydrase [23], and /-amylase [24]

Quantum dots

Cytochrome P450BSb [26], cytochrome C peroxidase, myoglobin, and the weak peroxidase CYP152A1 [62]

Upconversion nanoparticles

b-Galactosidase [63]

a

Italics indicate that the nanomaterial-incorporated catalyst has a new feature.

for regulating the stability and activity of biocatalysts. By contrast, there are only a few reviews on the aspects of enzyme–nanomaterial hybrid systems that integrate the catalytic properties of enzymes with the unique properties of diverse nanomaterials [10,11,30] Therefore, in this review, we focus on recent developments in endowing enzyme-catalytic systems with new and attractive features using nanoscale inorganic materials. We provide a general overview of the extra features of composite materials that are not possessed by enzymes themselves and then briefly describe the potential application and future development of nanomaterial-incorporated enzymes.

Unique Properties of Nanomaterial-Integrated Enzymes Nanoscale materials have the advantages of facile preparation, large surface area:mass ratios, the ability to tailor the surface with a broad range of functionalities, and other attractive electronic, optical, magnetic, and catalytic properties [2,14,15,31]. In some cases, nanomaterials-coupled enzymes can have not only the catalytic functions of natural enzymes, but also more advanced features provided by the nanomaterials. Here, we summarize and review several of these important extra features. Nanomaterial-Mediated Charge Transport Redox enzymes cannot directly establish electrical contact with electrode surfaces because of the insulation of their active sites by the protein shell. This limits their direct application in amperometric biosensors or biofuel cells [32]. Thus, the routing of these enzymes to electrodes has become the subject of extensive research [11,30,33,34]. Many methods have been reported to assemble integrated, electrically contacted enzyme electrodes, such as the use of diffusional electron mediators and the incorporation of the enzymes in redox-active polymers [33,34]. With the accompanying developments in nanotechnology, conductive nanomaterials

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has also been used to electrically contact redox enzymes with electrode supports; of the various material candidates, metal- and carbon-based nanomaterials are attracting growing interest as functional materials for charge transport [33,34] Nanomaterial-Mediated Delivery of A Membrane-impermeable Enzyme Intracellular delivery of enzymes with retention of activity is crucial in both therapeutic and fundamental applications [35,36]. However, most naked enzymes are poorly delivered to cells because of their low membrane permeability and stability. As a result, the delivery of active enzymes inside living cells is rare. To overcome these problems, one of the most effective strategies for enzyme delivery is to use nanoparticles as nanocarriers or transporters. Nanomaterial-Mediated Remote Control of Enzyme Activity Controlled initiation of biochemical events, in particular of enzyme activity, has become a powerful tool in biochemical research [37,38]. The use of trigger signals offers an approach for the temporary manipulation of protein function, which is crucial for understanding biochemical systems and for controlling biomolecular processes [37,38]. In this context, nanomaterials with unique energy absorbance (e.g., at radio and optical frequencies) and other unique physical features have recently emerged as a prominent class of materials for controlling biocatalytic processes. Fabrication of Catalytic Entities with Complementary Functions It is well known that many nanomaterials have shown great promise as nanocatalysts with high stability and easy recovery and/or recycling. The combination of different biocatalysts with nanocatalysts offers a simple route to the fabrication of catalytic ensembles with synergic and complementary functions [39].

Metal-Based Nanomaterials For ease of access, the examples treated in this review are classified according to metal-, carbon-, oxide-, sulfide-, and fluoride-based materials. Metal-based nanomaterials [especially the noble metals Au, platinum (Pt), and palladium (Pd)] show attractive physical and chemical properties owing to their small size, high surface, and quantum size effects. Metal Nanomaterial-Mediated Charge Transport The electrical contacting of redox enzymes with electrodes is a key process for the development of amperometric biosensors and biofuel cell elements [33,34]. Recently, due to their excellent conducting properties, Willner et al. incorporated redoxactive biocatalysts into metallic nanoparticles to construct electrochemical sensors using the nanoparticles as a current collector and an electron relay to an electrode (Figure 1) [13]. As shown in Figure 1A, they used two different routes to fabricate the reconstituted AuNP-GOx–monolayer electrode [13], and confirmed that AuNPs (marked with arrows in Figure 1B) had bound to the protein units [13]. Both electrodes constructed by the two routes exhibited bioelectrocatalytic activity toward the catalytic oxidation of glucose. In a similar fashion, the authors further demonstrated other cofactor-dependent enzymes using AuNPs to improve the electrical contact [13]. Since then, considerable research effort has been made in the construction of hybrid nanocomposites by combining the recognition and catalytic features of enzymes with the electronic features of the nanomaterials [33,34]. Taken together, the incorporation of nanomaterials into those redoxactive biocatalysts is a new way to form an electrically active biomaterial, which brings a whole new facet to nanobioelectronic research. Metal Nanomaterial-Mediated Enzyme Delivery The very high surface area and tunable functionality of AuNPs make them a promising nanocarrier for protein surface recognition and delivery. Rotello and colleagues explored peptide-capped AuNPs as an enzyme transporter for the effective delivery of membrane-impermeable

