Recyclable cellulose-containing magnetic nanoparticles: Immobilization of cellulose-binding module-tagged proteins and a synthetic metabolon featuring substrate channeling

Article abstract of DOI:10.1039/c3tb20482k

Easily recyclable cellulose-containing magnetic nanoparticles were developed for immobilizing family 3 cellulose-binding module (CBM)-tagged enzymes/proteins and a self-assembled three-enzyme complex called the synthetic metabolon. Avicel (microcrystalline cellulose)-containing magnetic nanoparticles (A-MNPs) and two controls of dextran-containing magnetic nanoparticles (D-MNPs) and magnetic nanoparticles (MNPs) were prepared by a solvothermal method. Their adsorption ability was investigated by using CBM-tagged green fluorescence protein and phosphoglucose isomerase. A-MNPs had higher adsorption capacity and tighter binding on CBM-tagged proteins than the two control MNPs because of the high-affinity adsorption of CBM on cellulose. In addition, A-MNPs were used to purify and co-immobilize a three-enzyme metabolon through a CBM-tagged scaffoldin containing three different cohesins. The three-enzyme metabolon comprised of dockerin-containing triosephosphate isomerase, aldolase, and fructose 1,6-bisphosphatase was self-assembled because of the high-affinity interaction between cohesins and dockerins. Thanks to spatial organization of the three-enzyme metabolon on the surface of A-MNPs, the metabolon exhibited a 4.6 times higher initial reaction rate than the non-complexed three-enzyme mixture at the same enzyme loading. These results suggested that the cellulose-containing MNPs were new supports for immobilizing enzymes, which could be selectively recycled or removed from other biocatalysts by a magnetic force, and the use of enzymes immobilized on A-MNPs could be very useful to control the On/Off process in enzymatic cascade reactions. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3tb20482k

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Materials Chemistry B
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Recyclable cellulose-containing magnetic nanoparticles:
immobilization of cellulose-binding module-tagged
proteins and a synthetic metabolon featuring substrate
channeling
Cite this: J. Mater. Chem. B, 2013, 1,
4419
a
abcd
Suwan Myung,ab Chun You and Y.-H. Percival Zhang
*
Easily recyclable cellulose-containing magnetic nanoparticles were developed for immobilizing family 3
cellulose-binding module (CBM)-tagged enzymes/proteins and a self-assembled three-enzyme complex
called the synthetic metabolon. Avicel (microcrystalline cellulose)-containing magnetic nanoparticles (A-
MNPs) and two controls of dextran-containing magnetic nanoparticles (D-MNPs) and magnetic
nanoparticles (MNPs) were prepared by
a solvothermal method. Their adsorption ability was
investigated by using CBM-tagged green fluorescence protein and phosphoglucose isomerase. A-MNPs
had higher adsorption capacity and tighter binding on CBM-tagged proteins than the two control MNPs
because of the high-affinity adsorption of CBM on cellulose. In addition, A-MNPs were used to purify
and co-immobilize a three-enzyme metabolon through a CBM-tagged scaffoldin containing three
different cohesins. The three-enzyme metabolon comprised of dockerin-containing triosephosphate
isomerase, aldolase, and fructose 1,6-bisphosphatase was self-assembled because of the high-affinity
interaction between cohesins and dockerins. Thanks to spatial organization of the three-enzyme
metabolon on the surface of A-MNPs, the metabolon exhibited a 4.6 times higher initial reaction rate
than the non-complexed three-enzyme mixture at the same enzyme loading. These results suggested
that the cellulose-containing MNPs were new supports for immobilizing enzymes, which could be
selectively recycled or removed from other biocatalysts by a magnetic force, and the use of enzymes
immobilized on A-MNPs could be very useful to control the On/Off process in enzymatic cascade reactions.
