Multifunctional HollowâShell Microspheres Derived from Cross

Article Cite This: Chem. Mater. 2018, 30, 1625−1634

pubs.acs.org/cm

Multifunctional Hollow−Shell Microspheres Derived from CrossLinking of MnO2 Nanoneedles by Zirconium-Based Coordination Polymer: Enzyme Mimicking, Micromotors, and Protein Immobilization Jian Sun, Yaqi Fu, Rong Li, and Wei Feng* Department of Biochemical Engineering, Beijing University of Chemical Technology, Beijing, China S Supporting Information *

ABSTRACT: MnO2 microspheres were prepared with nanoneedles vertically growing on their surfaces. The MnO2 microspheres were used as templates for the growth of zirconium-based coordination polymer (Zr-CP)/metal−organic-framework, which was synthesized by coordination between Zr(IV) ions and 2-methylimidazole in methanol at room temperature. The nanoneedles were enwrapped by Zr-CP, and the bases of the MnO2 nanoneedles were cross-linked with Zr-CP. Etching of the core MnO2 produced a hollow−shell structure h-MnO2@Zr-CP. The shell, consisting of a loose outer layer and a dense inner layer, possesses oxidase- and catalase-like activities. h-MnO2@Zr-CP can work as a micromotor with H2O2 as the fuel. Hexahistidine-tagged enzymes can be selectively and strongly absorbed onto the hollow−shell microspheres with a large capacity by taking advantage of the coordinative interactions between the hexahistidine-tags and Zr-CP. DAmino acid oxidase (DAAO) was immobilized on the hollow−shell microspheres, and the conjugate DAAO/h-MnO2@Zr-CP mimicked two-enzyme catalysis, achieving a catalysis efficiency 225% times that of free DAAO. R-ω-Transaminase (RTA) and DAAO were coimmobilized on the hollow−shell microspheres, and the conjugate RTA/DAAO/h-MnO2@Zr-CP mimicked three-enzyme catalysis. For converting (R)-1-phenylethylamine and (R)-1-aminoindan to corresponding ketones, RTA/DAAO/h-MnO2@ZrCP achieved the conversions 99 and 95%, respectively, in contrast to corresponding conversions 48.6 and 32.3% by the mixed free enzymes RTA+DAAO. Potentially, the hollow−shell microspheres can be applied to immobilize other enzymes to establish multienzyme systems.

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INTRODUCTION Porous metal−organic-frameworks (MOFs) or coordination polymers (PCPs), which are coordinated metal ions linked by anionic carboxylate or imidazolate ligands,1,2 have attracted great interest. Zeolitic imidazolate frameworks (ZIFs), which are formed via coordination between metal centers and organic imidazole linkers, have been extensively investigated. Hollowtype PCPs particles with thin shells and large internal cavities are the ideal materials for mass transfer due to their fascinating properties, i.e. high surface-to-volume ratio, low density, short diffusion paths, and shell permeability.3−8 Hollow-type PCPs particles have a great potential for a wide range of technological applications, including enzyme immobilization, drug delivery, photonic devices, catalysis, and chemical sensing.9−14 Several methods have been developed for the synthesis of hollow PCPs particles such as sacrificial templates, spray deposition, and colloidal approaches.15−17 Polystyrene beads have been used as sacrificial materials for the fabrication of welldefined hollow PCPs.4,12 A hollow structured ZIF-8 nanosphere as an efficient heterogeneous catalyst for cycloaddition reactions was constructed with polystyrene beads as sacrificial templates.12 HKUST-1 enwrapped the polystyrene sphere, forming the first shell.18 After Pt particles were embedded, the © 2018 American Chemical Society

secondary shell was formed by a zinc imidazolate framework. The hollow Pd/MOF nanosphere was used as catalyst for hydrogenation reaction.18 Hollow ZIF-8/Pt hybrids were fabricated using polystyrene microspheres as templates.19 The hybrids were used to detect volatile organic compounds. ZIF-71 membrane on the ceramic hollow fiber support was prepared, and the composite was used for recovery of ethanol from ethanol−water mixtures.20 Using an in situ solvothermal synthesis method, zirconium(IV)-based metal−organic framework (Zr-MOF) membranes were fabricated on alumina hollow fibers, and the membrane can reject the multivalent ion.21 Without using template materials, hollow metal−organic framework microparticles were fabricated through a one-step solvothermal reaction of Zn(NO3)2 and 3,5-benzenetricarboxylic acid.22 Nanosized emulsion droplets were used as templates with polyvinylpyrrolidone (PVP) as stabilizing agent; hollow ZIF-8 nanospheres were prepared, and Pd nanocubes can be encapsulated into the cavities of the ZIF-8 nanospheres.23 A metal−organic framework/polyelectrolyte hollow hybrid nanoReceived: November 27, 2017 Revised: February 12, 2018 Published: February 12, 2018 1625

