Biocatalytic synthesis of gold nanoparticles with cofactor regeneration in recombinant Escherichia coli cells

Article abstract of DOI:10.1039/c1cc12302e

Here we report the enzymatic synthesis of gold nanoparticles (Au NPs) by an engineered Escherichia coli harboring an NADH cofactor regeneration system coupled with glycerol dehydrogenase, which can be triggered by the addition of exogenous glycerol.

Full text of DOI:10.1039/c1cc12302e

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Biocatalytic synthesis of gold nanoparticles with cofactor regeneration
in recombinant Escherichia coli cellsw
Teppei Niide,a Masahiro Gotoab and Noriho Kamiya*ab
Received 20th April 2011, Accepted 17th May 2011
DOI: 10.1039/c1cc12302e
Here we report the enzymatic synthesis of gold nanoparticles
(Au NPs) by an engineered Escherichia coli harboring an
NADH cofactor regeneration system coupled with glycerol
dehydrogenase, which can be triggered by the addition of
exogenous glycerol.
E. coli cells as tunable bioreactors for the synthesis of diverse
NPs; however, the exact reduction mechanism of metal ions
inside the cells was not elucidated.
Herein, we report a new approach of bioreduction of gold
ions by a natural electron-donating cofactor, nicotinamide
adenine dinucleotide (NADH), using recombinant E. coli
cells. The catalytic growth of Au NPs has been achieved by
regeneration of the NADH cofactor by NAD+-dependent
lactate dehydrogenase (LDH).15 In fact, enzyme-mediated
growth of metallic NPs has been successfully demonstrated.16
Inspired by the coupling of Au NPs synthesis with an enzymatic
NADH regeneration system, the controllable bioreduction
function of recombinant E. coli cells by genetically incorporating
bacterial glycerol dehydrogenase (GLD) has been applied to
regenerate NAD+ to NADH with exogenous glycerol inside
the cells.17 In the present study, we demonstrate that the
cofactor regeneration system in E. coli can be exploited for
the in vivo synthesis of Au NPs with the inexpensive reducing
agent glycerol (Fig. 1).
Gold nanoparticles (Au NPs) exhibit unique physical properties,
such as size- and shape-dependent optical and electronic
features.1–3 Au NPs have been extensively studied in the field
of nanobiotechnology, because unique optical characteristics
can be feasibly coupled with both biological and synthetic
molecules including proteins, DNA, lipids and polyethylene
glycol by surface modification.4–6
In general, preparation of Au NPs is conducted by a
chemical reduction method. On the other hand, various types
of metal NPs have been prepared in the presence of biological
entities including peptides, proteins and nucleotides.7–9 Such
biomolecules control not only the nucleation, but also the
crystal structure and size under aqueous environments. Living
biological systems have also been exploited to prepare
metal nanoparticles and for nanocrystal synthesis. Microbial
synthesis of Au NPs with Verticillium sp. produced Au NPs of
approximately 20 nm in diameter from an aqueous solution of
We first tested the potential of the GLD-mediated cofactor
regeneration system in Au NPs synthesis in vitro. The plasmid
encoding the gld gene was constructed for the expression of
GLD in the E. coli strain BL21.18 The recombinant E. coli
harboring the plasmid was cultured under appropriate conditions
(see ESIw) and expression of the GLD protein was induced
by the addition of isopropyl-b-D-1-thiogalactopyranoside
(IPTG). Identification of GLD was conducted by SDS-PAGE
analysis and the enzymatic activity measurement (Fig. S1 and
S2 in ESIw). Fig. 2 shows the results of Au NPs preparation
in vitro when GLD (1 mM), NAD+ (400 mM) and glycerol
AuCl4 under microbial reducing conditions.10 However, the
À
microbial components that facilitate the synthesis of Au NPs
are not fully understood. The synthesis of Fe3O4 magnetite
nanoparticles was also demonstrated with magnetotactic
bacteria.11,12 Recently, the synthesis of various metal NPs,
including semiconductor NPs and Au NPs, by recombinant
Escherichia coli expressing phytochelatin (PC) and/or metallo-
thionein (MT) was reported.13,14 It has been proposed that
these biomolecules possessing free thiol and sulfide groups
which accumulate in recombinant E. coli cells can collect and
increase the metal ion concentration inside the cells. This
process therefore promotes mineralization. These reports
also demonstrated the excellent capability of recombinant
À
(50 mM) were added to an aqueous solution of AuCl4
(0.5 mM) in 50 mM Gly–NaOH buffer, pH 9.5, and incubated
at 37 1C for 48 h. The solution was intense reddish in color
when all the components required for NADH regeneration
were present (Fig. 2(a)–i). In contrast, the absence of either the
a Department of Applied Chemistry, Graduate School of Engineering,
Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan.
E-mail: nori_kamiya@mail.cstm.kyushu-u.ac.jp;
Fax: +81 (0)92-802-2810; Tel: +81 (0)92-802-2807
b Center for Future Chemistry, Kyushu University, 744 Motooka,
Fukuoka 819-0395, Japan
Fig. 1 Schematic illustration of Au NP formation coupled with the
cofactor regeneration system in a recombinant E. coli cell.
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc12302e
c
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Fig. 2 (a) UV/Vis spectra of Au NPs synthesized by the GLD-
mediated enzymatic NADH regeneration. Inset: an optical image of
the Au NPs (i). (b) TEM image of Au NPs synthesized in the GLD
enzymatic reaction. (c) The particle size distribution of the Au NPs.