Received 4th April 2013
Accepted 1st July 2013
DOI: 10.1039/c3tb20482k
www.rsc.org/MaterialsB
tags for the purication and immobilization of CBM-tagged
fusion proteins on cellulosic supports because of their high
Introduction
Enzyme immobilization has been widely used for prolonging
the lifetime of immobilized enzymes and separating and recy-
cling immobilized enzymes from soluble substrates and prod-
ucts.1,2 Numerous enzyme immobilization technologies have
been developed, such as physical adsorption,3 covalent
bonding,4,5 encapsulation/entrapment,6 cross-linking enzyme
aggregate (CLEA),7 and so on. The physical adsorption of an
enzyme onto an insoluble support is attractive because it is
simple, quick, cheap, and causes no or little damage to enzyme
activity. However, this technology may suffer from the leakage
of the enzyme from the support, non-specic binding, and
steric hindrance by the support. Cellulose-binding module
(CBM) tags, in particular, family 3, have been applied as affinity
affinity to cellulose, which is less costly, inert, stable, and
biodegradable.8,9 A one-step protein purication and immobi-
lization method has been developed to selectively adsorb CBM-
tagged proteins on cellulose.10,11
Magnetic nanoparticles (MNPs) are widely used in
biotechnological and biomedical applications.12–14 Numerous
synthesis and surface functionalization methods for iron oxide
MNPs have been developed, such as co-precipitation,15 thermal
decomposition,16 hydrothermal synthesis,17 microemulsion,18
and sonochemical synthesis.19 Also, MNPs can be applied to an
easy separation of target materials in a liquid phase reaction.
By using a magnetic force, MNPs are considered as control-
lable carriers for target materials, such as enzymes,20 drugs,21
antibodies,22 and so on. For enzyme immobilization on MNPs,
it is important to functionalize the surface of MNPs for the
selective attachment of target biomolecules. Huang et al.
studied the properties of surface functional groups, biocom-
patibility, and bioapplication of three MNPs prepared using
dextran, chitosan, or polyacrylic acid as surfactants while not
including cellulose.23
aBiological Systems Engineering Department, Virginia Tech, 304 Seitz Hall,
Blacksburg, Virginia 24061, USA. E-mail: ypzhang@vt.edu; Fax: +1-540-231-3199;
Tel: +1-540-231-7414
bInstitute for Critical Technology and Applied Science (ICTAS), Virginia Tech,
Blacksburg, Virginia 24061, USA
cCell Free Bioinnovations Inc., Blacksburg, Virginia 24060, USA
dGate Fuels Inc., Blacksburg, Virginia 24060, USA
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Cell-free biosystems comprised of (non-natural) synthetic cellulose, was purchased from FMC (Philadelphia, PA).
enzymatic pathways can implement complicated biochemical Thermotoga maritima (ATCC 43589) genomic DNA was
reactions that microbes and catalysts cannot do,24,25 for purchased from the American Type Culture Collection (Mana-
example, high-yield hydrogen generation from sugars,26 enzy- ssas, VA). Oligonucleotides were synthesized by Integrated DNA
matic conversion of cellulose to starch.27 For the purpose of Technologies (Coraville, IA). Liquid glucose reagent based on
biomanufacturing, these cell-free biosystems could be used to hexokinase/glucose-6-phosphate dehydrogenase was purchased
produce high-yield hydrogen,28–30 alcohols,31 organic acids,32 jet from Pointe Scientic Inc. (Canton, MI). Escherichia coli BL21
fuel,33 proteins,34 electricity,35,36 ne chemicals,37 saccharide Star (DE3) (Invitrogen, Carlsbad, CA) containing a protein
drugs,38 and even to x CO2.39,40 Cell-free biomanufacturing expression plasmid was used for producing the desired
could be economically competitive with microbial fermentation recombinant protein. Luria–Bertani (LB) medium supple-
for the production of biocommodities only when all of the mented with 100 mg mLꢀ1 ampicillin or 50 mg mLꢀ1 kanamycin
enzymes in systems have high total turn-over numbers and low- was used for E. coli cell growth and recombinant protein
cost bulk enzyme production and purication are available.25 expression.
However, it is impossible that all enzymes in cell-free bio-
systems have the same lifetime because their turn-over Plasmid construction
numbers oen vary by several orders of magnitude. It could be
too costly to eliminate all enzymes when only a fraction of them
The plasmids used are summarized in Table 1. Plasmid pNT02
encoding the thioredoxin-GFP-CBM (TGC) fusion protein was
with a short lifetime is deactivated. Therefore, it is essential to
described elsewhere.53 Plasmid pET20b-tim has an expression
selectively remove deactivated enzymes or re-use active enzymes
cassette containing the T. maritima tim gene.33 Plasmid pET28a-
ald whose expression cassette contains T. maritima ald gene was
from other enzymes.