DOI: 10.1021/acs.chemmater.7b04945 Chem. Mater. 2018, 30, 1625−1634

Article

Chemistry of Materials material was prepared by first encapsulating ZIF-8 crystallites with poly(vinylphosphonic acid) and then by fragmenting the crystallites.24 The hybrids exhibit good proton conductivity. Core−shell ZIF-67@ZIF-8 was fabricated using ZIF-67 as template. Hollow Zn/Co ZIF particles were obtained from ZIF-67@ZIF-8 and utilized as catalyst for acetylene hydrogenation.25 Through utilization of the strong interaction between Fe3+ species and catechols, hollow metal−organic nanoparticles were prepared.26 A series of well-defined metal− organic-frameworks, including MOF-5, FeII-MOF-5, FeIIIMOF-5, and FeIII-ICP with hollow nanocages were fabricated for catalytic reactions involving gases.27 Various materials and methodology have been investigated for immobilization of enzymes.28,29 Enzyme immobilization can enhance stability, reusability, and enzymatic activity.30−32 Metal−organic-frameworks have been extensively used to immobilize enzymes. MIL-88A framework hollow spheres were fabricated via interfacial reaction in-droplet microfluidics and used for enzyme immobilization.9 Microperoxidase was immobilized into a mesoporous MOF consisting of nanoscopic cages to improve enzymatic activity.33 Uricase and horseradish peroxidase were immobilized on hierarchical micro/mesoporous MOFs,34 and glucose oxidase and horseradish peroxidase were immobilized on a hierarchical porous MOF35 and ZIF-8.36 A nerve agent hydrolyzing enzyme, organophosphorus acid anhydrolase, was immobilized on a water-stable zirconium MOF for nerve agent hydrolysis.37 Soybean epoxidehydrolase was immobilized onto UiO-66-NH2 MOF by glutaraldehyde cross-linking for the synthesis of valuable vicinal diols.38 Catalase was embedded into ZIF-90 and ZIF-8 to improve the enzyme stability,39 and a mesoporous catalase@ZIF composite was synthesized to improve catalase activity.40 Fabricating hollow nano- and microarchitectures with delicate compositions is still a challenge. Manganese oxide is a good candidate for fabrication of nano- and micromaterials due to its low cost and abundant supply.41 Herein, we report the fabrication of hollow−shell microspheres using MnO2 microsphere as template, as illustrated in Scheme 1. MnO2

were used to immobilize enzymes, mimicking two-enzyme and three-enzyme catalysis.

