Subscriber access provided by CARLETON UNIVERSITY
Article
Stoichiometrically Controlled Immobilization of Multiple Enzymes on Magnetic Nanoparticles by the Magnetosome Display System for Efficient Cellulose Hydrolysis Toru Honda, Tsuyoshi Tanaka, and Tomoko Yoshino Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01174 • Publication Date (Web): 16 Nov 2015 Downloaded from http://pubs.acs.org on November 21, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 23
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
1
2
Stoichiometrically Controlled Immobilization of Multiple Enzymes on Magnetic
3
Nanoparticles by the Magnetosome Display System for Efficient Cellulose Hydrolysis
4
5
Toru Honda, Tsuyoshi Tanaka, and Tomoko Yoshino*
6 7
Division of Biotechnology and Life Science, Institute of Engineering, Tokyo University of
8
Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan
9 10
*Corresponding author: Tomoko Yoshino; Division of Biotechnology and Life Science,
11
Institute of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho,
12
Koganei, Tokyo 184-8588, Japan
13
Telephone: +81-42-388-7021
14
Fax: +81-42-385-7713
15
E-mail:
[email protected] 16 17
Keywords: magnetosome display system; cellulosome; protein complex; magnetic nanoparticle
18 19
ACS Paragon Plus Environment
1
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 23
20
Abstract
21
The immobilization of multiple cellulase complexes receiving attention for use in the efficient
22
hydrolysis of celluloses. In this study, the magnetosome display system was employed for the
23
preparation of systems mimicking natural multiple cellulase complexes (cellulosomes) on
24
magnetic nanoparticles (MNPs). Initially, two fluorescent proteins, namely green fluorescent
25
protein and mCherry, were immobilized on MNPs. Fluorescence analysis revealed the close
26
proximity of two different proteins on the MNPs. Enzyme-linked immunosorbent assay analysis
27
showed that stoichiometrically equivalent amounts of the proteins were immobilized on the
28
MNPs. Next, endoglucanase (EG) and β-glucosidase (BG) were immobilized on MNPs to give
29
EG/BG-MNPs. The resulting MNPs were applied for the hydrolysis of celluloses, with rapid
30
hydrolysis of carboxymethyl cellulose being observed. Furthermore, the fusion of the cellulose-
31
binding domain to EG/BG-MNPs promoted improved hydrolysis activity against the insoluble
32
cellulose. We could therefore conclude that the magnetosome display system can expand the
33
possibilities of mimicking natural cellulosome organization on MNPs.
34 35 36 37 38 39 40
ACS Paragon Plus Environment
2
Page 3 of 23
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
41
1. Introduction
42
Cellulose is the most abundant and promising biomass for bioethanol production. Its hydrolysis
43
by cellulase is key for this process, however, the large consumption of cellulase makes it costly,
44
resulting in a bottleneck in cellulose-based bioethanol production 1. An alternative approach is
45
the utilization of immobilized cellulase on solid materials, as immobilization provides
46
advantages in terms of handling, reusability, and cellulase stability2-6.
47
Although a number of studies have been reported for the preparation of immobilized cellulase,
48
the immobilization of multi-enzyme complexes, known as cellulosomes7, has also attracted
49
considerable attention. Cellulosomes has been discovered in several cellulolytic bacteria, such as
50
Clostridium cellulolyticum, Clostridium thermocellum, and Ruminococcus flavefaciens8. It is
51
composed of a scaffold protein containing a cohesin domain, a cellulose-binding domain (CBD),
52
and several cellulases with the dockerin domain (i.e., endoglucanase, exoglucanase, and β-
53
glucosidase). The dockerin domain can specifically bind to the cohesin domain on the scaffold
54
protein, allowing the construction of highly organized multi-enzyme complexes in close
55
proximity to one another. This close proximity leads to synergistic effects in enzymatic activity,
56
resulting in efficient cellulose hydrolysis9. Recently, to mimic the cellulosome on solid materials,
57
the immobilization of multiple cellulases onto magnetic particles has been attempted10,
58
However, reproduction of the highly organized multiple enzyme assembly was difficult, as the
59
enzymes were randomly immobilized on the magnetic particle surfaces through chemical cross-
60
linking or Au-S bonding.