Spatial organization of cascade enzymes could greatly
described elsewhere.54 The pCIF plasmid encoding the CBM-
accelerate reaction rates.41,42 This phenomenon is called
intein-FBP fusion protein45 and pCIP plasmid encoding the
substrate channeling, a process of transferring the product
CBM-intein-PGI fusion protein10 were described elsewhere.
catalyzed by one enzyme as the substrate to the adjacent
pET20b-mini-scaf, pET20b-tim-ctdoc, pET20b-ald-ccdoc, and
pET20b-p-rfdoc made by simple cloning55 were described
cascade enzyme without fully equilibrating the bulk phase.41
For example, the optimized distance between the two enzymes
elsewhere.51
controlled by DNA scaffolds results in over 20-fold improvement
of the reaction rate compared to the free enzyme mixture.43
Recombinant protein expression and purication
Enzyme complexes on specic DNA origami tiles with the
controlled inter-enzyme spacing and position enhanced the
activity more than 15 times higher than the free enzyme
mixture.44 In addition to facilitating reaction rates, synthetic
enzyme complexes called metabolons may avoid the degrada-
tion of labile metabolites.41,45
As natural cellulase complexes called cellulosomes were
formed through the high-affinity interaction between cohesins
and dockerins, Bayer et al.46 proposed to construct designed
enzyme complexes by utilizing species-specic dockerins and
cohesins, where they can bind tightly at a molar ratio of 1 : 1.
Later, a few synthetic mini-cellulosomes containing various
extracellular glycoside hydrolases were constructed.47–50 Few
efforts were made for constructing synthetic enzyme complexes
containing cascade enzymes from a metabolic pathway by using
dockerins and cohesins.51,52
For the preparation of recombinant proteins: TIM and ALD, two
hundred milliliters of the LB culture containing 50 mg mLꢀ1 of
kanamycin was incubated in 1 L Erlenmeyer asks with a rotary
ꢁ
shaking rate of 250 rpm at 37 C. When the absorbance (A600
)
reached ca. 1.2, the recombinant protein expression was
induced by adding isopropyl beta-D-1-thiogalactopyranoside
(IPTG, 0.1 mM, nal concentration). The culture was induced at
37 ꢁC for 4 h. The cells were harvested by centrifugation at 4 ꢁC,
washed twice with 50 mM Tris–HCl buffer (pH 7.5), and re-
suspended in 15 mL of 30 mM Tris–HCl buffer (pH 7.5) con-
taining 0.5 M NaCl and 1 mM EDTA. The cell pellets were lysed
using a Fisher Scientic Sonic Dismembrator Model 500 (5 s
pulse on and off, total 360 s, at 20% amplitude) in an ice bath.
Aer centrifugation, the target proteins (TIM and ALD) were
puried through heat treatment at 60 ꢁC for 20 min followed by
gradient ammonium sulfate precipitation.56 The expression and
purication of tag-free FBP,45 PGI and CBM-PGI10 were
described elsewhere.56 The expression and purication condi-
tions of mini-scaffoldin, TIM-CtDoc, ALD-CcDoc, and FBP-
RfDoc were described elsewhere.51 The TGC protein containing
thioredoxin, green uorescent protein, and cellulose-biding
module was puried as described elsewhere.53
In this paper, we prepared cellulose-containing MNPs for the
immobilization of CBM-tagged proteins and
a synthetic
metabolon for the rst time. Avicel-containing MNPs (A-MNPs),
dextran-containing MNPs (D-MNPs), and MNPs were prepared
by a solvothermal method. Also substrate channeling of the
synthetic metabolon on the surface of A-MNPs was investigated.