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EXPERIMENTAL SECTION

Materials. Ethylenediaminetetraacetic acid disodium salt (EDTA· 2Na), 2-methylimidazole, Zr(NO3)4·5H2O, MnSO4·H2O, K2S2O8, K2SO4, AgNO3, methanol, ethanol, D-alanine, pyridoxal-5-phsophate (PLP), (R)-1-phenylethylamine, (R)-1-aminoindan, sodium pyruvate, o-phenylenediamine (OPD), polyvinylpyrrolidone (PVP), and other chemicals were purchased from Sinopharm Chemical Reagent (Shanghai, China) or Sigma-Aldrich (Shanghai, China) and used without additional purification processes. Double-distilled water was used in making solutions. The Ni−NTA resin was purchased from QIAGEN (Shanghai, China). Restriction enzymes, DNA polymerase, and DNA ligase were obtained from New England Biolabs and Fermentas. R-ω-transaminase (RTA) and D-amino acid oxidase (DAAO) were expressed and purified as described in the Supporting Information. Synthesis of Solid MnO2 Microspheres. MnO2 microspheres were prepared according the methods reported.42 In a typical synthesis, 2.028 g of MnSO4·H2O, 3.24 g of K2S2O8 and 2.091 g of K2SO4 were dissolved in 300 mL of distilled water, followed by addition of 12 mL of H2SO4 and 0.3 mmol of solid AgNO3. The mixture was stirred continuously until they formed a clear solution. Then, the solution was incubated at 45 °C for 12 h. The precipitate was filtered through a 450 nm polycarbonate membrane and rinsed with deionized water and ethanol. The collected MnO2 was dried at 80 °C for 8 h under vacuum. Synthesis of Core−Shell MnO2@Zr-CP. The synthesized MnO2 microspheres (100 mg) were added to a solution of PVP dissolved in methanol (50 mL, 1 mg/mL) and then sonicated in a bath-type sonicator for 60 min. Then, the mixture was centrifuged at 5000g for 10 min, and the supernatant was removed. The precipitate was collected and dispersed in a solution of 2-methylimidazole (2-MeIm) in methanol (25 mL, 40 mM) under sonication. Then, a separate solution of Zr(NO3)4·5H2O in methanol (25 mL, 40 mM) was added to the solution under stirring. After 10 s, the generated precipitate was filtered immediately and rinsed thoroughly with methanol solution. The precipitate was dried at 80 °C for 8 h. Synthesis of Hollow−Shell h-MnO2@Zr-CP. MnO2@Zr-CP (30 mg) was dispersed in 8 mL of distilled water under sonication for 15 min. Then, 0.186 mg of EDTA·2Na was added to the mixtures and sonicated for 1, 2, 3, 4, and 5 min, respectively. After centrifugation at 5000g for 10 min, the supernatant was removed, and the precipitate was washed with distilled water and methanol. Typically, six washes were performed. The finally collected precipitate was dried at 80 °C for 8 h. Immobilization of Enzymes on h-MnO2@Zr-CP. Six milligrams of h-MnO2@Zr-CP was dispersed in 10 mL of distilled water under sonication for 10 min. Then, 8 mL of DAAO solution (1.0 mg/mL) was added. The mixtures were incubated at 4 °C under shaking (150 rpm) for 5, 10, 20, 30, 60, and 120 min, respectively. The mixtures were then centrifuged at 5000g at 4 °C for 15 min, and the supernatants were collected. Typically, five washes were carried out, with fresh buffer added each time to remove unbound DAAO. The concentrations of DAAO in the solutions were determined using the micro bicinchoninic acid (BCA) assay.43 By measuring the protein concentrations of the original DAAO solution, supernatant, and washing solution, the amount of DAAO immobilized on h-MnO2@ZrCP was determined. Average values were obtained from triplicate measurements of three immobilization operations. RTA was immobilized on h-MnO2@Zr-CP similarly. For coimmobilization of RTA and DAAO on h-MnO2@Zr-CP, 10 mg of h-MnO2@Zr-CP was dispersed in 20 mL of distilled water under sonication for 10 min. Then, 2 mL of DAAO solution (1.0 mg/mL) and 2 mL of RTA solution (1.0 mg/mL) were added. The mixtures were incubated at 4 °C under shaking (150 rpm) for 2 h. The amount of two enzymes immobilized was determined as described above.

Scheme 1. Illustration of the Process for Fabricating Hollow−Shell Microspheres EDTA· 2Na:Ethylenediaminetetraacetic Acid Disodium Salt

microspheres were synthesized with vertically growing nanoneedles on their surfaces. The MnO2 microspheres were used as templates for the growth of zirconium-based coordination polymer (Zr-CP). The nanoneedles were enwrapped by Zr-CP, and the bases of the MnO2 nanoneedles were cross-linked with Zr-CP. Etching of the core MnO2 produced a hollow−shell structure h-MnO2@Zr-CP. The hollow−shell microspheres 1626


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Chemistry of Materials

Figure 1. SEM and TEM images for the solid MnO2 microsphere Left: SEM; right: TEM. (a and c) Overall look of the microsphere. (b and d) Manifested images for observation of the nanoneedles.

Figure 2. SEM and TEM images for the MnO2@Zr-CP core−shell microspheres. Left: SEM; right: TEM. (a and c) Overall look of the microsphere. (b and d) Manifested images for observation of the Zr-CP-enwrapped nanoneedles.

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Figure 3. SEM and TEM images for the hollow−shell microsphere h-MnO2@Zr-CP. Left: SEM; right: TEM. (a and c) Overall look of the microsphere. (b and d) Manifested images for observation of the Zr-CP-enwrapped nanoneedles. Mimicking Two-Enzyme Catalysis. The enzyme kinetics for DAAO/h-MnO2@Zr-CP was investigated at 30 °C. DAAO/hMnO2@Zr-CP was dispersed in 30 mL PBS buffer (pH 8.0, 50 mM) with a protein concentration of 0.002 mg/mL. The substrate was D-alanine, which was dissolved in the PBS buffer with a concentration range from 0.5 to 40 mM. The concentrations of D-alanine and the product pyruvic acid were monitored by HPLC (Shimadzu LC-10A) with a Diamonsil C18 column. The mobile phase was a solution of PBS (0.05 M, pH 2.5) and methanol (95/5 v/v) at a flow rate of 0.8 mL/min, and the injection volume was 20 μL. Detection was performed at 220 nm. All the solutions were filtered using a 0.22 μm polycarbonate membrane (Millipore) prior to the injection. The initial rate of reaction was determined by measuring the change of substrate concentration every 1 min for 5 min. At each substrate concentration, triplicate measurements were performed, and averaged data were obtained. Kinetics parameters were determined by fitting the Michaelis−Menten equation to the initial rates of reaction. Mimicking Three-Enzyme Catalysis. The transformation of (R)2-aminooctane and (R)-1-phenylethylamine was carried out at 30 °C. (R)-1-phenylethylamine (5 mM)/(R)-2-aminooctane (5 mM), pyridoxal-5′-phosphate (1 mM), and sodium pyruvate (0.5 mM) were dissolved in Tris-HCl (pH 8.5, 100 mM). The mixed free enzymes RTA (0.125 mg/mL) + DAAO (0.125 mg/mL) and the coimmobilized enzymes RTA/DAAO/h-MnO2@Zr-CP (RTA 0.125 mg/mL, DAAO 0.125 mg/mL) were used for transformation of the amines. The concentrations of the corresponding ketones were quantified by HPLC (Shimadzu LC-10A) with a Diamonsil C18 column. The mobile phase consisted of acetonitrile/acidic acid/H2O (60/0.1/39.9 by vol) at 1.0 mL/min. The injection was 20 μL. Detection was carried out at 280 nm. All the solutions were filtered using a 0.22 μm polycarbonate membrane (Millipore) prior to the injection.

