EnzymeâNanowire Mesocrystal Hybrid Materials ... - ACS Publications

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Enzyme-nanowire mesocrystal hybrid materials with extremely high biocatalytic activity Galong Li, Pei Ma, Yuan He, Yifan Zhang, Yane Luo, Ce Zhang, and Haiming Fan Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b02620 • Publication Date (Web): 23 Aug 2018 Downloaded from http://pubs.acs.org on August 24, 2018

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Enzyme-nanowire mesocrystal hybrid materials with extremely high biocatalytic activity 1

1

Galong Li,†,‡, Pei Ma,‡, Yuan He,† Yifan Zhang,†,‡ Yane Luo,§ Ce Zhang,ǁ and Haiming Fan*,† †

Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of the Ministry of

Education, College of Chemistry and Materials Science, Northwest University, Xi’an, 710069, PR China ‡

School of Chemical Engineering, Northwest University, Xi’an, 710069, PR China

§

College of Food Science and Engineering, Northwest University, Xi’an, 710069, PR China

ǁ

School of Physics, Northwest University, Xi’an, 710069, PR China

1

These authors contributed equally to this work

Corresponding Author: Haiming Fan *

E-mail: [email protected]

Tel: +86 29 81535040 Fax: +86 29 81535040

1 Environment ACS Paragon Plus

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ABSTRACT The laccase-Cu2O nanowire mesocrystal hybrid materials were developed with superior catalytic activity inspired by natural biocatalysis processes in living cells that highly resembles the metal ions activation and the well-organized spatial structure of the natural rough endoplasmic reticulum. The enzyme and nanobiocatalyst activities of the obtained hybrid material exhibited approximately 10-fold and 2.2-fold increase than the free enzyme, surpassing the currentlyavailable nanobiocatalysts. The comprehensive catalytic performance of the hybrid materials has been further demonstrated using a prototype continuous-flow reactor for the bioremediation of 2,4-dichlorophenol contaminated water, which showed high degradation efficiency and remarkable reusability. These new high-efficient nanobiocatalysts are expected to be used for diverse applications in biotechnology, biosensing and environmental remediation.

KEYWORDS Nanowire mesocrystal, enzymes, hybrid material, biocatalysis, wastewater treatment


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Inorganic nanomaterials have emerged as competitive carriers for enzyme immobilization owing to the fascinating properties related to the reduced dimensions of inorganic materials.1-4 While a plethora of immobilization methods including the covalent/noncovalent binding, entrapment and cross-linking have been well-established,5 recent interests in this field have focused on the development of enzyme-nanomaterials mutualistic system with high biocatalytic activity.5,6 For example, traditional mesoporous silica, nanofiber and nanooxide immobilized enzymes have increased the thermal/chemical stability and reduced mass transfer limitations,7-9 but have reduced apparent activity mainly due to unfavorable enzyme-carrier interactions which further lead to denaturation and deactivation of the enzymes.7,10,11 In contrast, the bio-active inorganic nanocarriers can dramatically enhance the apparent specific enzyme activity, where the enzyme-carrier interaction is effectively modulated.12,

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Consequently, a large number of

enzyme-nanomaterial hybrid materials with enhanced specific enzyme activity have been reported, where diverse enhancement mechanisms such as interfacial ion activation, allosteric effect, electron transfer, and synergistic effect have been proposed.12-16 Despite their apparent higher enzyme activity over the conventional immobilized enzymes, nanobiocatalysts are still beset by certain unfavorable catalytic features. In most cases, immobilized enzymes are dispersed disorderly at both the nanoparticle surface and the three-dimensional (3D) spatial arrangement in a partially closed system, which adversely affects the promotion of diffusion and partition.17 In addition, conventional method assesses specific activity of the immobilized enzyme based on the amount of enzyme immobilized on the carrier,18 which might not be suitable for the nanobiocatalysts as bulk carriers are still needed in most cases for recovering them from the reaction solution. In this context, the reported nanobiocatalysts with high specific