Materials and methods
Preparation of MNPs
Chemicals and strains
A-MNPs and D-MNPs were synthesized by the solvothermal
All chemicals were of reagent grade, purchased from Sigma- method.19,23,57 Briey, 0.338 g of iron chloride (FeCl3$6H2O,
Aldrich (St. Louis, MO) and Fisher Scientic (Pittsburgh, PA), 1.25 mmol) was completely dissolved in 10 mL of ethylene glycol
unless otherwise noted. Avicel PH105, microcrystalline to yield a clear yellow solution, followed by the addition of 1.36 g
4420 | J. Mater. Chem. B, 2013, 1, 4419–4427
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Table 1 Plasmids and purification methods
Puried protein and purication
Plasmid
pNT02
Characteristics
methods
Ref.
53
AmpR, gfp-cbm expression cassette
cloned, green uorescence protein
(gfp) gene, cellulose-binding
module (cbm) gene from
GFP-CBM (TGC), bio-specic
adsorption of CBM-tagged GFP on
RAC followed by ethylene glycol
elusion
C. thermocellum
pCP
AmpR, cbm-pgi expression cassette
cloned, cbm gene from
CBM-PGI, bio-specic adsorption of
CBM on RAC followed by ethylene
glycol elusion
10
51
C. thermocellum, pgi gene from
C. thermocellum
pET33b-tim
KanR, tim expression cassette
cloned, tim gene from
TIM, heat treatment and
ammonium sulfate precipitation
T. thermophilus
pET20a-ald
pCIF
KanR, ald expression cassette
cloned, ald gene from T. maritima
AmpR, cbm-intein-p expression
cassette cloned, p gene from
T. maritima
ALD, heat treatment and
33
51
ammonium sulfate precipitation
FBP, bio-specic adsorption of CBM
tagged intein-FBP on RAC followed
by intein self-cleavage
PGI, bio-specic adsorption of CBM
tagged intein-PGI on RAC followed
by intein self-cleavage
CBM-CtCoh-CcCoh-RfCoh (mini-
scaf), bio-specic adsorption of
CBM on RAC followed by ethylene
glycol elusion
pCIP
AmpR, cbm-intein-pgi expression
cassette cloned, pgi gene from
T. maritima
45
10
pET20b-mini-scaf
AmpR, mini-scaffoldin expression
cassette cloned, containing a CBM
module from C. thermocellum and
three different cohesins from
C. thermocellum, C. cellulovorans
and R. avefaciens
pET20b-tim-ctdoc
pET20b-ald-ccdoc
pET20b-p-rfdoc
AmpR, tim-ctdoc expression cassette
cloned, tim gene from
TIM-CtDoc, bio-affinity interaction
between CtDoc and mini-scaf
followed by CBM adsorption on RAC
and ethylene glycol elusion
ALD-CcDoc, bio-affinity interaction
between CcDoc and mini-scaf
followed by CBM adsorption on RAC
and ethylene glycol elusion
FBP-RfDoc, bio-affinity interaction
between RfDoc and mini-scaf
followed by CBM adsorption on RAC
and ethylene glycol elusion
51
51
51
T. thermophiles, the C. thermocellum
dockerin module
AmpR, ald-ccdoc expression cassette
cloned, ald gene from T. maritime,
the C. cellulovorans dockerin
module
AmpR, p-rfdoc expression cassette
cloned, p gene from T. maritime
FBP, the R. avefaciens dockerin
module
of sodium acetate (NaAc$3H20, 10 mmol) and 0.125 of Avicel or room temperature for 10 min. The protein mass concentration
dextran. The mixture was stirred vigorously for 30 min. The of the unbound protein and total protein was measured by the
mixture was sealed in a pressure container and then heated at Bradford assay. The maximum adsorption capacity of MNPs was
200 ꢁC for 12 h. The container was cooled down slowly at room calculated following the Langmuir isotherm.53
temperature. The products collected by a magnet were washed
The leakage of adsorbed CBM-PGI on MNPs was investigated
with ethanol and then dried at 60 ꢁC for 6 h. The MNPs without as follows. The adsorbed CBM-PGI on MNPs was mixed by
cellulose were synthesized as described above.
vortexing for 3 seconds in a 100 mM HEPES buffer (pH 7.5) at
room temperature. Aer MNPs were removed by using a
magnet, the supernatant was decanted. The MNPs were re-
suspended with a fresh HEPES buffer at a v/v ratio of 50. These
washing steps were repeated several times. A small amount of
the re-suspended MNPs containing adsorbed enzyme was
withdrawn for the PGI activity assay.