Solid MnO2 microspheres were prepared, and Figures 1a and b show the scanning electron microscopy (SEM) images for one solid MnO2 microsphere. MnO2 nanoneedles vertically grew on the surface of the microsphere, forming an aligned MnO2 nanoneedle array structure. The length of the nanoneedles is in the range from 50 to 200 nm (Figures 1c and d). The solid MnO2 microspheres were used as templates. Zr-CP grew on the surface of the solid MnO2 microspheres by coordination between Zr(IV) ions and 2-methylimidazole in methanol at room temperature, forming a core−shell microstructure MnO2@Zr-CP (Figure 2). Zr-CP itself exhibited amorphous morphology (Supplementary Figure S1). With polyvinylpyrrolidone as stabilizing agent, Zr-CP grew on the solid MnO2 microspheres, and the nanoneedles were enwrapped with ZrCP as evidenced by the amplified SEM and transmission electron microscopy (TEM) images (Figures 2b and d), in comparison to the SEM and TEM images for non-enwrapped nanoneedles (Figures 1b and d). Etching of the core MnO2 of core−shell MnO2@Zr-CP occurred during the incubation treatment in EDTA·2Na, resulting in a transformation to hollow−shell microspheres h-MnO2@Zr-CP. The morphology of the hollow−shell structure was experimentally realized (Figure 3); the TEM image clearly shows the hollow space (Figure 3c). Photographs show that h-MnO2@Zr-CP exhibited a light brown color (Supplementary Figure S2), in contrast to Zr-CP in white, MnO2 in black, and MnO2@Zr-CP in black. The color comparison confirmed that the shell of h-MnO2@ZrCP is the composite of MnO2 and Zr-CP. The opening on the hollow−shell microsphere shows that the shell is composed of a dense inner layer and a loose outer layer (Figure 3a). The magnified SEM and TEM images show that the MnO2 nanoneedles were retained after the EDTA·2Na etching due to the protective layer of enwrapped Zr-CP (Figures 3b and d).

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RESULTS AND DISCUSSION Hollow−Shell Microspheres. The approach to synthesizing the hollow−shell microspheres is illustrated in Scheme 1. 1628


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Figure 4. TEM images showing the etching process from the core−shell MnO2@Zr-CP to the hollow−shell h-MnO2@Zr-CP. All scale bars are 2 μm.

zine exhibited yellow color.44 The characteristic absorption peaks of 2,3-diaminophenazine centered at 420 nm are shown in Figure 5. The N2-saturated system exhibited the lowest