61
We previously proposed a novel process for the immobilization of recombinant proteins onto
62
magnetic nanoparticles (MNPs) using the magnetosome display system with a magnetotactic
11
.
ACS Paragon Plus Environment
3
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
12
Page 4 of 23
63
bacterium, Magnetospirillum magneticum strain AMB-1
64
in magnetotactic bacteria, consisting of a magnetite core covered by a lipid bilayer membrane,
65
and an average diameter of 75 nm 12 (see also Figure 1(A)). In the magnetosome display system,
66
foreign proteins can be immobilized on magnetosomes using the mms13 gene as a fusion partner
67
by genetic engineering13. Mms13, a protein kwon to double-pass transmembrane protein and
68
tightly bind to the magnetite core. Therefore, M. magneticum AMB-1 was transformed by the
69
fusion gene of mms13 and target protein gene, and then desired target protein can be efficiently
70
expressed (or immobilized) on magnetosomes via Mms13 in M. magneticum. Until now, various
71
proteins, including single-chain variable fragments
72
complex
73
have been successfully immobilized on MNPs using the magnetosome display system. The
74
genetically engineered MNPs can be easily extracted and purified from the M. magneticum
75
AMB-1 transformant cell lysate by magnetic separation. The complexes of foreign proteins with
76
MNPs have been applied to immunoassays, ligand screening assays, and cell separation systems
77
as magnetic carriers18-23. The magnetosome display system can therefore enable us to construct
78
highly organized multi-enzyme complexes on MNPs.
79
We report the preparation of highly organized protein complexes on MNPs using the
80
magnetosome display system, for application in the hydrolysis of celluloses. Initially, two
81
fluorescent proteins, namely green fluorescent protein and mCherry, will be immobilized on
82
MNPs for the proof-of-concept study. Fluorescent microscopy and fluorescence resonance
83
energy transfer analysis will be used to determine the proximity of two different proteins on the
84
MNPs based on the interaction between the cohesin and dockerin domains. Furthermore, ELISA
85
analysis will be used to determine the quantities of proteins immobilized on the MNPs.
15
, a tropomyosin receptor kinase A
16
14
. Magnetosome is a unique organelle
, the class I major histocompatibility
, and a thyroid-stimulating hormone receptor
17
,
ACS Paragon Plus Environment
4
Page 5 of 23
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
86
Subsequently, endoglucanase and β-glucosidase immobilized on MNPs will be prepared using
87
the magnetosome display system and tested for cellulose hydrolysis activity. We expect to
88
confirm synergistic effects when using two close proximity enzymes and the CBD on MNPs,
89
resulting in the efficient degradation of celluloses. We predict that the magnetosome display
90
system will expand the possibilities to mimic natural cellulosome organization.
91 92
2. Experimental Section
93
2.1 Bacterial Strains and Culture Conditions
94
Escherichia coli TOP10 (Life technologies, CA, US) and EPI300 (ARBROWN, Tokyo, Japan)
95
were used for gene cloning. E. coli transformant cells were cultured at 37 °C in Luria-Bertani
96
(LB) agar plates or in ampicillin-containing media (50 µg mL-1). The Magnetospirillum
97
magneticum AMB-1 (ATCC 700264) mms13 gene deletion mutant (strain AMB-1 ∆mms13) was
98
used for the production of magnetic nanoparticles (MNPs)24. Strain AMB-1 ∆mms13 was
99
microaerobically cultured in magnetic spirillum growth medium (MSGM) containing
100
gentamycin (2.5 µg mL-1) at 28 °C as described previously
101
constructed by electroporation
102
and ampicillin (5 µg mL-1). Furthermore, the expression of target proteins was induced by the
103
addition of anhydrotetracycline hydrochloride (500 ng mL-1, Kanto Chemical, Tokyo, Japan) at
104
the mid-log phase, and the mixture cultured for a further 12 h.