Fig. 3 UV/Vis spectra of recombinant E. coli BL21 cells under
different conditions. (a) Aqueous solution of the recombinant cells
(1 mg mLÀ1) with IPTG induction for the over-expression of GLD
(IPTG+). (b) The same recombinant E. coli BL21 cells without IPTG
induction.
substrate for the enzymatic reaction or GLD did not result in
an increase in the absorbance at ca. 530 nm, which is attributed
to the surface plasmon band of Au NPs (Fig. 2(a)–ii–iv). In
addition, an increase in the absorbance at 340 nm derived
from NADH was observed. The in vitro results indicate that
the GLD reaction is essential to stimulate the formation of Au
NPs, and that AuCl4À received electrons from NADH present
in excess in the system. Therefore, formation of Au NPs can be
triggered by the GLD-mediated NADH regeneration reaction.
For further analysis of the Au NPs, transmission electron
microscopy (TEM) and dynamic light scattering (DLS) were
conducted (Fig. 2b and c). Fig. 2b shows the TEM image of
the Au NPs formed during the GLD cofactor regeneration
reaction. The formation of Au NPs was evident, and the TEM
analysis showed that the particle size was widely distributed
with a range between 4 and 30 nm (Fig. S3 in ESIw). The
average particle size diameter was 10.4 Æ 6.4 nm (n = 100).
The DLS measurement of the same sample showed an average
diameter of 30.8 (Æ0.2) nm (Fig. 2c). The discrepancy in
particle sizes between TEM and DLS analyses suggests that
the Au NPs surface was coated with biological entities,
possibly with GLD.
(Fig. 3(a)–iii) suggests that the endogenous NAD+ in E. coli
was employed for Au NPs synthesis. Other control samples
including the lack of IPTG induction showed negligible levels
of Au NPs formation. These results indicate that the Au NPs
formation inside the E. coli cells was dominated by GLD-mediated
NADH regeneration. The NADH cofactor regeneration by
GLD thus drives Au NPs formation even in the recombinant
E. coli cells.
To further validate the formation of Au NPs in E. coli cells,
we conducted TEM analysis for sample (i) in Fig. 3. The TEM
image of a recombinant E. coli cell suggests that the Au NPs
accumulate in cells (Fig. 4a). We also observed an image which
showed leakage of the Au NPs from the cytoplasm together
with the contents of E. coli cells (Fig. S4 in ESIw). This
observation implies the formation of Au NPs in the cytoplasm.
The particle size ranged from 7.2 to 29 nm from the TEM
analysis and 475% of the Au NPs have 415 nm diameter
(Fig. S5 in ESIw): the average particle size diameter was
19.5 Æ 5.8 nm (n = 100). Fig. 4b and c show the TEM image
of Au NPs after disruption of the E. coli cells (see ESIw). The
results indicate that larger Au NPs were formed in the E. coli
cells when compared to those prepared by in vitro cofactor
regeneration (Fig. 2(b)). Although the reason for this observation
is unclear, we postulate that slower nucleation of gold ions
inside the cells due to non-specific interaction with a variety of
biomolecules in the cytoplasm is a possible reason. This leads
to the formation of a smaller number of nuclei compared to
We next explored the incorporation of the Au NPs synthetic
scheme into recombinant E. coli cells. The resting cells of
E. coli strain BL21 in which GLD was over-expressed by IPTG
induction were prepared (see ESIw). For control experiments,
the recombinant E. coli cells without IPTG induction were
prepared. The Au NPs synthesis was conducted with the
recombinant E. coli cells (1 mg mLÀ1) supplemented with or
without NAD+ (400 mM) and glycerol (50 mM) in an aqueous
À
solution of AuCl4 (0.5 mM) in 50 mM Gly–NaOH buffer,
pH 9.5, at 37 1C for 72 h.
Fig. 3 shows the results of the preparation of Au NPs by the
recombinant E. coli cells. Only the E. coli sample with IPTG
induction and exogenous glycerol addition showed a clear
absorption spectral change at ca. 540 nm. The supplementation
of both exogenous NAD+ and glycerol generated the intense
absorption maximum at 538 nm derived from the surface
plasmon band of Au NPs (Fig. 3(a)–i). The characteristic
peak observed without the supplementation of NAD+
Fig. 4 TEM images of a recombinant E. coli cell in which Au NPs
were produced (a) and Au NPs synthesized via enzymatic NADH
regeneration in the cells with different magnification (b & c).
c
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the in vitro system, and this may result in the growth of larger
Au NPs.
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In conclusion, we have designed and validated a biocatalytic
system for Au NPs synthesis in recombinant E. coli cells. The
GLD-mediated enzymatic NADH regeneration afforded
E. coli cells that act as controllable (via glycerol input)
bioreactors for Au NPs synthesis inside the cells, although
further characterization of biocatalytically synthesized Au
NPs is required. The methodology is simple and should be
applicable for the cost-effective production of Au NPs
and other metal NPs. Our results also suggest the plausible
participation of the NADH cofactor in metal NPs synthesis
with the aid of biological living systems.
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We are grateful to Dr Tsuyoshi Mouri for his help in the
preparation of recombinant E. coli cells. This work was
supported by a Grant-in-Aid for the Global COE program,
‘Science for Future Molecular Systems’ from the Ministry of
Education, Culture, Sports, Science and Technology of Japan.
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7352 Chem. Commun., 2011, 47, 7350–7352
This journal is The Royal Society of Chemistry 2011