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Chemical Engineering Science 135 (2015) 21–32

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Chemical Engineering Science


journal homepage: www.elsevier.com/locate/ces

Advances in carrier-bound and carrier-free


immobilized nanobiocatalysts
Mengfan Wang a,c, Wei Qi b,c,d,n, Rongxin Su b,c,d, Zhimin He b
a
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China
b
State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China
c
Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin 300072, PR China
d
The Co-Innovation Center of Chemistry and Chemical Engineering of Tianjin, PR China

H I G H L I G H T S

 Nanobiocatalysts have been developed as an attractive strategy for enzyme immobilization in biocatalysis and biosensor fields.
 The carrier-bound nanobiocatalysts exhibit specific performances by combining enzymes with nanomaterials.
 Cross-linked enzyme aggregates shows higher activity and stability than native enzymes as carrier-free nanobiocatalysts.

art ic l e i nf o a b s t r a c t

Article history: Although the immobilization of enzymes with improved activity and stability has been the subject of
Received 21 December 2014 growing interest for many years, new opportunities arose by implementing nanomaterials as immobiliz-
Received in revised form ing carriers. The nano-carriers not only offer larger surface area and lower mass-transfer limitation but
22 March 2015
also endow the native enzyme with specific performances due to their various physical or chemical
Accepted 29 March 2015
Available online 3 April 2015
properties. A carrier-free nanobiocatalyst, cross-linked enzyme aggregates (CLEAs), has been developed
for applications in industrial bioprocesses. This review illustrates the recent achievements in nanobio-
Keywords: catalysts, including the carrier-bound and carrier-free enzymes. These nanobiocatalysts provide enzymes
Immobilized enzyme with broad properties to serve in the fields of biocatalysis, biomedicine and biosensors.
Nanobiocatalyst
& 2015 Elsevier Ltd. All rights reserved.
Nanomaterial
Cross-linked enzyme aggregates
Biocatalysis
Biosensor

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2. Inorganic nanomaterial as carrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.1. Gold nanoparticles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.2. Magnetic nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.3. Carbon-based scaffold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.4. Ordered mesoporous nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.5. Enzyme–inorganic hybrid nanoflowers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3. Organic nanomaterial as carrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.1. Peptide-based scaffold. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.2. Stimuli-responsive polymer nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.3. Single enzyme nanoparticles/nanogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4. Carrier-free immobilized enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

n
Corresponding author at: State Key Laboratory of Chemical Engineering, School
of Chemical Engineering and Technology, Tianjin University, Tianjin 300072,
PR China. Tel.: þ 86 22 27407799; fax: þ 86 22 27407599.
E-mail address: qiwei@tju.edu.cn (W. Qi).

http://dx.doi.org/10.1016/j.ces.2015.03.051
0009-2509/& 2015 Elsevier Ltd. All rights reserved.
22 M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32

Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

1. Introduction simple preparation process, low cost, high unit activity and intrinsic
stability, CLEAs have been used in many unconventional biocatalysis
Enzymes, the biocatalysts in chemical reactions, have numerous processes, such as non-aqueous media (Kartal et al., 2011; Yang et al.,
advantages, including high efficiency, specificity, regioselectivity, enan- 2012; Yu et al., 2014) and one-pot cascade reactions (Chmura et al.,
tioselectivity, mild reaction conditions, and environmentally friendly 2013; Jung et al., 2013; Touahar et al., 2014).
processes (de Carvalho, 2011; Illanes et al., 2012). In the biosensor In this review, we focus on the recent developments of nanobio-
field, enzymes play important roles, including in enzyme electrodes catalysts including nanocarrier-bound and carrier-free enzymes. Inor-
and enzyme-linked immunosorbent assay (ELISA) (Sassolas et al., ganic nanomaterial immobilization with gold nanoparticles, magnetic
2012). However, native enzymes that dissolve in aqueous solution nanoparticles, carbon-based scaffolds, ordered mesoporous nanoma-
are sensitive and fragile to temperature, pH and organic media, which terials and enzyme–inorganic hybrid nanoflowers, and organic nano-
limit their applications in biocatalysis and biosensors. Therefore, the material immobilization, including peptide-based scaffolds, stimuli-
strategies developed for stabilizing enzymes have attracted increasing responsive polymers and single-enzyme nanoparticles/nanogels, are
attention. Soluble enzymes increase the cost because they cannot be presented. Typical examples of enzymes benefiting from nanomater-
recycled such as most chemical catalysts (DiCosimo et al., 2013). ials are illuminated. In addition, several improved preparation strate-
Immobilization of enzymes onto solid carriers is an effective method gies for CLEAs are presented to provide a new avenue for the
to stabilize enzymes because the carrier provides superior physical application of enzymes in chemical engineering, biotechnology, bio-
stability to the soluble counterparts, and the carrier is easier to recover sensors and materials.
from the medium at the end of a reaction (Cao et al., 2003). In a strict
sense, the carrier can be considered as a modifier that improves the
performance of enzymes. Accordingly, a significant number of syn- 2. Inorganic nanomaterial as carrier
thetic or natural carriers with different shapes, sizes, porous/nonpor-
ous structures, hydrophobic/hydrophilic properties and functionalized 2.1. Gold nanoparticles
surfaces have been designed for various enzyme immobilizations
(Garcia-Galan et al., 2011; Tran and Balkus, 2011). The high surface-to-volume ratio and easily functionalized surface
In the past decades, nanomaterials have become the most flourish- of gold nanoparticles (AuNPs) make them excellent carriers for
ing subject in the fields of optics, electronics, catalyst engineering and protein/enzyme immobilization. Multipoint Au–S bond formation is
biomedicine (Choi et al., 2014; Hubbell and Chilkoti, 2012; Lee et al., a straight-forward way to fix functional molecules in the desired
2014a; Ma et al., 2009; Zhang and Dai, 2012). The small unit size and geometry (Dreaden et al., 2012; Keighron et al., 2014). In some cases,
high surface-to-volume ratio make them an extraordinary option for the tight chemisorption of proteins on AuNPs originates from the
enzyme immobilization with higher protein loading and less mass- binding of thiol and amino groups that exist in proteins (Zhao et al.,
transfer limitation. Furthermore, the unique optical, electronic, mag- 2008). Anchoring groups are usually utilized for attachment of an
netic or mechanical properties of nanomaterials endow enzymes with enzyme to the AuNPs surface. These anchoring groups include
special properties and broaden the application fields of enzymes. thiolate, dithiolate, dithiocarbamate, amine, carboxylate, selenide,
Cross-linked enzyme aggregates (CLEAs), which are well-known as isothiocyanate and phosphine (Brown et al., 2010; Hou et al., 2009;
carrier-free immobilized enzymes, are another newly developed Knight et al., 2009; Zhao et al., 2005). Additionally, some of the
nanobiocatalyst with size ranging from tens to hundreds of nan- thiolate ligands in alkanethiolate-stabilized AuNPs can be substituted
ometers (Sheldon, 2011b; Sheldon et al., 2013). The preparation of by other thiols through a thiol–thiol exchange reaction. The reaction
CLEAs consists of two steps: precipitation and cross-linking. Due to the rate depends on the chain length and steric bulk of the leaving thiolate

Fig. 1. Schematic of thermophilic enzyme photothermal gold nanoparticle (TE–PGNs) synthesis and laser-induced activation (adapted from Blankschien et al. (2013)).
M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32 23