26
25
. AMB-1 transformants were
and cultured in MSGM containing gentamycin (2.5 µg mL-1)
105 106
2.2 Plasmid Construction
ACS Paragon Plus Environment
5
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 6 of 23
107
A schematic diagram representing the co-expression plasmids in Figure S1. Co-expression
108
plasmids were comprised of two fusion gene sets, namely “mms13-cohesin” genes and “target-
109
dockerin” genes. Cohesin domains from Clostridium thermocellum (CohC: CipA 364-702 amino
110
acids) and Ruminococcus flavefaciens (CohR: ScaB 29-186 amino acids) were fused to mms13.
111
Dockerin domains from C. thermocellum (DocC: CelS 673-741 amino acids) and R. flavefaciens
112
(DocR: ScaA 787-879 amino acids) were fused to target proteins (GFP and mCherry, or
113
endoglucanase celCCA (EG) and β-glucosidase Ccel_2454 (BG). EG and BG were removed
114
signal sequence and native dockerin. The gene sequences of two cellulases and the cellulose
115
binding domain (CBD) were also obtained from C. thermocellum. All genes were generated by
116
artificial gene synthesis (Integrated DNA Technologies, Coralville, IA, USA). Fusion genes were
117
cloned into pUMtOR which is a tetracycline-inducible vector for M. magneticum AMB-1 27. The
118
linker is a hydrophilic polypeptide linker containing repeating amino acid chains of 4 asparagine
119
residues and 1 serine residue (linker length: 10 amino acids)28. The thrombin cleavage site was
120
designed between the linker and CohC. All prepared transformants are listed in Table S1.
121 122
2.3 Preparation of MNPs
123
Cultured cells of M. magneticum AMB-1 transformants using MSGM were collected by
124
centrifugation at 9000 g for 10 min at 4 °C. Collected cells were re-suspended in HEPES-Ca
125
(40 mL, 10 mM, supplemented with 1 mM CaCl2, pH 7.4), and disrupted by passing through a
126
French press at 1500 kg cm-2 (Ohtake Works, Tokyo, Japan). MNPs were collected from the
127
disrupted cell suspension using a columnar neodymium-boron magnet. The MNPs were washed
128
10 times with HEPES-Ca (20 mL). The concentration of MNPs in suspension was determined by
ACS Paragon Plus Environment
6
Page 7 of 23
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
129
measuring the optical density at 660 nm using a UV-2200 spectrophotometer (Shimadzu, Kyoto,
130
Japan). A value of 1.0 corresponded to 172 µg MNPs (dry weight) per mL.
