Bioinspired Dual-enzyme Colloidosome Reactors for High

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Functional Inorganic Materials and Devices

Bioinspired Dual-enzyme Colloidosome Reactors for High-Performance Biphasic Catalysis Zhenning Liu, Bingdi Wang, Shenghui Jin, Zhida Wang, Lei Wang, and Song Liang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b14321 • Publication Date (Web): 07 Nov 2018 Downloaded from http://pubs.acs.org on November 8, 2018

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ACS Applied Materials & Interfaces

Bioinspired Dual-enzyme Colloidosome Reactors for High-Performance Biphasic Catalysis Zhenning Liu, a Bingdi Wang, ‡ a Shenghui Jin, c Zhida Wang, a Lei Wang, * b and Song Liang * a a

Key Laboratory of Bionic Engineering (Ministry of Education), College of Biological and

Agricultural Engineering, Jilin University, Changchun, Jilin 130022, P. R. China b

College of Life Science, Jilin University, Changchun, Jilin 130022, P. R. China

c

College of Food Science and Engineering, Jilin University, Jilin 130022, P. R. China

‡

Contributed equally

KEYWORDS: colloidosome, dual-enzyme, biphase, micro-reactors, pyridine oxidation ABSTRACT: In this paper, a novel method for the construction of colloidosome as a microreactor for dual-enzyme cascade biphasic reaction have been reported. A lipase-glucose oxidase enzyme pair is employed in this system. A water-soluble enzyme glucose oxidase (GOx) is compartmentalized inside the colloidosomes. A hydrophobic environment-favored enzyme, Candida Antarctica lipase B (CalB) is adsorbed on the outer surfaces of the colloidosomes.The catalysis system is set up by introducing these dual-enzyme-immobilized microcapsules into acetic ether. H2O2 is produced in the aqueous phase by the doped GOx, then H2O2 diffused out the microcapsules is utilzized by CalB to catalyze the oxidation of ethyl acetate. Finally, the formed peracids oxidized N-heteroaromatic in situ. Furthermore, no obvious yield decline is observed in four reaction cycles. Thus, our work provides a new strategy for the design of high performance

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biomimicking reactors for multiple-enzyme cascade reactions and further expends the potential application area of colloidosome.

INTRODUCTION Multi-step enzymatic reactions, interrelated to each other, are crucial to living organisms, in which billions of cells and organelles function as micro-compartments to ensure that various enzymatic biochemical cascades occur in confined spaces to avoid mutual interference. Meanwhile, the biomembranes of cells and organelles also provide interfaces with appropriate polarity for enzymatic catalysis. Owing to these cellular or sub-cellular structures, an organism can remain homeostasis with enormous complex biochemical reactions taking place inside.1-5 Inspired by these phenomena, synthetic cell-like micro-compartments with various functions to mimic one or more characteristics of the biological counterparts have been constructed, such as liposomes,

6-8

polymersomes,

9-13

polyelectrolyte capsules,

14-16

inorganic colloidosomes,

17-25

membrane-free, and membrane-bounded coacervate microdroplets. 26-32 Colloidosomes are a kind of shell-like microstructures formed spontaneously by nanoparticles at water/oil interfaces in Pickering emulsions. 33 Such colloidosomes hold good potentials in a wide range of applications, particularly for microcapsulation and microreactor, due to their characteristics of robustness, semipermeability and convenient self-assembly preparation.34-35 It is known that most biochemical reactions in living cells engage multiple enzymes confined in certain structures of favorable polarity to lower the activation energy and allow the reaction to proceed with high efficiency under mild conditions. 22-23, 36-39 To this point, colloidosomes formed on biphasic interfaces have unique advantages for the design and construction of synthetic cell-mimicking structures. Nevertheless, no work of immobilizing dual-enzymes to colloidosomes in Pickering emulsions for biphasic catalysis has been reported to date.


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Heterocyclic N-oxides are important compounds for their wide applications in inorganic and organic chemistry. 40-42 They can be utilized as protecting groups, auxiliary agents, 43oxidants or energy materials.

40, 44

In our previous work, a novel lipase-glucose oxidase cascade reaction

system has been designed and proven to be an efficient system for the oxidation of Nheteroaromatic compounds.

45

As show in scheme 1, in this system, glucose oxidase was

applied for the in situ generation of H2O2, which was used for the lipase-catalyzed perhydrolysis of acetic ether. Then, N-heteroaromatic compounds were oxidized to the final products by the preacid generated in the duel-enzyme catalyzed reactions. However, this dual-enzyme system encountered some drawbacks of poor stability and difficulties in recovery and recycling for enzymes. Inspired by the natural cascade reactions and cellular structures, a dual-enzyme colloidosome reactor was designed for the high performance biphasically catalyzed oxidation of Nheteroaromatic compounds (scheme 1). An organic phase (acetic ether), water phase, watersoluble catalyst glucose oxidase (GOx), and Candida antarctica lipase B (CalB) that could catalyze at the oil-water interface are converted to a water-in-oil Pickering emulsion by adding a small amount of silica emulsifiers. Being soluble in water, GOx is thus compartmentalized into water droplets with the emulsifiers surrounded outside. Lipase molecules are adsorbed on the colloidosome surfaces, which provide unique reaction interfaces for the oxidation of the Nheteroaromatic substrates dissolved in the oil phase. The dual-enzyme colloidosome reactors (DECRs) are obtained by crosslinking the silica emulsifiers with TMOS which the membrane is semipermeable (scheme 1). The biphasic catalysis system was setup by introducing these colloidosome reactors into acetic ether, hydrogen peroxide (H2O2) was produced in the aqueous phase by the doped GOx which consumes glucose as the energy source. The H2O2 diffused out the


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microcapsules was employed by CalB to catalyze the perhydrolysis of ethyl acetate. Finally, the generated peracids could catalyze the oxidation of N-heteroaromatic compounds in situ (scheme 1).