and incoming thiols as well as on the charge of the AuNPs (Hostetler 2.2. Magnetic nanoparticles
et al., 1999; Saha et al., 2012; Templeton et al., 1998). Lv et al. (2014)
described a reusable bioreactor that consists of porous polymer Magnetic metal nanoparticles (MNPs), which usually have sizes
monolithic supports with the pore surface coated with AuNPs. The ranging from 10 to 100 nm, have been used in enzyme immobilization
AuNPs serve as an intermediate ligand to immobilize lipase via the due to their unique superparamagnetism, high surface area, large
non-covalent bond between the thiol and amine functionalities with surface-to-volume ratio and easy separation under external magnetic
gold. After several cycles of operation, inactive lipase can be stripped fields (Garcia et al., 2011; Reddy et al., 2012). The ferrite colloids
from the nanoparticles using 2-mercaptoethanol and subsequently magnetite (Fe3O4) and maghemite (γ-Fe2O3) are the main representa-
replaced by fresh enzyme. tives of MNPs. Immobilized enzymes based on iron magnetic nano-
Metal nanoparticles with localized surface plasmon resonance particles have been applied in certain fields, such as immunoassays
(LSPR) properties at the specific incident wavelength generate (Yang et al., 2013b; Zhang et al., 2013), biosensors (Baratella et al.,
strong collective oscillations of electron plasma in metals, which 2013; Garcia et al., 2011), bioseparations (Kannan et al., 2013; Ma et al.,
can be scattered in the form of light or heat (Daniel and Astruc, 2012) and drug delivery (Koppolu et al., 2012; Wang et al., 2014).
2004; Giljohann et al., 2010). Based on this, Blankschien et al. Wang et al. (2012) developed a simple and cost-effective method to
(2013) developed light-responsive, thermophilic enzyme photo- directly immobilize Candida rugosa lipase on alkyl silane modified
thermal gold nanoparticle complexes (TE–PGNs) using gold nanor- Fe3O4 magnetite nanoparticles through hydrophobic interaction. By
ods with a longitudinal resonance wavelength of 750–850 nm and tuning the chain length of alkyl silane, the highest catalytic activity
Aeropyrum pernix glucokinase (Glk) as the modal enzyme. By was obtained with trimethoxyl octadecyl silane (C18) modified
illuminating TE–PGNs with a continuous laser (λ ¼ 800 nm), nanoparticles, and 65% of the activity was retained after 7 recycles
photothermal heat was generated at the surface of the gold due to the easy separation under a magnetic field. Covalent bonding
nanorods. A calcium alginate matrix was used to encapsulate the using bi-functional agents as cross-linkers is common in MNPs-based
TE–PGNs to retain the heat around the TE–PGNs. As a result, enzyme immobilization (Alex et al., 2014; Kuo et al., 2012; Li et al.,
thermophilic Glk was activated, and the reaction rate increased 2007). Alex et al. (2014) modified MNPs with 3-aminopropyl triethoxy
60% compared with free enzyme (Fig. 1). silane (APTES) to change hydroxyl surface into amino groups. Then,
Moreover, the excellent conductivity and biocompatibility make enzymes were bound to MNPs through double Schiff base reactions
AuNPs an attractive material for biosensors. AuNPs-based enzyme between MNPs and glutaraldehyde and between glutaraldehyde and
electrodes can be prepared by immobilizing redox enzymes on AuNPs enzymes. Kuo et al. (2012) covalently immobilized lipase on Fe3O4–
and directly depositing them on the bulk electrode surface (Saxena et chitosan nanoparticles using N-(3-dimethylaminopropyl)-N'-ethylcar-
al., 2011). Horseradish peroxidase (Li et al., 2010), hydrogen peroxidase bodiimide (EDC) and N-hydroxysuccinimide (NHS) as cross-linkers.
(Li et al., 2012) and cholesterol oxidase (Saxena et al., 2011) have been After 20 cycles, the immobilized lipase retained over 83% of its original
immobilized onto AuNPs to provide an environment to improve the activity.
electrocatalytic activity and enhance electron transfer from enzymatic Based on the easy separation of magnetic nanoparticles, Le Nel
redox reactions to the electrode surfaces. et al. (2008) developed a microfluidic chip to realize online tryptic

Fig. 2. The microfluidic chip for online tryptic digestion and ESI-MS (adapted from Le Nel et al. (2008)). The experimental setup (A), the microdevice (B) and the diagram (C).
24 M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32

Fig. 3. The hybrid graphite film–carbon nanotube platform (A). SEM images of the top (B) and side (C) view of an as-prepared CNT array. Schematic of the enzyme protection
against microbiological attack (D). SEM images of the B. subtilis on the surface of the nanotube pattern (E) (adapted from Kondyurin et al. (2013)).