TEM and SEM of MNPs
Morphology and structure images of MNPs were examined with
a FEI TITAN 300 eld emission analytical electron microscope
(FEI, Hillsboro, OR) and the LEO 1550 as a high-performance
Schottky eld-emission scanning electron microscope (Carl
Zeiss Microscopy, Jena, Germany).
Enzyme activity assays
Adsorption of TGC or CBM-PGI on three MNPs
The activity assay of enzymes was conducted based on the
For determining the maximum adsorption capacity of MNPs, initial reaction velocity. For the TIM assay, glyceraldehyde-3-
the crude cell lysate containing TGC was mixed with MNPs at phosphate (G3P) was the substrate and dihydroxyacetone
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phosphate (DHAP) was the product. DHAP was measured by formation of nanocrystal particles.23,57 In this study, insoluble
using glycerol 3-phophate dehydrogenase (GPDH) in the Avicel (microcrystalline nano-cellulose) powders with an
presence of NADH and the consumption of NADH was average aggregated particle size of 20 mm were used instead
measured at 340 nm. Because thermophilic glycerol-3-pho- of water-soluble dextran. Under optimized preparation
phate dehydrogenase was not available and NADH was not conditions, the weights of A-MNPs, MNPs and D-MNPs were
stable at high temperatures, thermophilic TIM activity was 97.5 ꢃ 0.6, 94.4 ꢃ 0.6 and 95.1 ꢃ 0.8 mg, respectively, from
measured by using a discontinuous means. Specically, the 335 mg of FeCl3 (1.25 mmol). A-MNPs, D-MNPs, and MNPs
generation of DHAP by TIM was measured on 2 mM G3P in were characterized by using SEM (Fig. 1a, c and e) and TEM
100 mM HEPES buffeꢁr (pH 7.5) containing 10 mM MgCl2 and (Fig. 1b, d and f). D-MNPs and MNPs had typical spherical
0.5 mM MnCl2 at 60 C. The reaction was stopped by adding shapes, as described elsewhere.23,57 The particle sizes of A-
5.8 M HClO4 (nal, 0.65 M) in an ice-water bath for 5 min MNPs (Fig. 1a & b) were a little larger and they exhibited
followed by the addition of 5 M KOH until pH ꢂ 7 (ref. 51). broader size distribution compared to MNPs (Fig. 1c & d) and
Aer centrifugation, the supernatant was mixed with 0.2 mM D-MNPs (Fig. 1e & f). Their average sizes were ꢂ600 nm for A-
NADH and GPDH. The consumption of NADH was measured MNPs, ꢂ300 nm for MNPs, and ꢂ300 nm for D-MNPs (Fig. 1).
at 340 nm.
Small cracks/holes were observed on the surface of A-MNPs
The ALD activity was measured by a continuous cascade and D-MNPs while MNPs have a smooth surface. These
reaction along with sufficient TIM, FBP, and PGI.51 G3P and results implied that polymers (Avicel or dextran) were present
DHAP were substrates and F16P was the product. Aer the at both the surface and the interior of the particles, which
cascade reaction, the reaction was stopped by the addition of was reported in the cases of dextran, chitosan, or polyacrylic
HClO4.51 The nal product of G6P was measured by the Pointe acid as surfactants.23
Scientic liquid enzymatic glucose kit at 37 ꢁC. The absorbance
was read at 340 mM with a reference of the blank ALD solution.