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enzyme activity actually have unsatisfactory overall catalytic activity due to the presence of large “dead volume” which does not contribute to the biocatalysis process. Natural nanostructures in the living system offer exquisite architecture for enzymes to mediate various biochemical reactions in a very efficient way.19-23 For example, the rough endoplasmic reticulum (rER), possessing an open morphology constituted of closely packed sheets of ER membrane with a diameter of about 100 nm between the neighboring sheets, serves as a biomachinery for high-efficient biosynthesis of large amounts of proteins in eukaryotic cells.24 The high-efficient synthetic activity of rER system is facilitated by spatially well-ordered membrane-bound ribosomes whose activities are regulated by the Mg ion concentrations of rER microenviroments.25,26 Thus, we sought an artificial 3D well-organized nanostructure that can provide a similar structure and function to bridge the performance gap between the efficient substrate/product diffusion and favorable enzyme-carrier interaction in the current hybrid materials used for high-efficient nanobiocatalysts. With the evolution of synthetic structural materials, the nonclassical crystallization processes have birthed various self-organized microscale 3D superstructures (mesocrystals) with precisely controlled spatial orientation and arrangement of nonspherical nanoscale building blocks interspaced with organic additives,27-29 which offer the opportunity to imitate the natural structure of the organelles for enzyme immobilization. In particular, Cu2O nanowire mesocrystals with an open octahedral structure are of special interest for bioinspired nanocarriers due to their high nanoscale intracrystal porosity, highly oriented interpenetrating nanowires architecture, and ultrathin nanowire building blocks.30,31 The immobilized enzymes are expected to distribute in the well-ordered 3D Cu2O nanowire mesocrystals. Moreover, the inherent dissolution-crystallization dynamic equilibrium of a kinetically stable nanosystem would induce a copper ions-richened microenvironment


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within the Cu2O nanowire mesocrystals, which would potentially prompt the activity of Cucontaining enzyme.13 Furthermore, the Cu2O nanowires are capped by poly(o-anisidine) with abundant amine groups for covalent binding of enzyme. Therefore, Cu2O nanowire mesocrystals are an extraordinary carriers for enzyme immobilization, featuring both reduced mass transfer limit and regulated enzyme-carrier interaction. In this study, we demonstrated the ultrahigh nanobiocatalyst activity of laccase-Cu2O nanowire mesocrystals (LAC-NWMCs), and their application in high-efficient water treatment. LAC (EC 1.10.3.2) was chosen here because it is a copper-containing enzyme widely used in bioremediation of wastewaters.15, 16 A artificial LAC-NWMCs indeed mimic the merits of rER smartly because of the highly ordered 3D spatial structure of immobilized LAC whose activities were regulated by Cu+ and Cu2+ ions generated by NWMCs (Figure 1), giving an approximate 10-fold increase in the specific enzyme activity. More significantly, the LAC-NWMCs exhibited a remarkable nanobiocatalyst activity, approximately 220% higher than free LAC due to their super-high specific enzyme activity and substantially reduced dead volume. Such high catalytic activity of LAC-NWMCs consequently lead to the superior degradation ability of organic contaminants. The biocatalytic performance of LAC-NWMCs in wastewater bioremediation is further demonstrated using a prototype continuous-flow reactor. The reactor embedded with 60 mg LAC-NWMCs can remove 99.17% of the residual 2,4-dichlorophenol in one cycle, and its efficiency is maintained up to 96.32% by refreshing the LAC after every 10 cycles, which is crucial for the industrial-scale sustainable chemical process. To our knowledge, the comprehensive catalytic performance of LAC-NWMCs is the best amongst all the enzymenanostructure hybrid materials reported till date.


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The NWMCs were synthesized through a nonclassical crystallization process through a simple one-pot hydrothermal method (Supporting Information, Part 1, Figure S1).30, 31 The pore size of NWMCs is about 120 nm, much larger than the diameter of LAC,32 allowing the entry of LAC molecules into their interior pores for immobilization. The LAC-NWMCs hybrids were then obtained through the covalent binding of LAC to NWMCs by EDC/NHS coupling reaction (Supporting Information, Part 2, Figure S3).33 The loading amount of LAC was optimized according to the maximized activity (Supporting Information, Part 2, Figure S4). The optimum value is 0.44 mg enzyme/mg of NWMCs (44% w/w) with a loading yield of about 88%. The calculated LAC weight percentage in the LAC-NWMCs was around 30.6%, confirmed by the thermal gravimetric analysis (Supporting Information, Part 2, Figure S5). Conformational changes of free LAC and immobilized LAC were characterized by circular dichroism (Supporting Information, Part 2, Figure S6 and Table S1). Compared with that of free LAC, the contents of α-helix and random coil of LAC-NWMCs is decreased, which may provide much more active sites to be accessible to the substrate, leading to enhanced catalytic activity.34, 35 Figure 1 presents the schematic illustration of the rER and the corresponding bioinspired LACNWMCs. The opened porous structure of LAC-NWMCs indeed shows a relatively high degree of similarity to rER. The copper ion-richened microenvironment within NWMCs is expected to facilitate the oxidization of SYR by improving LAC activity, similar to the role of Mg cation in the biosynthesis of protein and lipid by the ribosomes in rER.25,26 The scanning electron microscope (SEM) image of an individual LAC-NWMC hybrid material shows that the LACNWMC has a well-defined octahedron morphology, bounded by eight triangular external faces with edge length around 15 µm (Figure 2A). The interpenetrated nanowire building blocks endow the hybrid material with highly ordered channel structures. Compared with the pristine