The loose outer layer consists of the array of Zr-CP enwrapped MnO2 nanoneedles. The bases of the MnO2 nanoneedles were cross-linked with Zr-CP (Figure 3d). The dense layer is the MnO2 that was left after etching, which is much thicker than the loose layer. The synthesizing process experienced core− shell, yolk−shell, and hollow−shell structures (Figure 4). The MnO2@Zr-CP yolk−shell structures from 1 to 3 min of incubation in EDTA·2Na were the intermediate structures. After 3 min, substantial etching of the core MnO2 took place. The hollow−shell structure was finally obtained after 4 min of incubation, building up a composite shell consisting of MnO2 and Zr-CP. Both SEM and TEM images show that at the fifth minute, an opening (about 1−2 μm) appeared on the shell. Longer incubation time can lead to broken shell (SEM image not shown). It is suggested that the incubation time using EDTA·2Na to etch core MnO2 was controlled within 5 min. Figure 4 shows that the EDTA·2Na etching can be controlled to obtain yolk−shell and hollow−shell structures at different etching times. Through element mapping analysis of one hollow microsphere, it is revealed that the hollow microsphere consists of Mn and Zr elements that were uniformly distributed in the microsphere (Supplementary Figure S3). Fourier transform infrared (FTIR) spectra for the samples are shown in Supplementary Figure S4. Solid MnO2 microspheres exhibited a characteristic peak at 724 nm. Both MnO2@Zr-CP (core− shell) and h-MnO2@Zr-CP (hollow−shell) exhibited the characteristic peak at 724 nm, confirming the existence of MnO2. In addition, the band at 1390 cm−1 is ascribed to the stretching of the imidazole ring, and the band at 1568 cm−1 is attributed to the C−N stretch mode, indicating the presence of Zr-CP. The FTIR spectra confirmed that the shell of the hollow−shell microspheres consists of Zr-CP and MnO2. To further gain insight into the phase structures, powder X-ray diffraction (XRD) patterns for as-prepared MnO2, Zr-CP, MnO2@Zr-CP, and h-MnO2@Zr-CP samples were measured. Supplementary Figure S5 shows that the solid MnO 2 microspheres exhibited the diffraction peaks at 2θ values of 12.8°, 18.1°, 28.8°, 37.5° and 49.9° and can be indexed to (110), (200), (310), (211), and (411) plane reflections of αMnO2 (JCPDS card 44-0141), respectively. TEM image (Supplementary Figure S1) shows that Zr-CP exhibited amorphous morphology, which is also confirmed by the XRD pattern for Zr-CP (Supplementary Figure S5). h-MnO2@Zr-CP exhibited an XRD pattern similar to that of MnO2, indicating that the shell retained the phase structure of MnO2. Hollow−Shell Microspheres Mimicking Oxidase and Catalase. The as-synthesized h-MnO2@Zr-CP was used to catalyze the oxidation of OPD, which is a typical oxidase chromogenic substrate.44 The systems included N2-, air-, and O2-saturated ones. The oxidation product 2,3-diaminophena-

Figure 5. UV−vis spectra of oxidase-like activity of h-MnO2@Zr-CP. O2-dependent (N2 → air → O2) catalytic oxidation of 10 mM OPD with 100 μg/mL h-MnO2@Zr-CP in 100 mM PBS (pH 8.0) and further incubated for 3 min at 30 °C before UV−visible spectroscopic measurements. Insets: the corresponding photo of the reaction solutions.

absorption with a light yellow color, in contrast to the O2saturated system with the highest absorption and exhibiting a heavy yellow color. The result of the air-saturated system is in between the two systems. It is indicated that the OPD oxidation was oxygen dependent. The results validated the oxidase-like activity of h-MnO2@Zr-CP. The catalytic efficiency of h-MnO2@Zr-CP was further quantitatively investigated by studying the kinetics. Supplementary Figure S6 shows the initial rate of reaction versus OPD concentration. The kinetics parameter Km was obtained based on Supplementary Figure S6 through regression using the function of Michaelis−Menten kinetics. The Km value 4.66 mM of h-MnO2@Zr-CP is smaller than 6.24 mM for MnO2@Zr-CP and much smaller than 15.25 mM for MnO2 (Supplementary Table S1). The kinetics results indicate that Zr-CP contributed significantly to improving the affinity toward substrate. Catalase-like activity of h-MnO2@ZrCP was also investigated. Supplementary Figure S7 shows the initial rate of reaction versus H2O2 concentration. The kinetics parameter Km value 51.62 mM of h-MnO2@Zr-CP is smaller than 84.60 mM for MnO2@Zr-CP and much smaller than 230.07 mM for MnO2 (Supplementary Table S2). The results of mimicking catalase further confirmed the significant contribution of Zr-CP to improving the affinity toward substrate and showed advantage of the hollow−shell structure over the core−shell structure. 1629


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Figure 6. (a) Illustration of an H2O2-powered h-MnO2@Zr-CP. H2O2 was provided as the fuel, entering into the cavity through the small opening on the shell. MnO2 of the shell catalyzed the decomposition of H2O2, and the evolved O2 drove the movement of h-MnO2@Zr-CP. (b) Microscopy image illustrating the bubble-propelled movement of h-MnO2@Zr-CP (captured in the Supplementary Video for the motor).

Figure 7. (a) Illustration of the coordinative interaction of the hexahistidine tags of DAAO with Zr(IV). (b) Selective immobilization of DAAO analyzed by SDS-PAGE. Lane 1 is for marker proteins; lane 2 is for the proteins after immobilization, and lane 3 is for the initial proteins before immobilization. (c) SEM image of DAAO/h-MnO2@Zr-CP. (d) Fluorescent microscope image of DAAO/h-MnO2@Zr-CP.