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(A) s

(a)

s

Au

O N H

FAD

GOx e–

Au (b)

O

Au

FAD

N H

s

s

s

Au

O N H

O N FAD H

N H

FAD

GOx

Gluconic acid

O

FAD

N

N

O N N H H HO H HO H NH HO H N H H N O O N N H O H O P –O P –O O O

H2N

Au

O

Au

ApoGOx s

Glucose

e–

Au55

(1)

H H OH OH

S– CH2

CH2

S–

Spacer (B)

S

S

S–

S–

S–

S–

(2) (3) (4)

5 nm

Figure 1. Construction of Electrically Contacted Enzyme Electrodes by the Reconstitution of Apoproteins on Cofactor-Functionalized Gold Nanoparticles (AuNPs) Associated with the Electrode. (A) Different strategies for the assembly of AuNP-reconstituted GOx electrode reconstitution of apo-GOx on functional AuNPs [13] Copyright 2003, Science. (B) A scanning transmission electron microscopy (STEM) image of an apo-GOx/FAD functional AuNP ensemble. Arrows show Au clusters [13] Copyright 2003, Science.

b-galactosidase (b-Gal) (Figure 2A), which is a negatively charged enzyme with high molecular weight (465 kDa) [40]. The peptide groups on the surface of AuNPs have multiple roles in cellular delivery processes, such as enzyme surface recognition, membrane association, ‘endosomal buffering’, and nanoparticle stability [40]. With the help of peptide-capped AuNPs, b-Gal could be transported into various cell lines. Importantly, the transported b-Gal was able to escape from endosomes and retain its enzymatic activity inside the cells [40]. Metal Nanomaterial-Mediated Control of Enzyme Activity Due to their energy-absorbing ability, gold nanomaterials can be applied to photothermally activate and/or deactivate enzyme activity. For instance, Plessen and co-workers described the use of AuNPs to control the activity of horseradish peroxidase (HRP) via laser irradiation at a wavelength of 532 nm [28]. By using 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as a peroxidase substrate, they found that the amount of enzymatic product (ABTS+) decreased with increasing temperature in the range 15–458C [28]. Under irradiation with a 532-nm laser, laser light energy

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(A)

Gold nanoparcles

β-Gal

β-Gal

β-Gal Substrate

Product

(C)

(B)

12 nm 6 nm

(D)

NO

O H2N

N H O

O H2N

OH

+

Pepde bond cleavage

1

Glutamic acid p-nitroanilide

O

NO2

H2N

OH

O

OH

NH2 OH

H2, Hydrogenaon

NH2

Glutamic acid p-nitroanilide

2

O

OH

+ NH2

Glutamic acid p-phenylenediamine

Figure 2. Application of Metal Nanomaterial for Effective Delivery of Membrane-Impermeable Enzymes and Constructing Catalytic Ensembles. (A) A facile strategy for delivering membrane-impermeable protein [e.g., b-galactosidase (b-Gal)] via gold nanoparticles (AuNPs) [40]. Copyright 2010, ACS. Structures of PepA (B) and PepA–platinum nanoparticle complexes (C) [43]. (D) Schematic of the bioinorganic nanohybrid catalyst-catalyzed two-step reactions [43]. Copyright 2011, Wiley.

was absorbed by the AuNPs and transformed into heat, which affected the enzymatic conversion of ABTS and H2O2 to ABTS+ and H2O, respectively. The authors speculated that this reduction of ABTS+ was caused by the thermal deactivation of the enzyme [28]. Previous work revealed that the ABTS+ product is relatively unstable, and decays to a colorless product through disproportionation [41]. Therefore, another reason for such reduction may be the insufficient stability of ABTS+. Apart from their use in the controlled deactivation of biochemical processes, metal nanomaterials (e.g., Au nanorods) can also be applied to photothermally activate the enzyme activity [42].