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NWMC (Supporting Information, Part 1, Figure S1A), the hybrid material still maintains its intact architecture after enzyme immobilization. As shown in the large-area SEM image (Supporting Information, Part 1, Figure 2B), the LAC-NWMC can be prepared in a large scale (Supporting Information, Part 1, Figure S1F),31 guaranteeing industrial scale-up of the nanobiocatalyst. Transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) analyses were employed to visualize the detailed structure of LAC-NWMCs hybrid materials. Figure 2C displays the TEM image of a phosphotungstic acid stained nanowire fragment detached from LAC-NWMCs.36 The distinct light spots (yellow arrows) along the nanowire is attributed to the low electron-scattering density of LAC and their slight aggregates (10-20 nm),36 which was not observed for the negative-stained pristine nanowires (Supporting Information, Part 1, Figure S1B). In addition, the Cu2O nanowire has a larger diameter of ~90 nm compared to LAC, and the enzyme can attain intense multi-interactions with the nanowire for high stability.16 The selected-area electron diffraction (SAED) pattern (inset of Figure 2C) indicated the single-crystalline nature of Cu2O nanowires, responsible for their robust mechanical structure, and can be used in future catalytic applications. Confocal laser scanning microscopy (CLSM) image of a fragment of fluorescent molecule (FITC)-labeled LAC-NWMCs is presented in Fig. 2D. The FITC-labeled LAC (green color) are distributed throughout the entire NWMC (Supporting Information, Part 2, Figure S6), and the interpenetrated nanowires render the LAC immobilization, forming high-ordered 3D spatial structures (Figure 2D). The porous structure of LAC-NWMCs was evaluated using nitrogen isothermal adsorption measurement. The hysteresis can be classified as a type IV isotherm (Supporting Information, Part 2, Figure S3B), implying the presence of both mesopores and macropores in LAC-NWMCs. The specific Brunauer–


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Emmett–Teller (BET) surface area of the LAC-NWMCs was found to be 13.2 m2 g-1, which is slightly lower than the pristine NWMCs (17.3 m2 g-1) due to enzyme immobilization. The pore size distribution of the LAC-NWMCs was similar to pristine NWMCs; both had broad pore size, centered at ~120 nm (Supporting Information, Part 2, Figure S3). Such a continuous openchannel structure of LAC-NWMCs comprised of mesopores and macropores would improve enzyme activity greatly by the promotion of diffusion and partition, and the reduction of blocking area in catalytic reactions. The specific enzyme activity of LAC-NWMCs was determined by a standard colorimetric assay using syringaldazine (SYR) (0.216 mM) in citric acid buffer as the substrate. Kinetics of the oxidation of SYR by LAC-NWMCs and LAC at their optimum conditions showed that LACNWMCs exhibited a faster reaction rate than that of free LAC (Supporting Information, Part 2, Figure S8). The relative enzyme activity of LAC-NWMCs is approximately 10-fold higher than that of free LAC (Figure 3A). This enhancement in specific activity is the highest value for immobilized LAC reported so far (Supporting Information, Part 2, Table S2). Moreover, the LAC-NWMCs also exhibited high thermal and chemical stability. The LAC-NWMCs maintained a higher residual activity of 96.42% even at 60oC in comparison to the 93.82% residual activity for free LAC (Supporting Information, Part 2, Figure S4C); it attained maximum enzymatic activity at pH 3 (high acid toleration), while free LAC lost 89.47% activity at that pH (Supporting Information, Part 2, Figure S4D). Reusability of the LAC-NWMCs was tested and plotted in Fig. 3B. The LAC-NWMCs retained 72.54% of initial enzymatic activity over 10 cycles of reaction, much better than laccase-Cu3(PO4)2·3H2O nanoflowers (Supporting Information, Part 3, Figure S10). Such a good reusability may benefit from both high stability of NWMC nanocarriers and intense enzyme-carrier multipoint covalent conjugation, which