Hollow−Shell Microspheres As Micromotors. hMnO2@Zr-CP possesses catalase-like activity with a high affinity toward H2O2. Through controlled etching, the hollow−shell structure with an opening was obtained as shown in Figure 3. The hollow−shell microspheres possessing catalase-like activity suggest that h-MnO2@Zr-CP can work as micromotors with H2O2 as the fuel, as illustrated by Figure 6a. By decomposing H2O2 into O2 and water, the motion of bubble-propelled h-MnO2@Zr-CP was observed under microscopy (Figure 6b and Supplementary Video). The movement was evaluated at the H2O2 concentrations 5, 10, and 15% (v/v), as suggested in the article.45 Supplementary Video shows that h-MnO2@Zr-CP micromotors moved faster at a higher concentration of H2O2 fuel solution. The results demonstrate that chemical energy can be rapidly converted into mechanical work by h-MnO2@Zr-CP. Hollow−Shell Microspheres As Support for Selective Immobilization of Enzymes. The formation of Zr-CP on the

MnO2 microspheres was controlled by addition of Zr(IV) and 2-methylimidazole with a molar ratio of 1:1. Thus, the metal sites Zr(IV) of Zr-CP on the hollow−shell microspheres were unsaturated, facilitating their coordinative interactions with the hexahistidine-tagged enzymes. Similar interactions are routinely used for the purification of recombinant enzymes by immobilized metal ion affinity chromatography.46 Herein, the coordinative interaction is the driving force for h-MnO2@ZrCP to selectively immobilize the hexahistidine-tagged enzymes DAAO, as illustrated in Figure 7a. h-MnO2@Zr-CP was dispersed in a solution containing bovine serum albumin (BSA) and DAAO and incubated for 30 min. After centrifugation, the supernatant was separated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as shown in Figure 7b. The initial protein solution exhibited two heavy bands corresponding to BSA and DAAO, respectively (lane 3 in Figure 7b). After immobilization, the supernatant also exhibited two bands at the same positions 1630


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Figure 8. Mimicking two-enzyme catalysis. (a) Schematic presentation for the formation of hydrogen peroxide → oxygen → hydrogen peroxide cycle under the catalysis of DAAO/h-MnO2@Zr-CP. D-Alanine is converted to pyruvic acid by DAAO with h-MnO2@Zr-CP catalyzing the decomposition of the evolved H2O2, and the evolved O2 oxidizes the reduced cofactor FAD of DAAO. (b) Qualitatively comparing between DAAO and DAAO/h-MnO2@Zr-CP for catalyzing the conversion of D-alanine. (c) Plots of the initial rate of reaction versus substrate concentration for DAAO/h-MnO2@Zr-CP.

50 h for the enzyme absorption.33 It is indicated that h-MnO2@ Zr-CP can efficiently absorb DAAO. The hollow−shell structure presented a loading capacity much higher than that of the core−shell structure (0.17 mg DAAO/mg MnO2@ZrCP), demonstrating the advantage of h-MnO2@Zr-CP over MnO2@Zr-CP for enzyme immobilization. The binding stability of DAAO on h-MnO2@Zr-CP was investigated. Imidazole was used to compete with DAAO, which had been coordinately bonded to the unsaturated Zr(IV) sites on hMnO2@Zr-CP. Supplementary Figure S9 shows that with an imidazole concentration of 100 mM, less than 2% of the immobilized DAAO was stripped off. When the imidazole concentration was increased to 500 mM, less than 5% of the immobilized DAAO was stripped off, while imidazole of 500 mM can efficiently elute proteins from Ni sepharose beads in a disposable chromatography column.41 Supplementary Figure S9 demonstrates that the coordination bonds between the hexahistidine tags and Zr(IV) on h-MnO2@Zr-CP are very strong. Mimicking Two-Enzyme Catalysis. As h-MnO2@Zr-CP possesses catalase-like activity, the conjugate DAAO/h-MnO2@ Zr-CP can mimic a two-enzyme system DAAO/catalase: DAAO catalyzes the deamination of D-alanine to pyruvic acid and hydrogen peroxide is evolved,48 and h-MnO2@Zr-CP catalyzes the decomposition of the evolved H2O2 into H2O and O2, eliminating its inhibitory effect on the reaction and deleterious effect on the enzyme. The generated O2 is used to oxidize the reduced flavin adenine dinucleotide (FAD) factor of DAAO to maintain its function. The cycle of H2O2 → O2 → H2O2 can be formed between DAAO and h-MnO2@Zr-CP, as