131 132
2.4 Enzyme-Linked Immunosorbent Assay (ELISA)
133
Sandwich ELISA analysis was performed for the quantification of GFP and mCherry on the
134
MNPs. Mouse anti-GFP IgG (Takara Bio, Otsu, Japan) and alkaline phosphatase (ALP)-
135
conjugated anti-GFP IgG (Rockland Immunochemicals, PA, USA), or mouse anti-mCherry IgG
136
(Takara Bio, Otsu, Japan) and ALP-rat anti-mCherry IgG (Life Technologies, CA, USA) were
137
used as primary and secondary antibodies, respectively. ALP conjugation to rat anti-mCherry
138
IgG was performed using the Alkaline Phosphatase Labeling Kit–NH2 (Dojindo, Kumamoto,
139
Japan). Primary antibodies (2 µg mL-1, 100 µL) in bicarbonate buffer (pH 9.8) were added to a
140
96 well palate (Costar 3925, Corning Inc., NY, USA) and incubated at 4 °C overnight to allow
141
sufficient time for adsorption to take place. After washing three times with PBST (phosphate-
142
buffered saline, PBS, containing 0.1% Tween-20, at pH 7.4), the plates were blocked with 0.2%
143
bovine serum albumin (BSA) in PBS for 1.5 h at room temperature. After washing with PBST,
144
the protein samples (100 µL) were added, and the plates incubated for 1 h at 37 °C. The protein
145
fractions on the MNPs were extracted according to a modified literature procedure
146
washing with PBST, secondary antibodies (1 µg mL-1 for GFP, 2 µg mL-1 for mCherry) were
147
added and incubated at 37 °C for 1 h. After washing once more with PBST, Lumi-Phos 530
148
(100 µL, Wako Pure Chemical Industries) was added as a luminescence substrate. After
149
incubation for 5 min, the luminescence intensity was measured using a microplate reader SH-
150
9000 (Corona Electric, Ibaraki, Japan). The quantities of GFP and mCherry were estimated using
29
. After
ACS Paragon Plus Environment
7
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 8 of 23
151
luminescent curves of the GFP and mCherry standards (Cell Biolabs, Inc., CA, USA). Finally,
152
the quantities of GFP (27 kDa) and mCherry (29 kDa) on a single MNP were calculated as
153
described previously28.
154 155
2.5 FRET Analysis
156
Prior to fluorescence measurements, the protein complexes were purified from MNPs by
157
thrombin digestion to avoid interference from MNP light scattering30. The MNPs (1 mg) were
158
suspended in thrombin solution (100 µL, 100 U mL-1 in PBS) and incubated at 4 °C overnight.
159
Following centrifugation at 18000 g for 1 min, the supernatants were collected and used for the
160
measurements. Fluorescence emission spectra using an excitation wavelength of 483 nm were
161
acquired between 540 and 680 nm to detect the fluorescence resonance energy transfer (FRET)
162
between GFP and mCherry using a microplate reader SH-9000 (Corona Electric, Ibaraki, Japan).
163 164
2.6 Hydrolysis of Soluble and Insoluble Celluloses using MNP-Immobilized Cellulases
165
MNPs (50 µg) were added to a substrate solution (0.5% carboxymethyl cellulose (CMC) or 1%
166
Avicel in 100 mM Tris-HCl containing 1 mM CaCl2, at pH 5.5). Following incubation at 30 °C,
167
the MNPs were removed from the suspension by magnetic separation, and the supernatant was
168
collected. The supernatant (5 µL) was added to tetrazolium blue chloride assay buffer (195 µL,
169
composition = 1 mg mL-1 tetrazolium blue chloride, 0.5 M potassium sodium tartrate, 50 mM
170
NaOH)
171
absorbance of the solutions at 704 nm was measured using a microplate reader SH-9000
31
. After boiling at 100 °C for 3 min, the solutions were cooled using an ice bath. The
ACS Paragon Plus Environment
8
Page 9 of 23
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
172
(Corona Electric, Ibaraki, Japan). The concentrations of reducing sugars present were calculated
173
from a standard curve using glucose.
174 175
3. Results and Discussion
176
3.1 Protein Proximity Analysis of Marker Proteins on MNPs
177
Prior to preparation of the cellulase-MNP complexes, two marker proteins, namely GFP and
178
mCherry, were immobilized onto MNPs for the evaluation of spatial protein-protein proximity.
179
Immobilization was achieved using the magnetosome display system. A schematic diagram of
180
the transformants used in this section is given in Figure 1(A). In this study, the dockerin domains
181
from C. thermocellum (DocC) and R. flavefaciens (DocR) were fused to GFP and mCherry,
182
respectively. Furthermore, the cohesin domains from C. thermocellum (CohC) and R.