Scheme 1 Schematic illustration of the fabrication of the dual-enzyme colloidosome reactor and biphasic catalysis of pyridine using it. EXPERIMENTAL SECTIONC chemicals and instruments Candida antarctica lipase B (CalB, 10000 U/mL.One unit of CalB activity was defined as the amount of enzyme required to hydrolyze 1 μmol of p-nitrophenyl acetate per minute at 30 ℃) and glucose oxidase from A. Niger (GOx, 200 U/mg. One unit of GOx activity was defined as the amount of enzyme required to oxidise 1 μmol of ß-d-glucose to dgluconic acid and hydrogen peroxide per min at 35 ℃) were purchased from Sigma Aldrich Co. Triton X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl) phenyl ether)),


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tetraethyl orthosilicate (TEOS, 99.999%), cyclohexane (A.R), n-hexanol (98%), tert-butyl methy

ether

(MTBE,

99.9%),

octadecyltrimethosysilane

(ODTMOS),

and

octyltrimethosysilane (TMOS) were purchased from Aladdin Reagent(Shanghai) Co. Ltd. Pyridine, acetic ether, ethanol, and aqueous ammonia solution (NH3·H2O, 29%), Disodium hydrogen phosphate (Na2HPO4·12H2O), sodium dihydrogen phosphate (NaH2PO4·2H2O) were purchased from Beijing Chemical Works. Ultrapurified water (18.3 MΩ·cm@25℃) used in the all experiments was obtained from a Milli-Q Millipore system. A 500 MHz Bruker AvanceIII was used to acquire 1H NMR spectra. A JEOL JSM 4800F scanning electron microscope (SEM) was used to take SEM images. An Olympus DP80 fluorescence microscope with a DP80 digital camera was used to obtain bright field and fluorescent images. A Shimadzu IR Affinity-1 spectrophotometer was used to acquire IR spectra. A Shimadzu UV-2550PC UV−visible spectrophotometer was used to acquire UV-vis absorption spectra. An Agilent technologies 7820A gas chromatograph equipped with a flame ionization detector (FID) was used to analyze the yields of the products. A MAL VERN Zetasizer Nano ZS with a 633 nm laser (ZEN3600) was used to acquire zeta potential. Synthesis of SiO2 nanoparticle: The SiO2 nanoparticles were synthesized using a reverse microemulsion method. A clear solution was prepared by mixing 7.5 mL of cyclohexane, 1.8 mL of n-hexanol, and 1.77 mL of Triton X-100 together. To provide the templates for the formation of SiO2 nanoparticles, a water-in-oil microemulsion was obtained by adding 480 μL of water and 70 μL of TEOS to the oil mixture. To initiate the hydrolysis of TEOS, 60 μL of NH3·H2O were added to the microemulsion. After 24 hours of continuous stirring,


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the formed pure silica nanoparticles were collected and washed using acetone for twice and ethanol for 3 times in order. Surface modification of the SiO2 nanoparticle: A suspension was prepared by dispersing 150 mg of SiO2 nanoparticles in 20 mL of ethanol. To modify the surface polarity of the pure SiO2 nanoparticles, 300 μL of ODTMOS was added into the suspension. The suspension was then refluxed for 10 h. After being washed using ethanol for 3 times, resultant amphiphilic SiO2 nanoparticles (amSiO2NP) were resuspended in ethanol for future use. Fabrication of amSiO2NP colloidosomes: Typically, 10 mg of amSiO2NP were dispersed in 1 ml of MTBE by sonication. Water was then added to the MTBE suspension according to a certain SiO2 to water (S/W) ratio in mass. After one-minute of vigorous shaking, an amSiO2NP stabilized Pickering emulsion formed. Then 90 μL TMOS was added into the Pickering emulsion to crosslink the nanoparticles. After 12 hour of crosslinking reaction, 30 μL of ODTMOS was added to modify the surfaces of the formed colloidosomes. 12 hours later, the colloidosomes, washed several times with ethanol /water mixtures and PBS buffer solution (pH = 7.4), was dispersed in 1 mL acetic ether. Fabrication of dual-enzyme colloidosome reactor (DECRs): GOx and CalB solutions were prepared with PBS buffer solutions (pH=7.4). The GOx contained colloidosomes were obtained using above method, simply replacing water with GOx solution. To form a Pickering emulsion, 33 μL of 0.5 mg (100 U) GOx was added to 1 mL of MTBE suspension containing 10 mg of amSiO2NP as the aqueous phase. After the crosslinking process, the colloidosomes were washed intensively with ethanol for 4 times to remove the residual