digestion and ESI-MS determination. Trypsin was covalently forest, the enzymes were defended from microbial attack and shear
immobilized onto magnetic nanoparticles and introduced into a forces without impeding the flow of reactants and products (Fig. 3).
microchannel. Two permanent magnets were placed to trap the CNTs-immobilized enzymes play an important role in electroche-
trypsin-magnetic particles remaining around the microchannel. mical sensors due to the enhanced electronic properties, large edge
When protein solution was pumped through the microchannel, it plane/basal plane ratio and rapid electrode kinetics (Vashist et al.,
was digested by the magnetic nanoparticles-bound trypsin in a 2011; Yang et al., 2015). Redox enzymes, such as glucose oxidase
short time, and the resulting peptide fragments were separated (Palanisamy et al., 2014; Zhu et al., 2013b), horseradish peroxidase
from the enzymes and delivered by a stream flowing directly to a (Gao et al., 2011; Periasarny et al., 2011), cellobiose dehydrogenases
mass spectrometer (Fig. 2). (Tasca et al., 2013), superoxide dismutase (Tian et al., 2002; Zhu et al.,
2015) and laccase (Bourourou et al., 2013) are often deposited on
electrode surfaces with modified CNTs through adsorption or covalent
2.3. Carbon-based scaffold attachment. This combination has contributed to the rapid develop-
ment of enzyme-based sensors with higher sensitivity and selectivity.
Carbon-based nanomaterials, such as carbon nanotubes (CNTs),
graphene and graphene oxide (GO), with extraordinary mechan- 2.4. Ordered mesoporous nanomaterials
ical, electrical, thermal and biocompatible properties are excellent
materials in biosensors, gene delivery vectors and drug delivery Ordered mesoporous silicas, such as SBA-15, MCM-41 and MCF,
(Singh et al., 2005; Sun et al., 2014a; Vashist et al., 2011; Zhou et have attracted particular interest in enzyme immobilization
al., 2014). Enzymes immobilized on carbon-based nanomaterials because of their large specific surface area, controlled porosity,
allow researchers to take advantage of the mechanical strength, high mechanical strength and ease of modification (Lin et al.,
conductivity or functionalization properties of carbon-based nano- 2014a; Zhou and Hartmann, 2013).
materials to improve enzyme behavior (Feng and Ji, 2011). Jun et al. (2012) immobilized lipase into a meso-structured
Pavlidis et al. (2012) reported the immobilization of several onion-like silica (Meso-Onion-S) and used it as a nanoscale enzyme
recombinant hydrolytic enzymes of significant industrial interest on reactor (NER). Meso-Onion-S has a 200–300 nm sized primary
amine-functionalized multi-wall CNTs and GO. Biochemical and meso-structured onion building unit, and each onion unit has
structural studies indicated that the immobilization approach and highly curved mesopores with a 10 nm diameter in a multishell
the curvature of the nanomaterials significantly affect the immobiliza- structure. Cross-linking was performed after absorbing enzymes
tion efficiency, activity, secondary structure and the operational into pores, which resulted in improvement of enzyme loading
stability of immobilized enzymes. Kondyurin et al. (2013) developed (3373.4%) and stability due to effective protection of the enzyme
a novel platform consisting of a multilayered silicon/silicon oxide/Fe from leaking under rigorous shaking (Fig. 4). The specific activity of
substrate, an activated graphite-like carbon film and a dense forest of NER-lipase was 23 and 10 times higher than free lipase and
vertically aligned multiwall carbon nanotubes through chemical vapor adsorbed lipase in transesterification reactions.
deposition. Horseradish peroxidase and catalase were covalently The catalytic properties of enzymes, such as the conversion
immobilized on the activated carbon film underneath the nanotubes. rate, selectivity, stability and recoverability, can be enhanced by
Because bacteria cannot penetrate into the tall densely packed CNTs ionic liquids (ILs). Huang's group (Yang et al., 2013a; Zou et al.,
M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32 25

Fig. 4. Preparation of nanoscale enzyme reactor (NER) in Meso-Onion-S and the enzyme leaching of adsorbed enzymes (ADS) under rigorous shaking (200 rpm) but not with
NER (adapted from Lee et al. (2014b)).