FBP and PGI activities were measured as described
elsewhere.10,45
Protein adsorption and immobilization on MNPs
The protein adsorption proles on MNPs were studied based on
For the 3-enzyme cascade reaction assay, G3P was the CBM-tagged proteins: thioredoxin-GFP-CBM protein (TGC)53
substrate and F6P was the product. The product F6P can be and CBM-tagged phosphoglucose isomerase (PGI).10 It is well-
measured by the liquid glucose reagent kit supplemented with known that family 3 CBM tags can be bound on the surface of
PGI at 37 ꢁC for 3 min. Specically, the generation of F6P can be cellulose through biospecic adsorption (Fig. 2a & c).11 The
done by using cascade enzymes with 2.5 mM of G3P in 200 mM green aqueous solution containing TGC turned colorless aer
HEPES buffer (pH 7.5) containing 10 mM MgCl2, 0.5 mM A-MNPs were added and a magnet was applied, suggesting that
MnCl2, 0.5 mM thiamine pyrophosphate and 1 mM CaCl2 at A-MNPs can bind with TGC (Fig. 2b). TGC adsorption by
60 ꢁC. The reaction was stopped by adding HClO4 in an ice- A-NMPs was very fast, reaching equilibrium within 5 min (data
water bath followed by the neutralization of 5 M KOH. Aer not shown).
centrifugation, the product F6P in the supernatant was assayed
by the liquid glucose reagent supplemented with PGI at 37 C MNPs, and D-MNPs. The TGC adsorption proles on MNPs and
The adsorption proles of TGC were examined on A-MNPs,
ꢁ
for 3 min.
D-MNPs obeyed typical Langmuir isotherms due to simple
physical adsorption (Fig. 3). The maximum binding capacity of
MNPs and D-MNPs was 9.6 and 5.4 mg gꢀ1, respectively. In
contrast, the adsorption of TGC onto the surface of A-MNPs
could be attributed to a combination of two adsorption forces:
(1) simple physical adsorption between a protein and the large
surface of MNPs and (2) high-affinity binding between the CBM
tag and cellulosic materials.10,59 The lumped adsorption prole
of TGC on A-MNPs may be tted into a simple Langmuir
isotherm with a relatively large deviation. The maximum
binding capacity of A-MNPs was 13.1 mg gꢀ1, nearly 1.4 and 2.4
times those of MNPs and D-MNPs.
Other assays
The mass concentration of protein was measured using the Bio-
Rad modied Bradford protein kit with bovine serum albumin
as a standard protein.58 12% SDS-PAGE was performed in the
Tris–glycine buffer as described elsewhere.11 The tting curves
of adsorption data in the Langmuir isotherm was obtained with
soware named Curve Expert 1.4 (http://www.curveexpert.net/).
The error bars in the gures represent standard deviations.
Because physically adsorbed proteins on MNPs may be
washed out, the leakage of CBM-PGI immobilized on MNPs was
investigated (Fig. 4). By using the same amounts of MNPs,
Results and discussion
Preparation and characterization of MNPs
Three types of magnetic particles (A-MNPs, D-MNPs, and excess cell lysate of CBM-PGI was mixed with different MNPs
MNPs) were synthesized by using a solvothermal method. (i.e., MNPs were saturated by proteins). The absolute specic
FeCl3, ethylene glycol, and sodium acetate were the source of activities of A-MNPs, MNPs and D-MNPs before the rst
magnetite, a solvent and high boiling point reducing agent, washing were 0.73 ꢃ 0.01, 0.38 ꢃ 0.02, and 0.30 ꢃ 0.01 U mgꢀ1
and an electrostatic stabilizer to prevent particle agglomera- of MNPs, respectively. It was found that the remaining PGI
tion, respectively.23,57 The addition of dextran as a surfactant activity on A-MNPs decreased by only 20% aer 8 washes, while
can prevent particle agglomeration and facilitate the the remaining PGI activities on D-MNPs and MNPs were 34 and
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Fig. 1 Images of different types of MNPs under SEM (a, c, and e) and TEM (b, d, and f). A-MNPs (a & b); MNPs (c & d); and D-MNPs (e & f). The bars indicate 100 nm in
SEM and 500 nm in TEM images, respectively.
46% of initial activities, respectively. This result suggested that MNPs. Similarly, immobilized CBM-tagged PGI on regenerated
CBM-tagged enzymes can be bound more tightly with A-MNPs amorphous cellulose was strong enough to retain enzyme
through the CBM tag than physical adsorption on D-MNPs and activity even aer a number of washes.10
Fig. 2 Scheme of CBM-tagged proteins adsorbed on A-MNPs. (a) TGC adsorbed on A-MNPs, (b) the process of collecting TGC adsorbed on A-MNPs by using a
magnetic field and (c) PGI adsorbed on A-MNPs.