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TMOS, methanol, and GOx bound on the outer surface of the colloidosomes. Then the GOx encapsulated colloidosomes were washed with PBS for several times to reestablish the pH 7.4 bioactive environments inside the colloidosomes. The obtained GOx encapsulated colloidosomes were dispersed in 1 mL acetic ether. Then, 20 μL (200 U) of CalB solution was added to the acetic ether suspension. The mixture was vigorously hand-shaken for one minute to ensure the adsorption of CalB on the surfaces of the reactor. Thus, DECR were prepared and ready for use. Biphasically catalyzed pyridine oxidation using dual-enzyme colloidosome reactors: The pyridine oxidation started by adding 20 μL of 1 mg/μL glucose buffer solution, and 1 μL of pyridine were added into the 1 mL of acetic ether with DECR(100 U GOx and 200 U CalB). After the mixture had been standing in water bath at 37 ℃ for 12 hours, the organic product was generated in acetic ether. At the end of reaction, the product was in the upper layer by centrifuging which could be easily isolated through liquid transfer, while the colloidosomes, contained GOx and CalB, were in the bottom. The catalysis were performed at 17 ℃, 27 ℃, 37 ℃, and 47 ℃ to study the temperature effect on the product yield. A citrate buffer (pH = 5.5) and a serious of phosphate buffers (pH = 6.5, 7.4, or 8.5) were adopted as the aqueous phase in the DERCs. In the reusability experiments, after each batch, the colloidosomes were washed with acetic ether for three times and dispersed in 1 mL of acetic ether again. Then fresh glucose buffer solution and pyridine were added for the next cycle. The yields of the reactions were determined by GC analysis. RESULTS AND DISCUSSION


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The SiO2NPs were prepared as the building blocks for the colloidosomes. To obtain evenly sized building blocks, a reverse microemulsion method was used to synthesize the pure silica nanoparticles. Using a reverse microemulsion method, the silica nanoparticles form in water droplets, which restrict the growth of nanoparticles, consequently, lead to a narrow particle size distribution. Various alkylsilane have been applied to manipulate the wetting properties of silica nanoparticles to fabricate colloidosomes22, 24, 46. ODTMOS was chosen to fabricate the amSiO2NPs from the SiO2NPs, because it provides not only hydrophobicity to form DECRs, but also an environment mimicking the nature conditions of CalB47. FTIR and SEM were employed to study the chemical properties and size distribution of the pure silica nanoparticles and amSiO2NPs. From the SEM images (Fig. S1A, B), it can be seen that both types of silica nanoparticles were uniform and monodisperse. The surface modification didn't cause significant aggregation or change of particle size, which were both 60 nm. The FTIR spectra (Fig. S1C) of both types of silica nanoparticles show broad peaks in the range from 3750 to 2500 cm-1, which corresponding to the stretching of the hydrogen-bond forming on the surfaces of the silica. The FTIR spectrum of amSiO2NPs shows peaks at 2855 cm-1, 2927 cm-1, and 2985cm-1, which represent C-H stretches of CH3 and -CH2- groups. The FTIR spectra suggests that the surfaces of the amSiO2NPs have been functionalized by octadecyl groups successfully. SiO2NPs prepared from reverse microemulsion methods are uniform nanospheres (Fig. S1B) with hydrophilic surfaces, on which silanol groups are densely distributed48. In layered MTBE/ water mixtures, the SiO2NPs only exist in the water layer because of their hydrophilicty, while the amSiO2NPs stabilize the oil-water interface (Fig. S1E). The Zeta potential of the amSiO2NPs exhibit similar trend as the pure silica nanoparticles (Fig. S1D). However, the Zeta potentials of the amSiO2NPs are higher than the ones


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of pure silica under most pH values. Above results indicated that the ODTMOS modification had tuned the wetting properties of the SiO2NPs from hydrophilicty to amphiphilicty. However, a large amount of silanol groups remains on the surfaces of the amSiO2NPs. Their dissociation in the neutral and alkaline aqueous solutions causes the negative charges on the particle surfaces. Therefore, the surfaces of the amSiO2NPs are still active for the crosslinking reaction.

A

B

C

D

E

F

Fig. 1 (A) Optical image of GDCs in water; (B) fluorescence microscopy image of GDCs in water ultraviolet fluorescence （excitation block: the passband of the excitation filter is 360–370 nm, the passband of the emission filter is 420–460 nm，exposure time = 121.2 ms）; (C) SEM image of air dried GDCs; (D) optical image of DECRs in water; (E) fluorescence microscopy image of DECRs in water under ultraviolet fluorescence excitation block; (F) SEM image of air dried DECRs. GOx doped colloidosomes (GDCs) were prepared from an amSiO 2NP stabilized Pickering emulsion, in which a GOx phosphate buffer solution (PBS) played the role of


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aqueous phase. Tetramethyl orthosilicate (TMOS) was applied as the crosslinking reagent due to their mild hydrolysis condition, which minimizes the damage caused by the crosslinking process to the activity of the enzyme. To adjust the wetting property of the colloidosomes, octadecyl- trimethoxysilane (ODTMOS) was added to introduce more hydrophobic groups to their surfaces. The spherical GDCs had an average size of 27 ± 4 μm in diameter. Encapsulating a large amount of autofluorescent GOx, the colloidosomes show dark yellow color in the bright field image (Fig. 1A), which coinciding with the light pattern in the fluorescent image (Fig. 1B). The SEM images of air dried colloidosomes were taken to study their morphology (Fig. 1C). Despite the high roughness, the surfaces of the GDCs were relatively clean. With CalB adsorbed on their surfaces, the colloidosomes look similar to their previous forms in the optical and fluorescent images (Fig. 1D, 1E). However, these reactors exhibit stronger fluorescence than the GDCs do. The fluorescence intensity change was studied from the grey scale data of the images. The fluorescence intensity of the DECRs is 154 % higher than the GDCs’ in average (Fig. 1B, 1E). The increase of fluorescent intense indicated that autofluorescent CalB had been adsorbed on the surfaces of the DECRs. The SEM image of DECRs also supports the successful immobilization of CalB. In the SEM image, enzyme residuals can be observed on the surfaces of the colloidosomes (Fig. 1F).