Fig. 5. Enzyme–inorganic hybrid nanoflowers (adapted from Ge et al. (2012)). The proposed hybrid mechanism where yellow spheres indicate the protein molecules (A). The
SEM images of the α-lactalbumin, laccase, carbonic anhydrase and lipase nanoflowers (column 1–4) under the protein concentrations of 0.5, 0.1 and 0.02 mg/mL (row 1–3)
(B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
26 M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32

2013) developed a strategy to graft functionalized ILs onto a SBA- flower-like copper phosphate nanocrystals gradually grew from
15 surface to immobilize enzymes through ion interactions or these nucleation sites with enzyme proteins serving as “glue” to
covalent bonding. Compared with plain SBA-15, ILs modified SBA- bind the petals together (Fig. 5). Compared with free enzyme,
15 had significantly promoted enzymatic properties, including enzyme–inorganic hybrid nanoflowers showed 650% increased
activity, thermal stability, reusability and storage stability. Lee activity for laccase in the oxidization reaction of syringaldazine
et al. (2014b) developed enzyme-/chemo-catalysts based on and  260% increased activity for carbonic anhydrase in the
mesoporous silica nanoparticles (MSN) for the cascading conver- hydration of CO2. To reveal the hyperactive mechanism of
sion of glucose into 5-hydroxymethylfurfural (5-HMF). Fe3O4 enzyme–inorganic hybrid nanoflowers, Wang et al. (2013a)
nanoparticles (20 nm) were added to the precursor to form designed three α-amylase–CaHPO4 hybrid nanocrystals with
Fe3O4-loaded MSN (Fe3O4@MSN) with a final particle size of nanoflower, nanoplate and parallel hexahedron structures. Both
500–600 nm and a pore size of approximately 20 nm. Then, they the nanostructure morphology and the allosteric effect between
used Fe3O4@MSN as the host for adsorbing cellulase and glucose Ca2 þ and the amine group of the enzyme notably influence the
isomerase to form cellulose–Fe3O4@MSN and isomerase– catalytic efficiency. Enzymes immobilized on nanoflowers are
Fe3O4@MSN. A maximum fructose yield of 51% was achieved more likely to contact substrate molecules due to the lower
under the cascading catalysis (cellulose-to-glucose-to-fructose) mass-transfer limitation and larger enzyme loading based on the
in aqueous media by cellulose–Fe3O4@MSN and isomerase– high surface area of nanoflowers.
Fe3O4@MSN. Due to the high stability and easy separation of Moreover, enzyme–inorganic hybrid nanoflowers have been
Fe3O4@MSN, the biocatalysts can be withdrawn at the end of each applied to bioanalysis (Li et al., 2014; Lin et al., 2014b; Sun et al.,
reaction step, followed by chemical conversion of fructose into 5- 2014b; Zhu et al., 2013c). Zhu et al. (2013c) used laccase–inorganic
HMF with HSO3-MSN in organic media. hybrid nanoflowers in the rapid detection of phenol through
catalyzing the generation of visible products that are convenient
2.5. Enzyme–inorganic hybrid nanoflowers for either quantitative measurement or direct observation. Sun
et al. (2014b) synthesized multi-enzyme–inorganic hybrid nano-
Hyperactive enzyme–inorganic hybrid nanoflowers is a novel flowers that included both glucose oxidase and peroxidase and
and simple enzyme stabilization method created by Ge et al. used them for the detection of glucose. Due to the close orienta-
(2012). In phosphate buffered saline (PBS) containing enzyme, tion of enzymes in the nanostructure, the two-enzyme cascade
primary crystals of copper phosphate (Cu3(PO4)2  3H2O) were reaction was conducted in one step to effectively convert glucose
formed as nucleation sites after the addition of Cu2 þ . As a result, into a chromogenic product.