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improve enzyme stability and prevent desorption of enzyme in an active form.10 As a result, the LAC-NWMCs also possess high storage stability, with only 12.69% loss of the initial activity after two months incubation in PBS (pH 6.5) at room temperature (Supporting Information, Part 2, Figure S9A). It is worth noting that no obvious morphological changes were observed in the LAC-NWMCs during all the tests (inset of Figure 3B; Supporting Information, Part 2, Figure S9B), suggesting high mechanical stability of the NWMC nanocarriers. For a fair comparison of the catalytic activities of various nanobiocatalysts reported previously, we defined specific nanobiocatalyst activity as A′=U/B, where B is the amount (mg) of both enzyme and nanocarrier. The relative nanobiocatalyst activities of various enzyme-nanostructure hybrid materials are presented in Fig. 3C. It was reported that the immobilized enzymes with high specific enzyme activity actually have low specific nanobiocatalyst activity relative to their native counterparts. In contrast, the LAC-NWMCs showed the highest nanobiocatalyst activity, 2.2-fold higher compared to the native LAC. These results again verified the prominent catalytic performance of LAC-NWMCs, for which the improvement of enzyme activity and high-density spatial distribution of enzymes were integrated perfectly. A series of experiments were further performed to unveil the mechanisms behind the ultrahigh biocatalytic activity of LAC-NWMCs. As a copper-containing enzyme, the activity of LAC has long been known to be activated by copper ions.13,37 However, most studies of immobilized LAC focused on the effect of Cu2+ ion on the activity of LAC,13,37 while the effect of Cu+ ion has not been given much importance, probably due to its low solubility in aqueous solution. The NWMCs, as a kinetically stable nanosystem undergo an inherent dissolution-crystallization dynamic process in solution,38 leading to the interfacial release of Cu+ ion. The released Cu+ ions may suffer from oxidation to become Cu2+ ions, eventually resulting in a copper (Cu+/Cu2+) ion-


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richened microenvironment within NWMCs. Therefore, we first measured the effect of monovalent and divalent copper ions on the activity of LAC. As shown in Figure 4A, both Cu+ and Cu2+ ions can improve the activities of LAC, but the Cu+ ions induced a higher enzyme activity, approximately 1.8-fold higher than Cu2+ ions. In addition, the optimal concentration for Cu+ is about 0.09 mM, which is far less than that of Cu2+ (0.9 mM) (Supporting Information, Part 4, Figure S11). The significant enhancement in Cu+ ions-induced activity is possibly due to the fact that only Cu+ ions can be efficiently incorporated into the type 2 copper-depleted active sites and reconstitution of LAC.39-41 In contrast, Cu2+ ions contributed to the enhancement in activity by their positive effect on the intramolecular electron transfer.13, 37 The concentrations of copper ions released from LAC-NWMCs were then measured at pH ranging from 2 to 8, where equal weight of LAC-NWMCs was separately dispersed in a 3 mL buffer solution. The pH dependent concentrations of Cu+/Cu2+ ions and enzyme activity are shown in Fig. 4B. As expected, the measured copper ions contained both Cu+ and Cu2+ ions, which increased with decreasing pH value. The maximum activity of LAC-MWMCs occurred when pH value is 3, and the corresponding Cu+ concentration was approximately 0.07 mM, a concentration very close to the optimized Cu+ concentration for free LAC. The effect of Cu2+ ions is identified by measuring LAC activity under different ratios of Cu+/Cu2+ with a fixed Cu+ ion concentration of 0.07 mM (Supporting Information, Part 4, Figure S11D). The optimized ratio for free LAC is around 0.12 that coincides with the ratio observed for LAC-NWMCs (0.13). These results suggested that the NWMCs act as a “solid ion reservoir” to produce a mixed Cu+ and Cu2+ ion-richened microenvironment, contributing to the remarkably enhanced enzyme activity of immobilized LAC over the Cu2+ compounds based nanocarriers.