a

b

c

d

e


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Fig. 2 (upper) Optical micrographs of GDCs with the different concentrations of emulsifier in water(S/W), scale bar is 100 μm: 0.05 (a), 0.1 (b), 0.2 (c), 0.3 (d), 0.4 (e). The relationship of GDCs diameter change with the concentrations of emulsifier: (lower left) GOx aqueous solution as water phase, (lower right) pure water as water phase. To achieve better catalytic activity, the fabrication of the DECRs was optimized. In an emulsion stabilized by traditional surfactants, the surfactant to water ratio is usually a key parameter to control the size of the droplets and the density of the surfactant layer. In a Pickering emulsion, particles work as the surfactant. Therefore, the GDCs were fabricated with the same concentration of GOx and various amSiO2NP to water (S/W) ratio (Fig. 2 upper). It could be found that the sizes of the GDCs were affected by the S/W ratio. However, their size alteration did not exhibit any clear trend related to the ratio change (Fig. 2 lower left). Interestingly, the color of the colloidosomes did turn darker, as the S/W ratio increased. To explore the fabrication mechanism, colloidosomes without enzyme immobilization were prepared as the control samples (Fig. S4). In contrast with enzyme doped colloidosomes, the sizes of these colloidosomes are dependent on the S/W ratio of the Pickering emulsion. As shown in Fig 2 lower right, the sizes of these colloidosomes


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decreased as the S/W ratio increased. Compared with the GDC with the same S/W ratio, control samples all have smaller sizes, except the one that S/W ratio is equal to 0.05. Additional to their sizes, the presence of enzyme might also affect the thickness of colloidosome shells. The SEM images show that these control samples have clear wrinkle patterns on their surfaces, which are not observed on enzyme-doped colloidosomes (Fig. S4, C). It indicates that the shells of these colloidosomes might be thinner than the GDCs’. In a typical surfactant stabilized emulsion, the increase of surfactant concentration leads to an increase of the total oil-water interface area, which causes the decrease of the droplet sizes. Since, the results of the control colloidosomes accord with the behaviours of typical reverse emulsions, the amSiO2NPs can work as common surfactant in a Pickering emulsion. Therefore, the unusual change of the colloidosome sizes indicates that the introduction of GOx affected the water droplet sizes and the formation of the colloidosomes in the Pickering emulsions. To understand the effect of GOx on the nanoparticle assembly at the interfaces, the adsorption of GOx on the amSiO2NPs was studied employing UV-Vis spectroscopy. GOx was added to 1mL water suspension containing 10 mg of amSiO2NPs. After five minute of quick eddying, the suspension was centrifuged. The absorption spectra were taken from the supernatant and a control GOx solution. As demonstrated in Fig. 3A, the absorption peak of GOx in the UV region reduced by more than 50% compared with the control sample. These results reveals the presence of the nonspecific binding between GOx and the amSiO2NPs, which could involve GOx in the formation of the colloidosomes. Therefore, the GOx might be not only present inside the colloidosomes, but also within their shells. When S/W is low, the large amount of GOx in the colloidosome shells leaded to defects of their crosslinking structures, consequently the leakage of the doped enzyme


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(Fig. S5). Considering GOx leakage and permeability of the colloidosome shell, 0.3 was select as the optimized S/W for the DECR fabrication. To minimize the leakage of GOx, the amounts TMOS and ODTMOS used in the crosslinking was also optimized. Based on an assumption that the mass of a DECR equals to the mass of its silica structure, the loading capacity of GOx and CalB in DECRs are determined to be 9.3 U/mg and 20 U/mg, respectively (Fig. S6).

A

B

Fig. 3 (A) UV−vis absorption spectra of a GOx aqueous solution (black curve) and a GOx aqueous solution stirred with SiO2 (blue curve). (B) Yields of the dual-enzyme biphasic catalysis pyridine oxidation with different amounts of CalB. As a model reaction, the oxidation of pyridine was studied to demonstrate the efficiency of DECR. Using 1H NMR (Fig S8), the product was identified to be pyridine-Noxide, which was found to be the product of the conventional GOx/CalB biphasic catalysis in our previous work45. It’s suggests that the immobilization of these enzyme pair doesn’t change the mechanism of the catalysis of pyridine oxidation. The yields were determined from the integral peak areas of the starting materials and products in their gas chromatography graphs (Fig S9). The effect of the amount of CalB introduced to the


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DECRs on this oxidation was investigated. Fig. 3B shows that the yield of pyridine N-oxide increase with the increasing of CalB immobilized on the outer surfaces of colloidosomes when the CalB amount was less than 200 U. Further increasing of enzyme amount may cause the aggregation of the colloidosomes and significant decline of reaction yield (Fig. S10).