Fig. 6. The self-assembly of BAmoc-peptides hydrogel and H2O2-triggered hydrogel degradation (A). Schematic representation of hybrid hydrogel responsive to various
analytes (B) (adapted from Ikeda et al. (2014)).
M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32 27

Fig. 7. The enzymogel nanoparticle (A) and the cryo-TEM images of the particle with silica core and swollen/shrunken poly(acrylic acid) brush at pH 7 and pH 4.5 (B–D).
Degradation of a 500 μm thick cotton floss with free cellulase solution for 14 h (E, F), enzymogel for 2.5 h (G, H) and with magnetic field-directed deposition for 1 h (I, J)
(adapted from Kudina et al. (2014)).

3. Organic nanomaterial as carrier to eight times) in the oxidation of pyrogallol in organic media
compared to unconfined methemoglobin in water. The improved
3.1. Peptide-based scaffold activity results from hydrophilic hydrogels promoting pyrogallol
across the microinterface and entering the hydrogel, similar to that
Peptide-based supramolecular hydrogels, formed by self- of reversed micelles. Moreover, the amphiphilic molecular structure
assembly of amphiphilic peptides, have served as scaffolds for facilitates the approach of substrate to enzyme and the release of
tissue engineering, the matrix for biomineralization, and as product. To prevent enzymes from leaking, Hickling et al. (2014)
biomaterials for wound healing (He et al., 2014; Huang et al., covalently attached octopeptides (VKVKVEVK) to pentaerythritol
2010; Khadka and Haynie, 2012). Due to the amphiphilicity, tetranitrate reductase (PETNR). Two synthesis strategies were
adjustability and biocompatibility of peptides, peptide-based explored for the preparation of peptide–enzyme conjugates: chemical
hydrogels are an attractive matrix for enzyme immobilization coupling (SPep-PETNR) of the peptide to the enzyme via click
(Gao et al., 2010; Huang et al., 2014). chemistry and genetic expression (CPep-PETNR) of the full peptide–
Wang et al. (2007) encapsulated methemoglobin into peptide enzyme conjugate. A small portion of peptide–enzyme conjugates
hydrogels via self-assembling of Fmoc-L-lysine and Fmoc-L-phenyla- were doped with pure VKVKVEVK peptide, where the peptide
lanine. The encapsulated methemoglobin exhibited higher activity (up components contribute to the formation of β-sheet fibers through
28 M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32

self-assembly. As a result, PETNR was steadily immobilized on the include pH, ions and specific molecular recognition (Kang et al., 2014;
fiber surface. Zhou et al., 2013). With the help of stimuli-responsive materials,
Ikeda et al. (2014) developed a redox-responsive peptide-based enzymes can be absorbed or released at a certain time during a
hydrogel by encapsulating redox enzyme into a H2O2-responsive reaction, which improves catalytic performance, stability and reusa-
peptide matrix. The hydrogel was composed of di- and tri-peptides bility of the enzyme.
that contained an H2O2-reactive boronoarylmethoxycarbonyl Gan et al. (2012) developed a temperature-sensitive cross-linked
(BAmoc) group at their N terminus. Once the enzymatic oxidation/ gelatin nanoparticles (CLGNs) immobilized enzyme. CLGNs were
elimination occurred, the generated H2O2 triggered the removal of prepared by coacervation, and glucoamylase was physically immobi-
BAmoc, which induced destabilization of the nanofibers and collapse lized onto CLGNs. Because the phase transition temperature of CLGNs
of the hydrogel. This responsive hydrogel can be used as a biomarker is 40 1C, the release of glucoamylase can be controlled when the
for responding to disease-related molecules, such as glucose, lactose, system temperature is above 40 1C. As most enzymes that offer high
uric acid, choline, acetylcholine and sarcosine (Fig. 6). For example, catalytic performances in vivo display limited activities in organic
glucose molecules (input) can be recognized by the corresponding solvents, Zhu et al. (2013a) developed temperature-responsive poly-
glucose oxidase and converted into H2O2, which causes morpholo- mer nanoconjugates by covalently grafting Pluronic onto protein
gical changes to the hydrogel (output). Thus, encapsulation of glucose molecules. This allows solubilization of nanoconjugates in toluene
oxidase in this peptide hydrogel converts the H2O2 response into a with enhanced apparent activity for homogenous catalysis at 40 1C as
glucose response. They built Boolean logic gates (AND and OR) by well as precipitation of nanoconjugates at 4 1C for recovery. Kudina
combining two types of enzymes, for example, nitroreductase and et al. (2014) designed core–shell structural enzymogel nanoparticles
choline oxidase, into hydrogel-enzyme hybrid materials, which were with a magnetic core and negatively charged poly(acrylic acid) brush
able to sense multiple specific biochemicals. scaffolds at pH 4–8. Cellulase, with a typical isoelectric of pH 4.9, can
be loaded onto the brush via electrostatic interaction at pH 4.5. By
3.2. Stimuli-responsive polymer nanomaterials adjusting the cellulose solution pH to 7, cellulase was released from
the polymer brush and maintained its activity as a free enzyme. The
Stimuli-responsive polymer nanomaterials, which respond to free enzyme can be subsequently reloaded onto the enzymogel
external stimulus changes, have attracted substantial attention in nanoparticles by adjusting the pH to 4 and can be recovered under
drug delivery (Gupta et al., 2002; Roy et al., 2010; Schmaljohann, a magnetic field. Furthermore, the enzyme in the brush exhibited
2006; Stuart et al., 2010). Many polymer materials have been designed high activity in the hydrolysis of cellulose fibers, which makes it a
to respond to physical or chemical/biochemical stimuli. Physical new type of phase boundary catalysis. The enzymogel nanoparticles
stimuli include temperature, solvent composition, light, pressure, that adsorbed on the surface of cellulose fibers can cleave cellulose
electric fields and magnetic fields (Baumann et al., 2013; Katsuno chains with free cellulase shuttling between the polymer brush
et al., 2013; Rauch et al., 2012), whereas chemical/biochemical stimuli interior and the brush–cellulose interface (Fig. 7).