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which could be used for high-yield hydrogen production from
sugars.28,29 TIM catalyzes the reversible conversion of glyceral-
dehyde-3-phosphate (G3P) to dihydroxyacetone phosphate
(DHAP) (eqn (1)). ALD catalyzes the reversible aldol condensa-
tion of G3P and DHAP to fructose 1,6-bisphosphate (F16P) (eqn
(2)). FBP catalyzes the irreversible conversion of F16P to fructose
6-phosphate (F6P) (eqn (3)).
G3P T4IM DHAP
(1)
(2)
(3)
G3P þ DHAP A4LD F16P
F16P þ H O FBP F6P þ P
ƒ!
2
i
Fig. 3 The adsorption profiles of TGC on A-MNPs, MNPs, and D-MNPs. The
curves were fitted by the Langmuir equations.
The immobilization of
a
self-assembled three-enzyme
complex called metabolon containing TIM, ALD and FBP
through a synthetic trifunctional scaffoldin was investigated on
A-MNPs (Fig. 5a). This synthetic metabolon was comprised of a
dockerin-containing Thermus thermophilus triose phosphate
isomerase (TIM), a dockerin-containing Thermotoga maritima
fructose bisphosphate aldolase (ALD), a dockerin-containing
T. maritima fructose bisphosphatase (FBP) and a mini-scaffold
containing a family 3 cellulose-binding module at the N-
terminus followed by three different types of cohesins from the
Clostridium thermocellum CipA, Clostridium cellulovorans CbpA
and Ruminococcus avefaciens ScaB.51 This synthetic three-
enzyme complex was assembled in vitro through the high-
affinity interaction between cohesins and dockerins at a molar
ratio of 1 : 1 : 1 : 1 when the cell extracts containing four
proteins were mixed.
When A-MNPs were applied, the synthetic three-enzyme
complex was adsorbed onto the surface of A-MNPs for their fast
purication and co-immobilization (Fig. 5a). The mini-scaffol-
din, TIM, ALD, and FBP ratio was approximately 1 : 1 : 1 : 1, as
shown in SDS-PAGE gel (Fig. 5b, Lane 1). The activities of
dockerin-containing TIM, ALD, and FBP in the presence of
mini-scaffoldin were similar to those of dockerin-free enzymes
(data not shown), suggesting that the dockerin addition did not
inuence the activity of each enzyme.51
Furthermore, the reaction rates of the synthetic metabolon
immobilized on A-MNPs, the non-immobilized synthetic
metabolon and theꢁfree enzyme mixture were investigated from
2.5 mM G3P at 60 C (Fig. 6). The puried three free enzymes
(Lanes 2, 3, and 4) are shown in Fig. 5. Another puried PGI
(Lane 5) was used to measure fructose-6-phosphate formation.
The metabolon immobilized on A-MNPs had an initial reaction
rate of 0.285 mmol Lꢀ1 s, 4.6 times that of the free enzyme
mixture (i.e., 0.062 mmol Lꢀ1 s). Such a rate enhancement was
attributed to the fact that DHAP generated by TIM can be
rapidly transferred to an adjacent ALD to yield F16P, as reported
previously.51 Also, the immobilized synthetic metabolon
exhibited 1.75 times higher initial reaction rate than that of the
free synthetic metabolon (i.e., 0.163 mmol Lꢀ1 s), which was
possibly due to shorter enzyme–enzyme distances when the
metabolon was immobilized on the surface of the solid adsor-
bent than those in the aqueous solution. A similar observation
Fig. 4 Leakage testing of CBM-PGI immobilized on A-MNPs, MNPs, and D-MNPs.
Remaining activities of immobilized CBM-PGI were measured after a number of
washing steps.
We further investigated the adsorption of CBM-tagged PGI
by A-MNPs, MNPs and D-MNPs. At the same amounts of MNPs
and CBM-PGI, the immobilized PGI on A-MNPs in terms of
enzyme activity was 2.46 times that on D-MNPs and 1.94 times
that on MNPs (data not shown). The above results suggested
that A-MNPs not only had higher enzyme-immobilization
capacity but also retained more enzyme activity compared to D-
MNPs and MNPs. It was mainly due to the bio-specic affinity
interaction between cellulose in MNPs and the CBM tag, which
could decrease the possibility of random non-active adsorption.
