(397ba) Catalysis of Gold Nanoparticles Within Cross-Linked Lysozyme Crystals

Preparation of
bio¨Cnano hybrid materials has been the focus of numerous studies because of
their broad range of potential applications in catalysis, drug delivery, medical
imaging, and chemical sensing [Wei, Wang et al., 2011]. Among various
biological components that could be used as templates for the fabrication of
bio-nanocomposites, protein assemblies with different nanostructures, such as
fibrils, tubes, and cages, have received special attention due to diverse structures
and promising functionalities [Abe, Tsujimoto et al., 2012]. Nevertheless, bridging
from the nanoscale to the macroscale and to create macroscopic hybrid materials
for practical application remains a challenge. Therefore, it is desirable to explore macroscopic protein assemblies
as templates for the fabrication of novel composite materials with functional properties [Liang, Wang et al., 2013].

Here we show that lysozyme molecules in their crystalline state
can be treated as macroscopic templates for the construction of novel composite
catalysts with good recyclability (Scheme 1). Firstly, tetragonal lysozyme
crystals were prepared according to our previous crystallization conditions [Liang, Jin et al.,
2013]. The stabilization of native crystals with glutaraldehyde resulted in
cross-linked lysozyme crystals (CLLC) that were pale yellow in color and robust
physically. Then, soaking the CLLC in HAuCl₄ solution allowed the accumulation of Au³⁺
in the interior pores of crystal. Subsequently, the coordinated Au³⁺ underwent
progressive reduction to form Au nanoparticles (AuNPs) in situ after the
addition of NaOH. Furthermore, we demonstrated that these prepared
AuNPs-at-CLLC (Au@CLLC) hybrid materials exhibited good catalytic activity
toward nitrophenol reduction with excellent recyclability. The main ideas and
conclusions are summarized as follows.

(1) Tetragonal lysozyme crystals that comprised four relatively
large (110) side faces capped by four inclined (101) faces can be readily crystallized
using our previous conditions. It is known that protein crystals present highly
ordered three-dimensional arrangements of protein molecules with the properties
of high porosity (0.5 to 0.8), large surface area (800 m²/g to
2000 m²/g), and a wide range of pore sizes (2 nm to
10 nm). In addition, various functional groups that responsible for
coordinating metal ions are exposed on the surface of solvent channels of crystal lattices. Given these advantageous
properties, protein crystals can be treated as promising matrices for nanoparticle
immobilization. Light microscope of CLLC did not show obvious structural
distortion originated from the stabilization treatment. Meanwhile, SAXRD
pattern of the CLLC shows characteristic diffraction at 2¦È = 2.88°, 3.47°, 4.51°, and 6.91°, indicating
the crystalline form of CLLC has remained largely unchanged.

(2) AuNPs
were synthesized within the solvent channels of CLLC using a reductant-free and
green synthetic route. The CLLCs were immersed in HAuCl₄
solution for the sequestration of metallic precursors. Reduction of the
coordinated Au³⁺ by tyrosine and tryptophan residues of lysozyme under
alkaline condition resulted in intact red metallized CLLC. Meanwhile, no characteristic
SPR of AuNPs was observed in the supernatant solution as shown in the UV-vis
spectrum. HRTEM measurements of the AuNPs formed within CLLC demonstrated that the particle
sizes were almost 1.5 nm to 3.0 nm with narrow size distribution. These
results indicated the crystalline porous structures of CLLC can effectively
limit the migration and aggregation of AuNPs. Moreover, the small sized
crystalline AuNPs was demonstrated by the relative weak characteristic diffraction
peaks as shown in XRD patterns. FTIR analysis confirmed the involvement of tyrosine
and tryptophan residues in the reduction of Au ions.

(3) we
choose the reduction of 4-nitrophenol as a model system to evaluate the
catalytic properties of the as-prepared Au@CLLC hybrid catalysts. The reaction over
Au@CLLC catalyst was almost completed within 360 s for the first round. The
pseudo-first-order kinetics with respect to 4-nitrophenol was used to evaluate
the kinetic reaction rate of the current reaction, and the apparent rate
constant was estimated from the slop to be 14.92 °Á 10^-3 s^-1.
The recovered hybrid catalysts exhibited similar catalytic activity in the first
three rounds of the reaction, and can be successfully recycled and reused in
twenty successive reactions with a conversion of >96%. These results clearly
indicate that the CLLCs are a good supporting material for AuNPs because of
their physical robustness, high specific surface area, and three-dimensional
nanostructure property.

Scheme 1. Schematic illustration of the lysozyme
crystallization, crosslinking and preparation of Au@CLLC composite materials
for the catalytic conversion of 4-nitrophenol.

This work was
supported by the Natural Science Foundation of China (Nos 21276192, 51173128
and 31071509), the Ministry of Science and Technology of China (Nos
2012YQ090194, 2012BAD29B05 and 2012AA06A303), and the Ministry of Education
(No. NCET-11-0372).

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