Robust and Recyclable Two-Dimensional Nanobiocatalysts for

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Materials and Interfaces

Robust and Recyclable Two-dimensional Nanobiocatalysts for Biphasic Reactions in Pickering Emulsions Jingjing Zhao, Dong Yang, Jiafu Shi, Jie Li, Shaohua Zhang, Yizhou Wu, and Zhongyi Jiang Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.8b01297 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 17, 2018

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Industrial & Engineering Chemistry Research

Robust and Recyclable Two-dimensional Nanobiocatalysts for Biphasic Reactions in Pickering Emulsions Jingjing Zhaob,c, Dong Yanga,c, Jiafu Shia,b,*, Jie Lic, Shaohua Zhangb,c, Yizhou Wub,c, Zhongyi Jiangb,c,* a

School of Environmental Science & Engineering, Tianjin University, Tianjin 300072, P. R. China

b

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, P.

R. China c

Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical

Engineering and Technology, Tianjin University, Tianjin 300072, P. R. China *

Corresponding author: Jiafu Shi ([email protected]); Zhongyi Jiang ([email protected])

ABSTRACT: In this study, a facile, yet effective surface-engineering method was reported to confer graphene oxide (GO) nanosheets with amphiphilic feature and numerous binding sites towards enzymes for biphasic reactions in Pickering emulsions. Briefly, the surface of GO nanosheet is firstly modified and simultaneously reduced by polydopamine to endow with catechol groups. A portion of catechol groups are utilized to anchor zeolitic imidazolate framework-8 (ZIF-8) nanoparticles onto the polydopamine-modified graphene oxide (P-rGO) nanosheets through Zn2+-catechol coordination. The remaining uncoordinated catechol groups in P-rGO nanosheets are utilized to immobilize lipase onto the P-rGO nanosheets through chemical conjugation. The resulting 2D P-rGO/ZIF-8/Lipase nanobiocatalysts with an enzyme loading percent of 34.05~48.75% could be spontaneously assembled at the oil/water interface, which were then utilized to catalyze the hydrolysis of water-insoluble p-nitrophenyl palmitate (p-NPP) into water-soluble p-nitrophenol (p-NP). The 1

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Pickering emulsions, which were robustly stabilized by P-rGO/ZIF-8/Lipase, facilitated the diffusion of p-NP from the oil/water interface to aqueous phase, acquiring an enzymatic activity recovery of ~60%. Moreover, P-rGO/ZIF-8/Lipase exhibited remarkably enhanced stabilities against multiple reuses and various harsh conditions compared with free lipase, GO/Lipase and P-rGO/Lipase, showing great potentials in practical applications.

KEYWORDS: Graphene oxide nanosheets; Nanobiocatalysts; Zeolitic imidazolate framework-8; Pickering emulsions; Biphasic reactions 1.

INTRODUCTION

Two-dimensional (2D) nanomaterials, e.g., graphene/graphene oxide (GO), molybdenum disulfide, phosphorene, etc., have attracted considerable interests in various applications, including adsorption, catalysis, sensing and a series of electron/photoelectron-related applications.1-4 Particularly, in the research field of heterogeneous catalysis, GO nanosheets with two accessible sides have the potential of offering large quantities of binding sites towards molecular catalysts and enriching reactants around the active catalytic sites, which favors to accelerating the catalytic reactions.5-7 However, the diversity in chemical compositions of different molecular catalysts commonly requires the surface engineering of GO nanosheets with catalyst-binding sites.8-9 Enzymes are one typical biological molecular catalyst, which could catalyze a broad range of reactions under ambient conditions with high chemo-, region-selectivity and stereo-specificity. Since enzymes are typically active in water, the enzymatic reactions are commonly categorized into aqueous reactions and organic/aqueous biphasic reactions. During the process of aqueous reaction, enzymes, substrates and products are all located in the aqueous phase, and no phase-transfer 2