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Besides the activation effect of environment-responsive ions, the structural architecture of nanocarriers also plays a vital role in prompting the catalytic activity of LAC-NWMCs, which are closely associated with the mass transfer process and 3D spatial arrangement of immobilized enzyme. To illuminate the impact of the unique structure of LAC-NWMCs on their catalytic activity, Cu2O nanocubes (NCs) and nanowires (NWs) were prepared to immobilize LAC for comparison study (Supporting Information, Part 5, Figure S12).42 Figure 4D and 4E show the SEM images of as-prepared LAC-NCs and LAC-NWs with respect to the LAC-NWMCs (Figure 4F). The LAC-NCs exhibited uniform cubic morphology of size ~120 nm, while the LAC-NWs showed a high-anisotropic wire structure of diameter ~90 nm. The Michaelis–Menten curves of LAC-NCs, LAC-NWs, and LAC-MWMCs are plotted in Fig. 4G. The plot lies on the premise of setting equivalent initial concentrations of immobilized LAC to 3.57×10-6 mM. Unlike the laccase-Cu3(PO4)2 ‧3H2O nanoflowers in which enzymes may undergo nanoscale entrapment resulting in non-Michaelis-Menten kinetics, all nanobiocatalysts in our study exhibited Michaelis-Menten kinetics. The detailed kinetic parameters are summarized in Table S3. It was found that the LAC-NWMCs attained the highest catalytic efficiency kcat/Km (75.6×10-3 s-1 µM1

), approximately 10 and 8 times higher than that of LAC-NCs (7.05×10-3 s-1 µM-1) and LAC-

NWs (8.94×10-3 s-1 µM-1). The catalytic efficiency of LAC-NWMCs was also much higher than Cu+-activated LAC (25.2×10-3 s-1 µM-1). The different morphologies of these nanobiocatalysts give rise to various diffusion pathways for the substrate (product) as schematically illustrated in the insets of Fig. 4(D-F). The nanoscale LAC-NCs and LAC-NWs were more likely to suffer from partial aggregation temporarily or permanently, during the catalytic reaction, which would inevitably increase the diffusion resistance of substrates and products, leading to reduced apparent enzyme activity. It is thus reasonable to attribute this high kcat/Km to the well-organized


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and open morphology of LAC-NWMCs with the interpenetrating nanoscale channels and 3D ordered spatial arrangement of the enzyme. These features not only shortened the diffusion distance and reduced the substrate/product inhibition effect, but also offered spatially abundant accessible sites for catalytic reactions.3,43 To determine the efficiency of the nanocarriers, the specific carrier activities (SCA=U/C, C is the amount of nanocarrier) of the nanobiocatalysts were calculated (Figure 4H). The SCAs were 0.27, 0.45 and 2.44 U mg-1 for LAC-NCs, LACNWs, and LAC-NWMCs, respectively. The high SCA of LAC-NWMCs implied that such a system can provide more accessible catalytic sites with minimized “dead volume”. The ironcontaining horseradish peroxidase (HRP),44 the metal-free enzyme uricases (URI)45, and βgalactosidase (GAL)46 were empoyed to replace LAC to further verify the favorable effect of the structure of the NWMC nanocarriers on enzyme activity (Figure 4I). All of them showed enhanced enzyme activity after immobilization, approximately 3.6, 1.5, and 2.3 times higher than that of their native enzyme counterparts, confirming that the extraordinary morphology of NWMCs indeed prompts catalytic activity. Additionally, since the enhancement ratio of for metal-containing HRP is larger than that for URI and GAL, the adaptive copper ions activation may also occur for immobilized HRP.47 Encouraged by the promising catalytic activity of LAC-NWMCs, we exploited their potential in the catalytic biodegradation of 2,4-dichlorophenol (2,4-DCP), phenol, m-cresol, and epinephrine, all of which are representative hazardous contaminants of industrial wastewater (e.g. paper and pulp, textile and dye, food, and distillery).48-53 Our results showed that the efficiency of LAC-NWMCs was considerably higher (~4.34 times) than free LAC in removing hazardous contaminants (Supporting Information, Part 8, Figure S14). This high efficiency benefited from the well-organized spatial structure and environment-responsive Cu+/Cu2+ ion