A

B

Fig. 4 (A) Temperature effect on the yield of the pyridine oxidation catalyzed by DECR in biphasic systems; (B) pH effect on the yield of pyridine oxidation catalyzed by DECR in biphasic systems. The thermal and pH stabilities of DECRs were investigated by studying the temperature and pH effects on the yields of pyridine oxidation. In the study of temperature effect, the pyridine oxidations were performed at 17 ℃, 27 ℃, 37 ℃, and 47 ℃. The scenarios of higher temperatures were ignored, because acetic ether is highly volatile at a temperature beyond 50℃. As shown in Fig 4A, the yield exhibits a maximum at 37 ℃. A higher or lower reaction temperature would cause a decline in the yield. Similar to the thermal stability study, the pH effect on this reaction also shows a maximum, which is at pH 7.4. These results reveal that DECRs cannot enhance the thermal and pH stability of this dual-enzyme biphasic catalysis (Fig. 4B). Since the GOx was dissolved in the aqueous phase of the DECRs, its conformation and catalytic activity would be


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influenced by the alteration of temperature and pH. Although the DECRs provide working conditions mimicking the natural environments of the enzymes, it cannot enhance their stabilities. Thus, optimized conditions of the catalysis using DECRs are pH 7.4 and 37℃, which are working conditions of most enzymes in nature. The catalytic performance of the DECRs was examined through comparison with control reactions. Previous research have proved that stirring does not lead to a significant increase in reaction activity for a Pickering emulsion reaction system, because a Pickering emulsion system has a high level of oil-water mixing, large reaction interfacial area, and short molecule diffusion distance, making the stirring unnecessary. 49 Thus, the comparison experiments were all performed in stir-free conditions (Fig. 5). As expected, no product could be detected without glucose or any of these two enzymes, which indicats that the enzymes and glucose (energy source of the reaction) are necessary to the oxidation of pyridine. In an acetic ether, the yield was very low (24.8%) because of the poor activity of GOx in hydrophobic environment. In a conventional biphasic system, the yield of this reaction increase to 33.5% with the presence of water. Using DECRs for the biphasic catalysis, the yield of pyridine-N-oxide further increased to 64.8%. The apparent Michaelis constants of GOx and GOx-CalB enzyme pair were determined using a reported method 50-51. To achieve fair comparisons, all experiments were performed in acetic ether/water biphasic systems, in which the apparent Michaelis constants of GOx, GOx-CalB without DECR, and GOx-CalB with DECR were determined to be 8.50 mg/mL, 5.11 mg/mL, and 3.26 mg/mL, respectively (Fig. S7). Since the apparent Michaelis constants of GOx-CalB pairs are higher than the value of GOx itself, the consumption of H2O2 catalyzed by CalB might enhance the affinity ability of GOx to glucose. The lowest apparent Michaelis constant was determined with the presence of DECRs. It suggested


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that the DECRs could enhance the affinity ability of GOx to glucose by providing more suitable working condition for CalB. These results demonstrate that the introduction of DECRs lead to significant enhancement of the catalytic performance of GOx and lipase, which should be attributed to the large oil-water interface and short molecule diffusion distance created by the colloidosomes.

a

b

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d

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Fig. 5 Catalytic performance comparison of the DECR involved cascade reaction and control reactions. Cartoon representation of various reaction systems: (a) DECR catalyzed biphasic reaction, (b) conventional biphasic reaction, (c) organic phase reaction, (d) colloidosome involved biphasic reaction without GOx, (e) DECR involved biphasic reaction without glucose.


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A

B

Fig. 6 (A) Yeilds of the pyridine oxidation catalyzed by the DECRs for 4 cycles, reaction time of each cycle was 12 h. (B) Optical image of the DECRs after four catalysis cycles, reaction time of reach cycle = 12 h. Generally, immobilization can improve the stability of enzyme and increase its reusability, which makes the enzymatic process economically viable. In this work, the reusability of DECRs was studied. The DECRs were recycled after each batch, which was initiated by adding glucose and lasted for 12 hours. The yields of products were quite similar in four cycles (Fig. 6A) respectively. In the fourth cycle, the DECR maintained over 90% of original activity. Furthermore, the DECRs exhibited good durability in the recycling. No cracked colloidosome was observed under microscope after four cycles (Fig. 6B). These results suggest that the DECRs are reusable and durable as designed, and exhibit a great potential as the biphasic reactors for efficient and sustainable biocatalysis. CONCLUSION In conclusion, we have reported a novel method for constructing colloidosomes as microreactors for dual-enzyme cascade biphasic reaction. By compartmentalizing GOx molecules inside and adsorbing CalB molecules on the outer surfaces, the DECRs were fabricated with simple


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enzyme immobilization approaches. Using pyridine as the model substance, DECRs exhibited high performance for biphasic catalysis of oxidation of N-heteroaromatic compounds. With the presence of DECR, the yield of pyridine-N-oxide was 93% higher than the conventional biphasic system at stirring-free conditions. Meanwhile, the crosslinked colloidosomes, achieving a reasonable mechanical robustness, could be collected and transferred into other phases. Recycled for four times, DECRs remain 90% of apparent activity for the catalysis of pyridine oxidation. To the best of our knowledge, this is the first report of such a dual-enzyme colloidosome reactor. Overall, our work provides a new strategy for design of high performance biomimicking reactors for biphasic multiple-enzyme involved cascade reactions and further expends the potential application area of colloidosomes. ASSOCIATED CONTENT Supporting Information. SEM images, FTIR spectra, the zeta potential of nanoparticles as the function of pH, picture of SiO2 nanoparticles and amSiO2 nanoparticles in layered MTBE/water mixtures; optical microscopy images, droplet size distributions, fluorescence microscopy and SEM images of the Pickering emulsions with water; optical microscopy images with GOx encapsulated and their droplet size distributions; the figure of the leakage ratio of GOx; the UV−vis absorption spectra of a GOx aqueous solution; the Michaelis constants; the 1H NMR spectrum of the final product, the gas chromatograph analysis and the picture of thin-layer chromagraphy ananlysis of the product; the picture of dual-enzyme biphasic catalysis system with the different amounts of CalB. AUTHOR INFORMATION Corresponding Author


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*E-mail: [email protected] *E-mail: [email protected] ORCID Song Liang: https://orcid.org/0000-0002-9728-0613 Lei Wang: https://orcid.org/0000-0002-9728-0613 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Notes There are no conflicts to declare. ACKNOWLEDGMENT The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (No. 21673098), Province Joint Fund (SXGJQY20171) and JLUSTIRT Program of Jilin University, and Education Department of Jilin Province (Grant Nos. 3D518M502418).