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phenomenon occurs. By contrast, during the organic/aqueous biphasic reaction, enzymes are located in the aqueous phase, while substrates of interests are usually water-insoluble. The phase transfer of substrates or products across the oil/water interface is inevitable. No matter which type of reaction, enzymes should be used in an immobilized form as free enzymes are prone to denature under harsh conditions (extreme pH, high temperature, etc.) and quite difficult to recycle. Of various carriers,10-11 GO nanosheets as one typical 2D nanomaterial have been explored for enzyme immobilization and both sides of GO nanosheets could be engineered to endow with reactive groups towards enzymes. Up to now, several enzymes, including β-amylase, xylanase, glucose oxidase, etc., have been chemically conjugated onto GO nanosheets through surface activation by glutaraldehyde, cyanuric chloride, polydopamine, etc.12-14 Desirable activity and elevated operational stabilities were achieved for a series of aqueous reactions, e.g., hydrolysis of starch, reduction of xylan, oxidation of glucose, etc.12-14 Alternatively, lipase as one typical interfacial enzyme has also been physically adsorbed on GO nanosheets for organic/aqueous biphasic reactions.15 The hydrophobic domain of GO nanosheets could help lipase to open its lids, which is beneficial for the acceleration of the reactions. Nonetheless, the weak interaction between GO nanosheets and lipase would result in the leaching of lipase from GO nanosheets, while the high hydrophilicity of GO nanosheets would confine majority of lipases within the aqueous phase rather than the organic/aqueous interface. The hydrophilicity of lipase-adsorbed GO nanosheets and hydrophobicity of water-insoluble substrate would significantly lower the contact opportunities of the substrate and enzyme at the organic/aqueous interface, causing unsatisfied activity recovery. Thus, more effective strategies to implement organic/aqueous biphasic

3



enzymatic reactions based on GO nanosheets and/or other 2D nanomaterials are still urgently required. Pickering emulsions, which refer to oil/water emulsions stabilized by colloidal nanomaterials instead of organic surfactants,16-17 have attracted broad interests as biphasic systems and shown great potentials for biphasic reactions.18-19 The surface wettability of the colloidal nanomaterials plays a critical role in stabilizing Pickering emulsions. Colloidal nanomaterials with more hydrophilic (or hydrophobic) surfaces tended to stabilize oil-in-water (or water-in-oil) emulsions. Stable Pickering emulsions could be obtained by regulating the surface wettability of colloidal nanomaterials. Since 2012, several research groups, including van Hest’s group20, Richtering’s group21, and our group22-23 have prepared various types of nanobiocatalysts through immobilizing enzymes on/in colloidal nanomaterials, e.g., polymersomes, nanogels, nanocapsules, etc., for biphasic reactions. These nanobiocatalysts could not only maximize the interfacial contacting areas of enzymes and substrates, but also facilitate the diffusion of substrates/products between the two immiscible phases, thus presenting enhanced operational stabilities against multiple reuses and harsh conditions by contrast with free enzymes. However, to the best of our knowledge, none of them were relevant to GO nanosheets or even 2D nanomaterials. In our study, graphene-based 2D nanobiocatalyst with ultrahigh enzyme loading and tunable surface wettability was prepared through surface engineering of GO nanosheets for efficient biphasic reactions in Pickering emulsions. Briefly, GO nanosheets were adopted as the supports.24-26 Dopamine was oxidative self-polymerized on the surface of GO nanosheets and simultaneously converted GO nanosheets into partially reduced GO (rGO) nanosheets, then forming 4


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polydopamine-modified rGO (P-rGO) nanosheets.27 Zeolite imidazolate framework (ZIF-8) nanoparticles were subsequently coordinated with P-rGO nanosheets to alter the surface wettability of P-rGO nanosheets for better stabilizing the Pickering emulsions.28 Finally, lipase was conjugated on the surface of P-rGO/ZIF-8 nanosheets through chemical conjugation by polydopamine. Due to the high surface area of GO nanosheets and physical/chemical interactions between enzyme and P-rGO/ZIF-8, the 2D nanobiocatalysts could have an enzyme loading percent up to 34.03~48.75%. By using lipase as a model interfacial enzyme, the as-prepared 2D P-rGO/ZIF-8/Lipase nanobiocatalysts were applied to catalyze the hydrolysis of water-insoluble p-nitrophenyl palmitate (p-NPP) into water-soluble p-nitrophenol (p-NP). The reactant, p-NPP in internal (oil) phase would diffuse to contact the nanobiocatalysts at the oil/water interface, where the reaction occurred. The as-produced p-NP would then diffuse into external (water) phase, ensuring that the reaction continuously proceeded in forward direction. The controlled assembly of our 2D P-rGO/ZIF-8/Lipase nanobiocatalysts at oil/water interface afforded the high catalytic activity, whereas the covalent binding between lipase and P-rGO nanosheets endowed the 2D nanobiocatalysts with desirable stabilities against multiple reuses and various harsh conditions. 2.