Although A-MNPs have larger particle sizes with more binding
capacity than MNPs and D-MNPs, this result suggested that a
signicant fraction of the binding capacity of A-MNPs is
internal, consistent with their porous structure observed in
Fig. 1. Similarly, most of the binding capacity of Avicel is
internal rather than external.53
Synthetic metabolon immobilized on A-MNPs
Triosephosphate isomerase (TIM, EC 5.3.1.1), aldolase (ALD, EC
4.1.2.13), and fructose 1,6-bisphosphatase (FBP, EC3.1.3.11) are
cascade enzymes in the glycolytic and gluconeogenic pathways,
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Fig. 5 (a) Scheme of the cascade reactions catalysed by the multi-enzyme complex including TIM, ALD, and FBP on A-MNPs for the generation of f6p from g3p. (b)
SDS-PAGE analysis of a purified multi-enzyme complex, three single enzymes, and additional enzyme for the product assay. Lane M, marker; Lane 1; enzyme complex
including mini-scaffoldin, TIM-CtDoc, ALD-CcDoc, and FBP-RfDoc, Lane 2; purified TIM, Lane 3; purified ALD, Lane 4; purified FBP; Lane 5 purified PGI.
immobilized enzymes by using a magnetic force. Therefore, it
was highly operative to selectively separate deactivated enzymes
from active enzymes. As a result, it could greatly decrease
enzyme costs in cell-free biosystems. On the other hand,
selective removal of enzymes immobilized on MNPs could be
very effective in stopping cascade enzymatic reactions within a
short time. For example, it will be important to stop and resume
enzymatic hydrogen generation of biotransformers in hypo-
thetical sugar-fuel cell vehicles.40 We envisioned that the
selective removal and addition of some key enzymes immobi-
lized on A-MNPs by a switchable magnetic force could stop and
resume reactions rapidly.
Fig. 6 Comparison of the reaction activities of the enzyme complex immobilized
Conclusions
on A-MNPs, the unbound enzyme complex, and the non-complexed enzyme
mixture at the same enzyme loading.
The cellulose-containing magnetic nanoparticles were
prepared for the immobilization of CBM-tagged proteins or
the metabolons. Although A-MNPs exhibited higher adsorp-
was reported for metabolons immobilized on regenerated
tion capacity, they did not impair the activity of immobilized
amorphous cellulose.11
enzymes, and retained more enzyme activity aer washing,
Whether substrate channelling among synthetic metab-
compared to MNPs and D-MNPs because of the high-affinity
olons comprised of an enzyme cascade was observed or not
binding of CBM tags on A-MNPs. Substrate channeling among
was complicated, depending on a number of factors: enzymes
the synthetic metabolons immobilized on the surface of
used and reaction conditions (e.g., substrate concentration,
A-MNPs could not only increase cascade reaction rates greatly
enzyme loading, temperature, pH, etc.).41,51 In our another
but also decrease enzyme costs in cell-free biosystems. The
study, a two-enzyme complex containing cellobiose phos-
use of enzymes immobilized on A-MNPs could be very useful
phorylase and photo glucan phosphorylase through the same
in controlling the On/Off process in cascade enzymatic
synthetic scaffoldin was constructed and immobilized on
reactions.
A-MNPs. We did not observe any substrate channelling among
adjacent cellobiose phosphorylase and potato glucan
phosphorylase.27
Acknowledgements
Cell-free biosystems comprised of synthetic enzymatic
pathways could become an innovative biomanufacturing plat-
form. It was essential to ensure that all enzymes reached their
maximum total turn-over numbers before their replacement.
Immobilized CBM-tagged enzymes or their complexes on
A-MNPs can be recycled easily from other free enzymes or
This work was supported by the Biological Systems Engineering
Department of Virginia Tech, and partially supported by the
Shell GameChanger Program and the CALS Biodesign and
Bioprocessing Research Center. SM was partially supported by
the ICTAS Scholar Program.
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