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Combination of Enzymes with Catalytic Metal Nanomaterials Compared with their bulky counterparts, metal nanomaterials have been discovered to have various catalytic activities. Recently, the strategy for fabricating catalytic entities that make use of the potential of both natural enzymes and catalytic metal nanomaterials has also been explored [39,43]. For instance, an early example of the catalytic growth of AuNPs was reported by utilizing the catalytic activities of GOx and gold seeds [39]. Additionally, for one-pot multistep synthesis of the desired compounds, Kim and coworkers prepared a biohybrid catalyst that combined a bacterial aminopeptidase from Streptococcus pneumonia (PepA) and Pt nanoparticles (Figure 2B) [43]. PepA can act as an ideal biotemplate, which allows the formation of Pt nanoparticles within PepA using an in situ biotemplate, reduction of K2PtCl4 with NaBH4 (Figure 2C). The resulting nanocomposite was successfully utilized in cooperative tandem catalysis, including PepA-mediated amide bond cleavage of Glu-p-nitroanilide and Pt-catalyzed hydrogenation of the produced p-nitroanilide (Figure 2D) [43]. A similar strategy was also utilized by Filice and coworkers to incorporate Pd nanoparticles into the interior of a lipase to synthesize a bioinorganic nanohybrid catalyst [44].

Carbon-Based Nanomaterials Carbon-based nanomaterials with unique advantages including high surface area, electronic and thermal properties, and extraordinary chemical stability, can serve as excellent supporting materials for tuning biocatalysis. Carbon Material-Mediated Charge Transport Owing to their conducting properties, carbon nanomaterials can be implemented to design electrically contacted enzyme electrodes [45–49]. For instance, Willner and coworkers presented a facile strategy to prepare integrated, electrically contacted NAD(P)+-dependent enzyme electrodes on CNTs, which could act as a conducting matrix with a large surface area [45]. First, the cofactors NADP+ and NAD+ were immobilized on Nile blue-functionalized CNTs via a phenyl boronic acid ligand [45]. The functional NAD(P)+ units present on the CNTs provide an anchoring surface, which allows the immobilization of glucose dehydrogenase or alcohol dehydrogenase on the CNTs due to the formation of affinity complexes between NAD(P)+ and the integrated enzymes [45]. The enzyme electrodes so obtained had bioelectrocatalytic activities and could be used as amperometric electrodes for sensing glucose or ethanol [45]. Furthermore, these electrically contacted enzyme–CNT electrodes could also be utilized to prepare biofuel cell elements [45]. In addition, Lacey and coworkers used CNTs grown on gold electrodes as a platform for the oriented and stable attachment of a NiFe hydrogenase; the unique features of this hydrogenase-based hybrid electrode are beneficial for hydrogen biosensing and biofuel applications [47]. Recently, mesoporous carbon nanoparticles as promising matrices for the assembly of electrical contacting of enzyme electrodes have attracted interest, because they not only have conductivity properties, but can also encapsulate and cap electron relay units in the pores [48,49]. For instance, the relay unit Fc-MeOH can be loaded in the pores of mesoporous carbon nanoparticles and the pores then capped with GOx (Figure 3A) [48,49]. The above functionalized nanocomposites were assembled on a glassy carbon electrode for the bioelectrocatalyzed oxidation of glucose. Carbon Material-Mediated Control of Enzyme Activity Photothermal Control Carbon materials have high photothermal conversion efficiency due to high absorbance in the near-infrared (NIR) region. By utilizing the heating properties of CNTs, Miyako et al. reported an early example of the remote control of thermostable enzyme-catalyzed reactions [50]. For instance, they were able to control the activity of Taq DNA polymerase and cyclomaltodextrin glucanotransferase in a microspace. The working principle of the first enzyme-based system is schematically represented in Figure 3B [50]. The activity of the anti-Taq DNA polymerase