Fig. 8. The synthesis and cellular uptake of cationic single-protein nanocapsules with degradable and non-degradable polymeric shell (A). TEM (B) and AFM (C) images of
HRP nanocapsules. The formation of single-core nanoscale architecture (D) (adapted from Yan et al. (2010)).
M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32 29

3.3. Single enzyme nanoparticles/nanogels enzyme, crystalline enzyme and physically aggregated enzyme, result-
ing in formation of cross-linked enzymes (CLEs), cross-linked enzyme
Single enzyme nanoparticles (Hegedus and Nagy, 2009; Kim and crystals (CLECs), and cross-linked enzyme aggregates (CLEAs) (Cao
Grate, 2003; Kim et al., 2006) and single enzyme nanogels were deve- et al., 2003; Roessl et al., 2010). CLEAs, as a nano-scaled biocatalyst,
loped to stabilize enzymes by encapsulating single enzyme mole- have attracted attention in recent years due to their comparable
cules within a polymer network. The sizes of single enzyme nano- activities to native enzymes, easier preparation and lower cost. The
particles/nanogels are usually several to several tens of nanometers preparation of CLEAs consists of two steps: precipitation and cross-
with very thin polymer layers, which provide higher surface area and linking. Firstly, soluble enzymes are precipitated into enzyme aggre-
less mass transfer limitation to the substrate. gates by adding water miscible organic solvents, salts or nonionic
Single enzyme nanoparticles were first presented by Kim (Kim and polymers. Secondly, the formed aggregates are cross-linked by a
Grate, 2003; Kim et al., 2006). The preparation involves enzyme bifunctional agent to produce CLEAs (Talekar et al., 2013).
surface modification, vinyl polymer growth from the enzyme surface, Because most CLEAs are prepared in batch processes, the particle
and a silanol condensation reaction to form a polymer network size of CLEAs and the cross-linking degree may vary widely, which
around each enzyme molecule. Later, Yan et al. (2010, 2006) devel- will affect the enzyme activity and reaction kinetics (Nguyen and
oped a versatile procedure to synthesize single enzyme/protein Yang, 2014). Chen et al. (2006) synthesized CLEAs in CO2-expanded
nanogels. After a mild modification with N-acryloxysuccinimide to micellar solutions. By changing the water-to-surfactant ratio (w0) and
generate vinyl groups on the enzyme surface, in-situ polymerization the concentration of enzyme in the reverse micellar solution, dendritic
at room temperature was conducted in aqueous solution with CLEAs can be tuned to 7–38 nm in diameter and exhibit 200% activity
monomers and cross-linkers. By using glycerol dimethacrylate as the compared with conventional CLEAs. Nguyen and Yang (2014) applied
degradable cross-linker, a delivery platform was obtained with a Y-shape millifluidic reactors to prepare uniform-sized cross-linked
nanocapsules consisting of a protein core and a thin permeable cellulase aggregate by precisely controlling the mixing pattern and
polymeric shell. This structure not only resisted protein against spatial distribution of reactants. By tuning the parameters of the
protease but also enabled the collapse of the shell once inside and precipitant concentration, glutaraldehyde concentration and flow
the release of protein from the nanocapsule (Yan et al., 2010) (Fig. 8). rates, the particle size of CLEAs can be controlled between 200 and