(lane 2 in Figure 7b). However, the band for DAAO in lane 2 became much weaker compared to the initial band in lane 3. In contrast, the protein BSA still exhibited a heavy band after immobilization (lane 2), with no difference form the initial band in land 3, indicating that BSA was not absorbed onto hMnO2@Zr-CP in the presence of DAAO. The SDS-PAGE analysis indicated that h-MnO2@Zr-CP can selectively absorb DAAO through coordination bonds between the hexahistidine tags and Zr(IV). Figure 7c shows the SEM images for the saturation immobilization of DAAO on h-MnO2@Zr-CP, in which the absorbed enzyme DAAO can be clearly observed. To observe the immobilized DAAO under microscopy, DAAO was labeled with fluorescein isothiocyanate (green) and immobilized on h-MnO2@Zr-CP. The conjugate was washed with Triton X-100 and subjected to fluorescent microscopy (DeltaVision OMX V3). Luminescent cross-section of the conjugate DAAO/h-MnO2@Zr-CP was observed (Figure 7d), confirming the immobilization of DAAO. In the FTIR spectra (Supplementary Figure S4), DAAO/h-MnO2@Zr-CP exhibited the bands at 1644 and 1534 cm−1, they were assigned to amide I (CO) and amide II (N−H) of DAAO, respectively.47 The enzyme immobilization capacity of h-MnO2@Zr-CP was investigated. The amounts of immobilized DAAO versus time are shown in Supplementary Figure S8. A saturated loading amount 0.82 mg DAAO/mg h-MnO2@Zr-CP was reached after 1 h incubation. This enzyme loading capacity is much larger than that reported (0.087 mg enzyme/mg MOF).33 The enzyme absorption profile also indicated that after 30 min incubation, the enzyme loading reached more than 90% of the saturation loading. The absorption time is much shorter than 1631


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Chemistry of Materials illustrated in Figure 8a. This cycle can be a driving force for accelerating the deamination reaction. Qualitative experiment was carried out to test the catalytic capability of DAAO/h-MnO2@Zr-CP. 2,4-Dinitrophenylhydrazine (DNPH) was added to the systems after reaction. DNPH reacts with pyruvic acid, forming a colored product.49 The free DAAO system exhibited a yellow color (Figure 8b), in contrast to the DAAO/h-MnO2@Zr-CP system exhibiting a brownish color. The results demonstrated that more pyruvic acid was produced by DAAO/h-MnO2@Zr-CP, indicating that the conjugate exhibited a catalytic capability higher than that of free DAAO. Quantitative experiment was also performed for DAAO/h-MnO2@Zr-CP by investigating enzyme kinetics. Plots of initial rate of reaction (V0) versus substrate concentration are shown in Figure 8c. Corresponding Lineweaver−Burk plots are shown in Supplementary Figure S10. The kinetics parameters in Table 1 were obtained by regressing Table 1. Kinetic Parameters for the Free and Immobilized Enzymes vm (mM min−1) Km (mM) Kcat (min−1) Kcat/Km (mM−1 min−1) a

a

free DAAOa

DAAO/h-MnO2@Zr-CP

0.0287 15.37 1155.8 75.2

0.0396 9.42 1594.8 169.3

The data are from our previous work.51

the data using the equation of the Michaelis−Menten kinetics. The immobilized DAAO was compared to free DAAO with an equimolar amount of protein. The comparison of Km values indicates that DAAO/h-MnO2@Zr-CP has exhibited a higher affinity toward the substrate than free DAAO. The Kcat/Km ratio has been used commonly to measure catalytic efficiency.50 The Kcat/Km ratio of DAAO/h-MnO2@Zr-CP is 225% times that of free DAAO. It is indicated that DAAO/h-MnO2@Zr-CP has exhibited a catalytic efficiency much higher than that of free DAAO. Mimicking Three-Enzyme Catalysis. RTAs are widely used to synthesize optically pure amines.52 In the RTA catalysis process, an amino group is transferred from an amino donor (e.g., (R)-1-phenylethylamine) onto an amino acceptor (e.g., pyruvic acid), producing a coproduct (e.g., D-alanine). To minimize the amount of pyruvic acid, D-alanine can be converted to pyruvic acid in situ under the catalysis of DAAO.48 Herein, the two enzymes RTA and DAAO were coimmobilized on h-MnO2@Zr-CP. Figure 9a illustrates the conjugate RTA/DAAO/h-MnO2@Zr-CP mimicking three enzymes. RTA catalyzes the transformation of (R)-1-phenylethylamine into acetophenone, and DAAO catalyzes the conversion of D-alanine into pyruvic acid, achieving the recycling of pyruvic acid in situ (pyruvic acid → D-alanine → pyruvic acid). h-MnO2@Zr-CP catalyzes the decomposition of evolved H2O2 of DAAO, and the reduced FDA of DAAO is reoxidized by the evolved O2, resulting in the formation of the H2O2 → O2 → H2O2 cycle. To observe the coimmobilized enzymes under fluorescent microscopy, RTA was labeled with tetramethylrhodamine-6isothiocyanate (TRITC), and DAAO was labeled with fluorescein isothiocyanate (FITC). The TRITC-labeled RTA was immobilized on h-MnO2@Zr-CP, and the conjugate TRITC-RTA/h-MnO2@Zr-CP exhibited a red color (Figure 9b), in contrast to the conjugate FITC-DAAO/h-MnO2@Zr-