REFERENCES (1) Brangwynne, C. P. Phase transitions and size scaling of membrane-less organelles. J Cell Biol 2013, 203 (6), 875-881, DOI: 10.1083/jcb.201308087. (2) Budin, I.; Szostak, J. W. Physical effects underlying the transition from primitive to modern cell membranes. P Natl Acad Sci USA 2011, 108 (13), 5249-5254, DOI: 10.1073/pnas.1100498108.


19


Page 20 of 27

(3) Hammer, D. A.; Kamat, N. P. Towards an artificial cell. Febs Lett 2012, 586 (18), 28822890, DOI: 10.1016/j.febslet.2012.07.044. (4) Mann, S. Systems of Creation: The Emergence of Life from Nonliving Matter. Accounts Chem Res 2012, 45 (12), 2131-2141, DOI: 10.1021/ar200281t. (5) Pohorille, A.; Deamer, D. Artificial cells: prospects for biotechnology. Trends Biotechnol 2002, 20 (3), 123-128, DOI: 10.1016/S0167-7799(02)01909-1. (6) Kumar, R. K.; Yu, X. X.; Patil, A.; Li, M.; Mann, S. Cytoskeletal-like Supramolecular Assembly and Nanoparticle-Based Motors in a Model Protocell. Angew Chem Int Edit 2011, 50 (40), 9343-9347, DOI: 10.1002/anie.201102628. (7) Kurihara, K.; Tamura, M.; Shohda, K.; Toyota, T.; Suzuki, K.; Sugawara, T. Selfreproduction of supramolecular giant vesicles combined with the amplification of encapsulated DNA. Nat Chem 2011, 3 (10), 775-781, DOI: 10.1038/Nchem.1127. (8) Pontani, L. L.; van der Gucht, J.; Salbreux, G.; Heuvingh, J.; Joanny, J. F.; Sykes, C. Reconstitution of an Actin Cortex Inside a Liposome. Biophys J 2009, 96 (1), 192-198, DOI: 10.1016/j.bpj.2008.09.029. (9) Wang, L.; Wen, P.; Liu, X. M.; Zhou, Y. T.; Li, M.; Huang, Y. D.; Geng, L.; Mann, S.; Huang, X. Single-step fabrication of multi-compartmentalized biphasic proteinosomes. Chem Commun 2017, 53 (61), 8537-8540, DOI: 10.1039/c7cc04180b. (10) Marguet, M.; Bonduelle, C.; Lecommandoux, S. Multicompartmentalized polymeric systems: towards biomimetic cellular structure and function. Chem Soc Rev 2013, 42 (2), 512529, DOI: 10.1039/c2cs35312a. (11) Peters, R. J. R. W.; Louzao, I.; van Hest, J. C. M. From polymeric nanoreactors to artificial organelles. Chem Sci 2012, 3 (2), 335-342, DOI: 10.1039/c2sc00803c.


20

Page 21 of 27 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) van Dongen, S. F. M.; Nallani, M.; Cornelissen, J. L. L. M.; Nolte, R. J. M.; van Hest, J. C. M. A Three-Enzyme Cascade Reaction through Positional Assembly of Enzymes in a Polymersome Nanoreactor. Chem-Eur J 2009, 15 (5), 1107-1114, DOI: 10.1002/chem.200802114. (13) Wilson, D. A.; Nolte, R. J. M.; van Hest, J. C. M. Autonomous movement of platinumloaded stomatocytes. Nat Chem 2012, 4, 268-274, DOI: 10.1038/NCHEM.1281. (14) Chandrawati, R.; Hosta-Rigau, L.; Vanderstraaten, D.; Lokuliyana, S. A.; Stadler, B.; Albericio, F.; Caruso, F. Engineering Advanced Capsosomes: Maximizing the Number of Subcompartments, Cargo Retention, and Temperature-Triggered Reaction. Acs Nano 2010, 4 (3), 1351-1361, DOI: 10.1021/nn901843j. (15) Hosta-Rigau, L.; Shimoni, O.; Stadler, B.; Caruso, F. Advanced Subcompartmentalized Microreactors: Polymer Hydrogel Carriers Encapsulating Polymer Capsules and Liposomes. Small 2013, 9 (21), 3573-3583, DOI: 10.1002/smll.201300125. (16) Stadler, B.; Price, A. D.; Chandrawati, R.; Hosta-Rigau, L.; Zelikin, A. N.; Caruso, F. Polymer hydrogel capsules: en route toward synthetic cellular systems. Nanoscale 2009, 1 (1), 68-73, DOI: 10.1039/b9nr00143c. (17) Chen, Y. K.; Li, Z. Y.; Wang, H. Y.; Pei, Y. C.; Shi, Y. L.; Wang, J. J. Visible LightControlled Inversion of Pickering Emulsions Stabilized by Functional Silica Microspheres. Langmuir 2018, 34 (8), 2784-2790, DOI: 10.1021/acs.langmuir.7b03822. (18) Rodriguez-Arco, L.; Li, M.; Mann, S. Phagocytosis-inspired behaviour in synthetic protocell communities of compartmentalized colloidal objects. Nat Mater 2017, 16 (8), 857-863, DOI: 10.1038/NMAT4916.