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activation effect. The phenolic molecules diffused freely into the LAC-NWMCs and reached the active sites of immobilized LAC, followed by catalytic oxidization to the products as demonstrated in Supporting Information Part 8 Figure S17. The plausible mechanism of biocatalytic oxidation phenolic substances by LAC-NWMCs is as follows:51 2 (Cu+) LAC + 1/2 O2 + 2 H+ 2 (Cu2+) LAC + Phenol

2 (Cu2+) LAC + H2O 2 (Cu+) LAC + Radical + 2 H+

The efficiency of LAC-NWMCs in wastewater bioremediation was further demonstrated using a prototype continuous-flow reactor (Figure 5A; Supporting Information, Part 8, Figure S15). Characterized by the length of 35 cm and the diameter of 0.3 cm, the tubular biodegradation reactor embedded with 60 mg LAC-NWMCs was used to remove 2,4-DCP from simulated wastewater with initial concentration of 0.05 mg mL-1 and a pH value of 3. Concentrations of 2,4-DCP were measured from sampling windows (Cn, n is the corresponding distance from the pipeline entrance) during the process. As shown in Figure 5B, it was found that the biodegradation ability was proportional to the length of the pipeline under a fixed flow rate of 60 µL min-1, as the residual 2,4-DCP (Cn) quickly decreased along the pipeline. The reactor in this condition could remove 99.17% of the residual 2,4-DCP in one cycle (40 min from entrance to end). However, the efficiency went down to ~74.82% after 10 cycles mainly due to the detachment of LAC from NWMCs, evidenced by the CLSM images of FITC-labelled LAC immobilized on NWMCs (insets of Figure 5C). Owing to the high acidic stability of NWMCs (Supporting Information, Part 8, Figure S16), the reactor regained its efficiency, up to 96.32% by automated LAC refilling and re-conjugation to NWMCs (Figure 5C), showing their remarkable recycling ability and reusability. Foreseeing their potential, we believe that the scaling up of bioinspired LAC-NWMCs nanobiocatalysts with superior comprehensive performance for


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industrial applications would significantly improve, if not revolutionize, sustainable wastewater bioremediation. The ultrahigh catalytic performance of bioinspired LAC-NWMCs can be mainly attributed to the effective cooperation of well-organized 3D spatial structure and interfacial ion activation effect. This is quite different from the reported high-efficient nanobiocatalysts such as CaHPO4α-amylase hybrid nanostructures,14 in which either the structurable configuration, or the proteincarrier interactions dominated the enhancement in catalytic properties. Therefore, the composition and structural features of NWMCs is the crucial factors that determine their catalytic performance. To apply this system to a wider range of enzymes, the construction of NWMCs with various chemical components is required, which can be solved to some extent by ion doping60 or template growth.61 Here, the interfacial ion-activation effect contributed almost half of the enhancement in enzymatic activity. However, metal ions can either activate or deactivate enzymes,12 making it necessary to choose appropriate enzymes, in terms of the type of the NWMCs. Furthermore, LAC-NWMCs show only the single-enzyme system and pHresponsive copper ions activation, while enzyme activity in the living cell is regulated by a complex physiological environment. Future enzyme-NWMCs systems could be more efficient if multiple-enzyme system and multiple-stimuli regulation can be attained. In summary, we have successfully developed a bioinspired LAC-NWMCs hybrid material that exhibits ultrahigh catalytic activity, and is a new high-efficient nanobiocatalyst for wastewater bioremediation. The ultrahigh catalytic activity is ascribed to the unique features of NWMCs, which vividly imitate the characteristics of the rER biomachinery, such as by well-organized spatial structure and environment-responsive Cu+/Cu2+ ion regulated enzyme activity. The wastewater bioremediation experiment using a continuous-flow reactor further demonstrates the