Additionally, Wang et al. (2013c) reported that the hydrophilic envi- 400 nm.
ronment contributed by the polyacrylamide gel network can faci- Another problem of CLEAs in practical application is internal mass-
litate enzyme catalysis in organic media. They presented a sub- transfer limitation, which result from its intrinsic supramolecular
strate-imprinted lipase nanogel by first encapsulating lipase into structure. This results in an accessibility limit when the substrates
polyacrylamide nanogel, then lyophilizing the nanogel with palmitic are macromolecules, such as proteins and polysaccharides. To over-
acid, a substrate of lipase, and finally extracting palmitic acid from the come this problem, Wang et al. developed two strategies to construct
nanogel by petroleum ether. Compared with native lipase, substrate- porous CLEAs (Fig. 9) (Wang et al., 2011; Zhen et al., 2013). One
imprinted lipase nanogel showed 200% activity in the transesterifica- involves co-precipitating enzyme and starch, cross-linking and
tion reaction between paranitrophenyl palmitate and ethanol in n- removal of starch by amylase (Wang et al., 2011). Starch acts as the
heptane. Novel piezoelectric biosensor was also fabricated based on pore-making agent, which cannot be cross-linked, and can be easily
single urease nanoparticles by immobilizing them onto nanoporous removed via the hydrolysis of amylase. The other strategy uses
alumina membranes for quantitative urea determination (Yang and multi-aldehyde macromolecules (dextran polyaldehyde or dialdehyde
Zhang, 2013). starch) as cross-linkers instead of glutaraldehyde. CLEAs prepared
using linear dextran polyaldehyde (MW¼ 2000 kDa) presented a
porous structure with low steric hindrance and thus exhibited
4. Carrier-free immobilized enzyme excellent activity toward macromolecular substrates (Zhen et al.,
2013). Both strategies exhibited similar porous structures in CLEAs,
Unlike carrier-bound enzyme immobilization, the carrier-free which provided enhanced catalytic efficiencies on proteins (BSA and
immobilized enzyme is composed of pure proteins without extra ovalbumin) and polysaccharides (carob bean gum).
inactive mass as carrier, resulting in higher specific activity (Sheldon, Due to the high stability of CLEAs, CLEAs of trypsin (CLEAs-T) were
2011a, 2011b). The carrier-free immobilized enzymes are prepared by successfully applied in rapid protein digestion for proteomics analysis
directly cross-linking different enzyme preparations, such as dissolved (Wang et al., 2013b). Unlike other immobilized enzymes, it is

Fig. 9. Two strategies to prepare pouous CLEAs. Using starch as pore-making agent (A) and the structure of resulting p-CLEAs (B). Using multi-aldehyde macromolecule
(C) as cross-linker. The structure of p-CLEAs cross-linked by dextran polyaldehyde (D) and dialdehyde starch (E). (Adapted from Wang et al. (2011) and Zhen et al. (2013).)
30 M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32

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MS measurement. Compared with conventional in-solution digestion anthraquinone for direct electron reduction of oxygen. Chem.—Eur. J. 19,
9371–9375.
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Science Foundation of Tianjin (13JCQNJC09300) and the Beiyang and their technological applications. Prog. Chem. 22, 2328–2337.
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