21


Page 22 of 27

(19) Chen, T.; Colver, P. J.; Bon, S. A. F. Organic-inorganic hybrid hollow spberes prepared from TiO2-stabilized pickering emulsion polymerization. Adv Mater 2007, 19 (17), 2286-2289, DOI: 10.1002/adma.200602447. (20) Li, S.; Moosa, B. A.; Croissant, J. G.; Khashab, N. M. Electrostatic Assembly/Disassembly of Nanoscaled Colloidosomes for Light-Triggered Cargo Release. Angew Chem Int Edit 2015, 54 (23), 6804-6808, DOI: 10.1002/anie.201501615. (21) Wang, H. L.; Zhu, X. M.; Tsarkova, L.; Pich, A.; Moller, M. All-Silica Colloidosomes with a Particle-Bilayer Shell. Acs Nano 2011, 5 (5), 3937-3942, DOI: 10.1021/nn200436s. (22) Wu, C. Z.; Bai, S.; Ansorge-Schumacher, M. B.; Wang, D. Y. Nanoparticle Cages for Enzyme Catalysis in Organic Media. Adv Mater 2011, 23 (47), 5694-5699, DOI: 10.1002/adma.201102693. (23) Chen, Z. W.; Zhao, C. Q.; Ju, E. G.; Ji, H. W.; Ren, J. S.; Binks, B. P.; Qu, X. G. Design of Surface-Active Artificial Enzyme Particles to Stabilize Pickering Emulsions for HighPerformance Biphasic Biocatalysis. Adv Mater 2016, 28 (8), 1682-1688, DOI: 10.1002/adma.201504557. (24) Li, M.; Harbron, R. L.; Weaver, J. V. M.; Binks, B. P.; Mann, S. Electrostatically gated membrane permeability in inorganic protocells. Nat Chem 2013, 5 (6), 529-536, DOI: 10.1038/Nchem.1644. (25) Li, M.; Huang, X.; Mann, S. Spontaneous Growth and Division in Self-Reproducing Inorganic Colloidosomes. Small 2014, 10 (16), 3291-3298, DOI: 10.1002/smll.201400639. (26) Qiao, Y.; Li, M.; Booth, R.; Mann, S. Predatory behaviour in synthetic protocell communities. Nat Chem 2017, 9 (2), 110-119, DOI: 10.1038/Nchem.2617.


22

Page 23 of 27 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


(27) Crosby, J.; Treadwell, T.; Hammerton, M.; Vasilakis, K.; Crump, M. P.; Williams, D. S.; Mann, S. Stabilization and enhanced reactivity of actinorhodin polyketide synthase minimal complex in polymer-nucleotide coacervate droplets. Chem Commun 2012, 48 (97), 11832-11834, DOI: 10.1039/c2cc36533b. (28) Koga, S.; Williams, D. S.; Perriman, A. W.; Mann, S. Peptide-nucleotide microdroplets as a step towards a membrane-free protocell model. Nat Chem 2011, 3 (9), 720-724, DOI: 10.1038/Nchem.1110. (29) Tang, T. Y. D.; Antognozzi, M.; Vicary, J. A.; Perriman, A. W.; Mann, S. Small-molecule uptake in membrane-free peptide/nucleotide protocells. Soft Matter 2013, 9 (31), 7647-7656, DOI: 10.1039/c3sm50726b. (30) Tang, T. Y. D.; Hak, C. R. C.; Thompson, A. J.; Kuimova, M. K.; Williams, D. S.; Perriman, A. W.; Mann, S. Fatty acid membrane assembly on coacervate microdroplets as a step towards a hybrid protocell model. Nat Chem 2014, 6 (6), 527-533, DOI: 10.1038/Nchem.1921. (31) Williams, D. S.; Koga, S.; Hak, C. R. C.; Majrekar, A.; Patil, A. J.; Perriman, A. W.; Mann, S. Polymer/nucleotide droplets as bio-inspired functional micro-compartments. Soft Matter 2012, 8 (22), 6004-6014, DOI: 10.1039/c2sm25184a. (32) Williams, D. S.; Patil, A. J.; Mann, S. Spontaneous Structuration in Coacervate-Based Protocells by Polyoxometalate-Mediated Membrane Assembly. Small 2014, 10 (9), 1830-1840, DOI: 10.1002/smll.201303654. (33) Marina, P. F.; Xu, J.; Wu, X.; Xu, H. L. Thinking outside the box: placing hydrophilic particles in an oil phase for the formation and stabilization of Pickering emulsions. Chem Sci 2018, 9 (21), 4821-4829, DOI: 10.1039/c8sc00678d.