183
flavefaciens (CohR) were fused to mms13. The CohC and CohR were linked with a peptide
184
linker of 10 amino acids. GFP and mCherry were expected to be co-localized on magnetosomes
185
arranged in a chain through interactions between cohesin and dockerin in AMB-1/CohCR-GmC
186
(details of the M. magneticum AMB-1 transformants are given in Table S1). As expected,
187
fluorescence from both GFP and mCherry was observed at the same positions, with
188
magnetosome chains present in AMB-1/CohCR-GmC (Figure 1(B), top), although no
189
fluorescence was detected in the wild type (Figure 1(B), bottom). Furthermore, fluorescence
190
from mCherry was widely distributed in the cytoplasm of CohR-lacking AMB-1/CohC-GmC
191
(Figure 1(B), middle), indicating that two different proteins are co-immobilized onto the MNPs
192
through cohesin-dockerin interactions.
ACS Paragon Plus Environment
9
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 10 of 23
193
The spatial protein-protein proximity was then investigated by FRET analysis. The MNPs were
194
extracted from cell lysates of AMB-1 transformants by magnetic separation, followed by
195
purification of the MNP protein fractions by enzymatic cleavage, as described in the
196
Experimental Section (Section 5). Figure 2 shows the emission spectra of fluorescent proteins
197
under an excitation wavelength of 483 nm. No FRET signal was detected from the MNP/CohC-
198
GmC protein complexes (Figure 2, dashed line), as these protein complexes include only GFP. A
199
slight increase in the signal at ~610 nm was observed, even when free mCherry was added to the
200
GFP-containing protein complexes (Figure 2, gray solid line). In contrast, mCherry fluorescence
201
was detected in protein complexes purified from MNP/CohCR-GmC (Figure 2, black solid line,
202
indicated by an arrow). mCherry may therefore act efficiently as a fluorescence acceptor towards
203
GFP, which acts as a fluorescence donor in the FRET system on MNP/CohCR-GmC. FRET
204
occurs when GFP and mCherry are co-localized within 1-10 nm of each other, with the highest
205
efficiency reached between 4-6 nm
206
CohR) were linked with a peptide linker consisting of 10 amino acids. If the DocC and DocR
207
fused to fluorescence proteins were respectively bound to each scaffold protein, two fluorescence
208
proteins were located within about 4 nm corresponding to a peptide unit’s length of 0.38 nm33.
209
Therefore, this closer position led to the generation of FRET.
210
The quantities of GFP (1.3 ± 0.2 µg mg-1-MNP) and mCherry (1.4 ± 0.1 µg mg-1-MNP) present
211
on the MNPs were estimated using enzyme-linked immunosorbent assay (ELISA) analysis.
212
Based on these results, the number of GFP and mCherry molecules per single MNP were
213
calculated according to literature methods28, and found to be 33 molecules/MNP for both
214
proteins. This value was in agreement with previously reported results, i.e., 22 molecules-Protein
215
A/MNP
13
32
. The distance between two scaffold proteins (CohC and
and 20-34 molecules-Protein G/MNP23. These results indicate that stoichiometrically
ACS Paragon Plus Environment
10
Page 11 of 23
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
216
equivalent amounts of GFP and mCherry were successfully immobilized on MNPs using the
217
magnetosome display system. This demonstrates that if desired, the number of recombinant
218
proteins present on MNPs can be altered by genetic engineering.