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outstanding performance of LAC-NWMCs including superior catalytic efficiency, high stability, feasibility to be recycled and scaled up, all of which are desirable for sustainable industrial chemical process. Although we have demonstrated the regulation of LAC activity by NWMCs, this system can be extended to a wider range of enzymes, for example, NWMCs coupled with various metal dopants. Therefore, our work provides the possibility for constructing a variety of high-performance nanobiocatalyst based on enzyme-nanowire mesocrystal hybrid materials, which are expected to fulfill high-efficient biocatalysis for diverse applications in biotechnology, biosensing and environmental remediation in future. Supporting Information. Experimental method, materials characterization, and additional figures including SEM images, TEM images, DLS measurement, CLSM images. AUTHOR INFORMATION Corresponding Author *E-mail: (H. F.) [email protected], Tel: (86) 29 81535040. ORCID Haiming Fan: 0000-0002-0091-772X Notes The authors declare no competing financial interest. ACKNOWLEDGMENT


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The authors wish to acknowledge financial support provided by National Natural Science Foundation of China (Grant Nos. 21376192, 31400663, 81771981, 81571809), and Natural Science Foundation of Shaanxi Province (Grant Nos. 2015JM2063 and 2017JM2031).

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Figure 1. Schematic illustration of the natural rER and the bioinspired LAC-NWMCs hybrid material. The rER, possessing an open morphology, is constituted of closely packed sheets of ER membranes with a diameter of about 100 nm between the neighboring sheets, serving as a biomachinery for high-efficient biosynthesis of proteins. The LAC-NWMCs vividly imitate the characteristics of rER, with their well-organized spatial structure and environment-responsive Cu+/Cu2+ ion-regulated enzyme activity.


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Figure 2. (A, B) SEM images of a single and large-area LAC-NWMCs. (C) TEM image of a nanowire detached from the negative-stained LAC-NWMCs. Inset: the corresponding SAED image. (D) CLSM image of a fragment of LAC-NWMCs where LAC is labeled by FITC.


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Figure 3. (A) Relative enzyme activities of LAC-NWMCs compared to the activity of free LAC that is considered as 100%. (B) Reusability of LAC-NWMCs for catalytically oxidizing SYR. The SEM image (inset) show intact morphology of LAC-NWMCs after 10 cycles. (C) Nanobiocatalyst activity comparison of representative enzyme-nanostructure systems previously reported, including ChT/Ca3(PO4)2,55 GOx@HRP,56 LAC/Cu3(PO4)2,13 Lipase-hNF,57 Cyt c/ZIF8,36 HRP/Cu3(PO4)2,59 and SBP/Cu3(PO4)258. Relative nanobiocatalyst activities were calculated from the data shown in the publications. Error bar represents the standard error derived from 3 times repeated measurements.


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Figure 4. (A) Relative enzyme activities of LAC/Cu+, and LAC/Cu2+ systems compared to the activity of free LAC that is considered as 100%. (B) The specific enzyme activities of LAC and LAC-NWMCs under various pH values. And the pH-dependent release of Cu+ and Cu2+ ions from LAC-NWMCs. (C) The specific enzyme activities of free LAC under various ratios of Cu+/ Cu2+. (D-F) SEM images of the LAC-NCs, LAC-NWs, and LAC-NWMCs. Insets: schematic illustrations of the plausible substrate diffusion pathways for these hybrid materials. (G) Steadystate kinetic assays and (H) specific nanocarrier activities of LAC-NCs, LAC-NWs, and LACNWMCs. (I) Relative enzyme activities of HRP-NWMCs, URI-NWMCs, and GAL-NWMCs compared to the activities of the respective free enzymes that are considered as 100%..


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Figure 5. (A) Schematic workflow of a home-made prototype continuous-flow reactor. The 2,4DCP contaminated wastewater is driven through the pipeline filled with LAC-NWMCs and sealed by a porous membrane. (B) Concentrations of 2,4-DCP (Cn, n=0, 4, 8 to 32) were measured from different sampling windows along the pipeline during the biodegradation process. The biodegradation ratio of 2,4-DCP by LAC-NWMCs is defined as (1-Cn/C0)×100%. In total, 10 tests were run continuously to evaluate the biodegradation efficiency and each test takes about 40 min. (C) Recycling of biodegradation reactor is accomplished by flushing the pipeline with free LAC solution for the rebinding of enzymes on NWMCs. The C32/C0 of 2,4-DCP increases from 0% (t1) to 25% (t2) in 400 min, and the new biodegradation processes (600-1000 min and 1200-1600 min) are started after rebinding of LAC on NWMCs. Insets are the fluorescence images of LAC-NWMCs, showing the states of LACs detachment and rebinding during the process.


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