Figure 9. Mimicking three-enzyme catalysis. (a) Schematic presentation of RTA/DAAO/h-MnO2@Zr-CP mimicking three enzymes. Using the coimmobilized two enzymes for transformation of (R)-1phenylethylamine, RTA catalyzes the transformation of (R)-1phenylethylamine into acetophenone, and DAAO catalyzes the conversion of D-alanine into pyruvic acid, achieving the recycling of pyruvic acid in situ (pyruvic acid → D-alanine → pyruvic acid). hMnO2@Zr-CP catalyzes the decomposition of evolved H2O2 of DAAO, and the reduced FDA of DAAO is reoxidized by the evolved O2, resulting in the formation of the hydrogen peroxide → oxygen → hydrogen peroxide cycle. Fluorescent microscope images for RTA/hMnO2@Zr-CP (b) and RTA/DAAO/h-MnO2@Zr-CP (c). The conversion of (R)-1-phenylethylamine (d) and (R)-1-aminoindan (e) under the catalysis of RTA/DAAO/h-MnO2@Zr-CP.

CP exhibiting a green color (Figure 7d). When the TRITClabeled RTA and the FITC-labeled DAAO were coimmobilized on h-MnO2@Zr-CP, the conjugate exhibited yellow color due to the overlap of green and red on h-MnO2@Zr-CP (Figure 9c). The microscope images confirmed the simultaneous coimmobilization of the two enzymes on the hollow−shell sphere. The conjugate RTA/DAAO/h-MnO2@Zr-CP was used to catalyze the transformation of (R)-1-phenylethylamine and (R)1-aminoindan. Under the catalysis of the conjugate RTA/ DAAO/h-MnO2@Zr-CP, more than 99% of (R)-1-phenylethylamine was converted after 3 h, in contrast to 48.6% conversion by the mixed free enzymes (Figure 9d). Ninety-five percent of (R)-1-aminoindan was transformed after 5 h by the conjugate, in contrast to 32.3% by the mixed free enzymes (Figure 9e). The advantage of RTA/DAAO/h-MnO2@Zr-CP over the mixed free enzymes are obvious. The H2O2 → O2 → H2O2 cycle drove the deamination reaction, and the cycle of pyruvic acid → D-alanine → pyruvic acid was the driving force for the transamination reaction. The coordination of the cycles 1632



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enhanced the catalytic efficiency. Generally, enzymatic activity is affected by pH condition and temperature.28 The conjugate RTA/DAAO/h-MnO2@Zr-CP exhibited a smaller change in activity against the change of pH and temperature, as shown in Supplementary Figure S11.

CONCLUSIONS In summary, multifunctional hollow−shell microspheres were synthesized. The shell consists of a loose outer layer and a dense inner layer. The loose outer layer comprises the array of zirconium-based coordination polymer enwrapped MnO2 nanoneedles, and the dense inner layer is MnO2. The distinctive shell structure confers multiple functions to the hollow−shell microspheres, possessing oxidase- and catalaselike activities, working as micromotors with H2O2 as the fuel, and selectively and strongly immobilizing enzymes of various molecular weights with a large enzyme loading capacity. DAmino acid oxidase was immobilized on the hollow−shell microspheres, mimicking two-enzyme catalysis and achieving a catalysis efficiency 225% times that of free D-amino acid oxidase. R-ω-transaminase and D-amino acid oxidase were coimmobilized on the hollow−shell microspheres, mimicking three-enzyme catalysis. Conversion of (R)-1-phenylethylamine and (R)-1-aminoindan to corresponding ketones achieved the conversions 99 and 95%, respectively, in contrast to corresponding conversions 48.6 and 32.3% by the mixed free enzymes. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.7b04945. Description for enzyme expression; kinetics parameters for the catalysts mimicking oxidase; kinetics parameters for the catalysts mimicking catalase; TEM image for ZrCP; photographs for Zr-CP, MnO2, MnO2@Zr-CP, and h-MnO2@Zr-CP; images of SEM, Mn mapping, Zr mapping, and EDS spectra; FTIR spectra; XRD patterns; plots of the initial rate of reaction versus OPD concentrations; plots of the initial rate of reaction versus H2O2 concentration, amount of immobilized DAAO versus incubation time; elution of DAAO from DAAO/ h-MnO2@Zr-CP with imidazole; Lineweaver−Burk plots for the free and immobilized enzyme; effect of pH and temperature on retaining the enzyme activity (PDF) Video of the micromotors (AVI)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Wei Feng: 0000-0002-7046-6469 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by The National Science Foundation of China (Grants 21576018 and 21776011). 1633


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