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(A) OH

Nafion

Fe

Glucose

O

Gluconic acid

H N

N H

O

GOx GOx

e–

GOx

GOx

GOx

e– Glassy carbon (B)

Acvated Taq DNA polymerase

Template DNA and primer

Laser ON

An- Taq

Nonacvated Taq DNA polymerase

Denatured an-Taq Extended DNA

Figure 3. Schematic Illustration of Carbon-Materials-Mediated Charge Transport and Photothermal Control of Enzyme Activity. (A) Schematic representation of the relay-mediated bioelectrocatalytic oxidation of glucose on the FcMeOH/GOx CNP-functionalized electrode [48] Copyright 2013, ACS. (B) Schematic representation of the laser-induced DNA extension reaction [50] Copyright 2009, RSC.

antibody (anti-Taq)–Taq DNA polymerase conjugate was typically activated at around 708C owing to the denaturation of anti-Taq in the high temperature range. The authors chose the intercalating dye YOYO-1 as a reporter, which formed a stable, highly fluorescent complex with double-stranded DNA (dsDNA). By applying YOYO-1 to stain the resultant dsDNA, the DNA extension reaction can be real-time monitored by the change in fluorescence. After stopping NIR irradiation (5 W, 30 s), the apparent fluorescence of YOYO-1–extended dsDNA complexes was detected [50]. These results demonstrated that enzymatic functions can be remotely controlled by the powerful photothermal effect of CNTs. Control of Enzyme Activities by Photosensitive Compounds Functionalized Nanomaterials The remote control ability can also be ascribed to functional groups present on carbon nanomaterials. Qu and colleagues prepared spiropyran dye (SP)-functionalized CNTs to modulate HRP activity by light irradiation, using 3,3,5,5-tetramethylbenzidine (TMB) as a peroxidase substrate. Light can be used to regulate the surface properties of the functionalized CNTs, thus affecting the catalytic activity of HRP [27]. Under UV light exposure, the neutral closed form of CNT-SP was isomerized to the merocyanine form (CNT-MC). CNT-MC exhibited a significant

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enhancement in the catalytic activity of HRP, whereas the activity of HRP after introducing CNTSP almost remained the same as before [27]. Additionally, a lysozyme DNA aptamer bound to CNT-MC inhibited the interaction of CNT-MC with HPR. Consequently, the presence of aptamer decreased the activity of the CNT-MC/HRP system. More interestingly, upon addition of lysozyme, it could specifically bind to its aptamer. As a result, HRP bound to CNT-MC again and the catalytic activity of HRP was rescued [27]. Inspired by these phenomena, a sensing strategy for selective colorimetric detection of lysozyme was developed.

Oxide-, Sulfide-, and Fluoride-Based Materials Porous Silica Structures There is a long history of the incorporation of enzymes and porous silica structures through encapsulation, adsorption, or covalent linking [51]. The enzyme confinements inside the pore channels usually improve the stability and selectivity of the enzyme, and enable its easy separation and reuse. Porous silica materials can also be potential candidates for enzyme delivery. For instance, hollow silica nanospheres (HSN) with low densities, large interior spaces, and permeable silica shells are suitable for loading HRP in the cavity to carry out intracellular biocatalysis [36]. In addition to loading of a single enzyme, Mou et al. encapsulated superoxide dismutase (SOD) and catalase (CAT) in HSNs as cell-implanted nanoreactors for the cascade transformation of superoxide through H2O2 to water (Figure 4A) [52]. More importantly, these SOD/CAT-encapsulated composites exhibited strong cell protection and detoxification effects on superoxide radicals [52]. This research presents a general method to realize the intracellular implantation of catalytic nanoreactors with designed functions. In addition, Ortac and colleagues fabricated dual-porosity HSNs as carriers for enzyme delivery (Figure 4B) [53]. Dual-porosity HSNs encapsulate enzymes filled through mesopores (pore size, 550 nm) into a hollow interior. The mesopores are then sealed with nanoporous material (pore size

Using Inorganic Nanomaterials to Endow Biocatalytic Systems with Unique Features.

The rapid growth in nanotechnology and biotechnology offers a wealth of opportunities for the combination of natural enzymes with different kinds of n...
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