23


Page 24 of 27

(34) Cho, J.; Cho, J.; Kim, H.; Lim, M.; Jo, H.; Kim, H.; Min, S. J.; Rhee, H.; Kim, J. W. Janus colloid surfactant catalysts for in situ organic reactions in Pickering emulsion microreactors. Green Chem 2018, 20 (12), 2840-2844, DOI: 10.1039/c8gc00282g. (35) Douliez, J. P.; Martin, N.; Beneyton, T.; Eloi, J. C.; Chapel, J. P.; Navailles, L.; Baret, J. C.; Mann, S.; Beven, L. Preparation of Swellable Hydrogel-Containing Colloidosomes from Aqueous Two-Phase Pickering Emulsion Droplets. Angew Chem Int Edit 2018, 57 (26), 77807784, DOI: 10.1002/anie.201802929. (36) Yang, H. Q.; Fu, L. M.; Wei, L. J.; Liang, J. F.; Binks, B. P. Compartmentalization of Incompatible Reagents within Pickering Emulsion Droplets for One-Pot Cascade Reactions. J Am Chem Soc 2015, 137 (3), 1362-1371, DOI: 10.1021/ja512337z. (37) Zhang, M.; Wei, L. J.; Chen, H.; Du, Z. P.; Binks, B. P.; Yang, H. Q. Compartmentalized Droplets for Continuous Flow Liquid Liquid Interface Catalysis. J Am Chem Soc 2016, 138 (32), 10173-10183, DOI: 10.1021/jacs.6b04265. (38) Zhang, M.; Ettelaie, R.; Yan, T.; Zhang, S. J.; Cheng, F. Q.; Binks, B. P.; Yang, H. Q. Ionic Liquid Droplet Microreactor for Catalysis Reactions Not at Equilibrium. J Am Chem Soc 2017, 139 (48), 17387-17396, DOI: 10.1021/jacs.7b07731. (39) Zhou, W. J.; Fang, L.; Fan, Z. Y.; Albela, B.; Bonneviot, L.; De Campo, F.; Pera-Titus, M.; Clacens, J. M. Tunable Catalysts for Solvent-Free Biphasic Systems: Pickering Interfacial Catalysts over Amphiphilic Silica Nanoparticles. J Am Chem Soc 2014, 136 (13), 4869-4872, DOI: 10.1021/ja501019n. (40) Goetz, A. E.; Shah, T. K.; Garg, N. K. Pyridynes and indolynes as building blocks for functionalized heterocycles and natural products. Chem Commun 2015, 51 (1), 34-45, DOI: 10.1039/c4cc06445c.


24

Page 25 of 27 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


(41) Xi, P. H.; Yang, F.; Qin, S.; Zhao, D. B.; Lan, J. B.; Gao, G.; Hu, C. W.; You, J. S. Palladium(II)-Catalyzed Oxidative C-H/C-H Cross-Coupling of Heteroarenes. J Am Chem Soc 2010, 132 (6), 1822-1824, DOI: 10.1021/ja909807f. (42) Chen, J. R.; Hu, X. Q.; Lu, L. Q.; Xiao, W. J. Formal [4+1] Annulation Reactions in the Synthesis of Carbocyclic and Heterocyclic Systems. Chem Rev 2015, 115 (11), 5301-5365, DOI: 10.1021/cr5006974. (43) Hwang, H.; Kim, J.; Jeong, J.; Chang, S. Regioselective Introduction of Heteroatoms at the C-8 Position of Quinoline N-Oxides: Remote C-H Activation Using N-Oxide as a Stepping Stone. J Am Chem Soc 2014, 136 (30), 10770-10776, DOI: 10.1021/ja5053768. (44) Goetz, A. E.; Garg, N. K. Regioselective reactions of 3,4-pyridynes enabled by the aryne distortion model. Nat Chem 2013, 5 (1), 54-60, DOI: 10.1038/Nchem.1504. (45) Yang, F. J.; Zhang, X. W.; Li, F. X.; Wang, Z.; Wang, L. A lipase-glucose oxidase system for the efficient oxidation of N-heteroaromatic compounds and tertiary amines. Green Chem 2016, 18 (12), 3518-3521, DOI: 10.1039/c6gc00780e. (46) Yang, J. S.; Liu, L.; Han, S. Y. Preparation of octyl-grafted alginate-amide gel particle and its application in Pickering emulsion. Colloid Surface A 2017, 529, 320-327, DOI: 10.1016/j.colsurfa.2017.06.018. (47) Palomo, J. M.; Munoz, G.; Fernandez-Lorente, G.; Mateo, C.; Fernandez-Lafuente, R.; Guisan, J. M. Interfacial adsorption of lipases on very hydrophobic support (octadecylSepabeads): immobilization, hyperactivation and stabilization of the open form of lipases. J Mol Catal B-Enzym 2002, 19, 279-286, DOI: 10.1016/S1381-1177(02)00178-9. (48) Xu, S. P.; Hartvickson, S.; Zhao, J. X. Increasing Surface Area of Silica Nanoparticles With a Rough Surface. Acs Appl Mater Inter 2011, 3 (6), 1865-1872, DOI: 10.1021/am200052a.


25


Page 26 of 27

(49) Wei, L. J.; Zhang, M.; Zhang, X. M.; Xin, H. C.; Yang, H. Q. Pickering Emulsion as an Efficient Platform for Enzymatic Reactions without Stirring. Acs Sustain Chem Eng 2016, 4 (12), 6838-6843, DOI: 10.1021/acssuschemeng.6601776. (50) Dubey, N. C.; Tripathi, B. P.; Muller, M.; Stamm, M.; Ionov, L. Bienzymatic Sequential Reaction on Microgel Particles and Their Cofactor Dependent Applications. Biomacromolecules 2016, 17 (5), 1610-1620, DOI: 10.1021/acs.biomac.5b01745. (51) Farrugia, T.; Perriman, A. W.; Sharma, K. P.; Mann, S. Multi-enzyme cascade reactions using protein-polymer surfactant self-standing films. Chem Commun 2017, 53 (13), 2094-2097, DOI: 10.1039/c6cc09809f.


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Bioinspired Dual-enzyme Colloidosome Reactors for High

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