219 220
3.2 Hydrolysis of Carboxymethyl Cellulose (CMC) using Endoglucanase (EG) and β-
221
Glucosidase (BG) Immobilized on MNPs (EG/BG-MNPs)
222
According to GFP and mCherry immobilization conditions, endoglucanase (EG) and β-
223
glucosidase (BG) were immobilized onto MNPs via cohesin-dockerin interactions (Figure 3(A),
224
EG/BG-MNPs) for application in the hydrolysis of the soluble cellulose, carboxymethyl
225
cellulose (CMC). A schematic diagram of the transformants used in this section is given in
226
Figure 3(A). To evaluate the proximity effect of the two enzymes on CMC hydrolysis, the
227
mixture of EG-MNPs and BG-MNPs was used as a control (Figure 3(A), EG-MNPs and BG-
228
MNPs). It should be noted that equal quantities of the two enzymes were used for CMC
229
hydrolysis. When the CMC solution was treated with EG/BG-MNPs, the quantity of reducing
230
sugar increased with time, reaching a plateau at >3 h (Figure 3(B), closed circles). In contrast,
231
the hydrolysis rate of the EG-MNPs and BG-MNPs mixture was significantly slower than that of
232
EG/BG-MNPs, with the quantity of reducing sugar reaching a plateau beyond 7 h (Figure 3(B),
233
open circles). This indicates that the close proximity of EG and BG may contribute to the higher
234
hydrolysis rate in EG/BG-MNPs. The previous report revealed the higher activity of
235
immobilized three enzymes (EG, BG and exoglucanase) against CMC than that of free
236
enzymes9. The resulting concentration of reducing sugars was increased from about 3.2 mM to
237
3.9 mM. These values (3.2 to 3.9 mM) are similar level with the obtained value (1.6 mM) in this
ACS Paragon Plus Environment
11
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 12 of 23
238
study. Based on these results, we have concluded that the co-immobilization by our proposed
239
method has an advantage over using free enzymes. In general, cellulose is degraded to
240
oligosaccharides by EG, and the oligosaccharides are subsequently degraded to glucose by BG.
241
Thus, the spatial proximity of these two enzymes leads to synergistic enzyme reactions for the
242
efficient degradation of cellulose, suggesting that the efficient degradation of CMC by EG/BG-
243
MNPs may be caused by the oligosaccharides being unable to diffuse away from the reaction
244
center due to the proximity of EG and BG.
245
The reusability of the EG/BG-MNPs was then investigated (Figure 3(C)). Following hydrolysis
246
of CMC, the MNPs were magnetically collected, and reused in further CMC hydrolysis cycles
247
under the same conditions. The hydrolysis activity in the first trial was defined as 100%, and the
248
activity of EG/BG-MNPs was maintained at >70% after five trials. Previous reports using
249
chemically conjugated cellulases and magnetic particles indicate that enzyme activity dropped to
250
20-50% after five trials, even with the use of thermostable cellulases and optimal conditions for
251
CMC hydrolysis
252
system show good performance in terms of cellulase reusability. The slight decrease in
253
enzymatic activity could be explained by two enzymes being released from or inactivated on
254
MNPs during the hydrolysis process. It is also possible that the amount of MNPs used gradually
255
decreased during the magnetic separation process.
2, 5
. This suggests that the MNPs prepared using the magnetosome display
256 257
3.3 Effect of the Cellulose-Binding Domain (CBD) on Hydrolysis of Microcrystalline
258
Cellulose
ACS Paragon Plus Environment
12
Page 13 of 23
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
259
The EG/BG-MNPs were applied in the hydrolysis of the insoluble cellulose, Avicel, but little
260
hydrolysis was detected (Figure 4(B)). In order to improve the hydrolysis activity, the cellulose-
261
binding domain (CBD) was fused to the end of the scaffold protein attached to the MNPs (Figure
262
4(A), EG/BG-CBD-MNPs). The CBD plays an important role in the binding of cellulosome to
263
the crystalline cellulose34-36. When compared to the EG/BG-MNPs, the production of reduced
264
sugar was significantly enhanced when EG/BG-CBD-MNPs were used (Figure 4(B)). The
265
binding of the CBD to microcrystalline cellulose may promote the hydrolysis of EG and BG on
266
MNPs. Although the hydrolysis rate of Avicel was approximately 10 times lower than that of
267
CMC (Figure 3(B)), this tendency correlates with previous results.
268 269
4. Conclusions
270
We reported the construction of highly organized protein complexes on magnetic nanoparticles
271
(MNPs) using the magnetosome display system. A proof-of-concept study using green
272
fluorescent protein (GFP) and mCherry revealed the close proximity (