EXPERIMENTAL SECTION

2.1 Materials Graphite powder (purity 99.9995%), potassium permanganate (KMnO4), Zn(NO3)2 6H2O (99.0%), 2-methylimidazole (Hmim, 99.0%), lipase from Candida rugosa (CRL, L-1754, EC 3.1.1.3, Type VII), p-nitrophenyl palmitate (p-NPP), tris (hydroxymethyl) aminomethane (Tris) were purchased from Sigma-Aldrich Chemical Co. Ltd. (USA). Dopamine hydrochloride was purchased from 5



Yuancheng Technology Development Co. Ltd (China). n-heptane (98.5%), p-nitrophenol (p-NP), sodium dihydrogen phosphate dehydrate, sodium phosphate dibasic dodecahydrate, sodium carbonate and sodium acetate trihydrate was acquired from Tianjin Guangfu Fine Chemical Research Institute (China). All reagents were analytical grade and used without further purification. The water used in the experiments was purified by a Millipore Milli-Q purification system with a resistivity of ~15.0 MΩ cm. 2.2 Preparation of Polydopamine-Modified Reduced Graphene Oxides/Zeolitic Imidazolate Framework-8 (P-rGO/ZIF-8) Graphene was prepared from natural graphite by the modified Hummers method.29 10 mL, 2 mg mL-1 GO was dispersed by sonication for 30 min in ice bath. The pH value of the suspension was adjusted to 8.5. 10 mg dopamine was added into the suspension and sonicated for another 5 min. The solution was stirred vigorously at 60 oC for 6 h. The as-synthesized P-rGO was centrifuged at 10000 rpm for 3 min and washed for 3 times. Finally, ZIF-8 nanoparticles (1 mg, 1.5 mg and 2 mg) were added into P-rGO suspension at different mass ratio of ZIF-8: P-rGO (1:1, 1.5:1 and 2:1). After stirring for 5 min, P-rGO/ZIF-8 was obtained and collected by centrifugation at 10000 rpm for 3 min and washed for 3 times. In this study, the preparation of ZIF-8 was demonstrated as follow. Zn(NO3)2·6H2O (200 mg, 0.14 mM) in 4.8 mL of water was rapidly poured into 8 mL Hmim (2 g, 3 mM) with stirring (600 rpm) for 15 min. The as-obtained ZIF-8 nanoparticles were collected by centrifugation at 8000 rpm for 3 min and washed for 3 times. All process was conducted under room temperature (~25±2 oC). 2.3 Preparation of Pickering Interfacial Nanobiocatalysts Stabilized by P-rGO/ZIF-8/Lipase

6


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The P-rGO/ZIF-8 with different mass ratio of ZIF-8 to P-rGO (1:1, 1.5:1 and 2:1, respectively) were suspended in 2 mL of lipase solution (1.5 mg mL-1, 50 mM Tris-HCl solution, pH 7.0) with continuous shaking for 10 min, collected by centrifugation at 10000 rpm for 3 min and washed for 3 times. 2 mL aqueous dispersion of P-rGO/ZIF-8/Lipase was mixed with 1 mL n-hexane. The mixture was then vigorously shaken for 1 min to prepare Pickering emulsions. The loading percents referred to the ratio of the immobilized lipase to the weight of the supports. The immobilization efficiency referred to the ratio of the immobilized lipase to the initial introduced lipase in Tris-HCl solution. The concentration of lipase was determined using Bradford’s method. The calculation of enzyme loading percents and immobilization efficiency were based on eq. (1) and (2):

Loading percents ሺ%ሻ =

ሺ݉ −‫ܥ‬1 ܸ1 ሻ ܹ

Immobilization efficiency ሺ%ሻ =

× 100% ሺ݉ −‫ܥ‬1 ܸ1 ሻ ݉

(1)

× 100%

(2)

Where m (mg) was the amount of lipase introduced into Tris-HCl solution; C1 (mg mL-1) and V1 (mL) were the lipase concentration and the volume of supernatant, respectively; W (mg) was the weight of the supports. . 2.4 Activity Determination of Pickering Interfacial Nanobiocatalysts The Pickering interfacial nanobiocatalysts was used to catalyze the hydrolysis of p-NPP to p-NP. The p-NPP was used as the substrate in the internal oil phase and the p-NP was the product in the external water phase. Specifically, 1 mL of n-hexane containing 2 mg mL-1 of p-NPP and 2 mL of Tris-HCl solution (50mM, pH 7.0) containing P-rGO/ZIF-8/Lipase was mixed. After 10 min of stirring, the reaction was terminated by adding 1 mL of 0.5 M Na2CO3. The aqueous phase was 7



diluted 10 folds to measure the concentration of p-NP by UV-vis spectrophotometer (Hitachi U-3010) at 410 nm. Activity recovery was then defined and adopted to describe the success of the total immobilization process, which was calculated based on eq. (3):

Activity recovery (%) =

ܸ‫ܽ݋‬ ܸ‫ܽݏ‬

× 100%

(3)

Where through Vsa (µmol min-1) was the total starting activity of the free enzyme; Voa (µmol min-1) was the observed activity of the immobilized enzyme. Notably, the enzyme initially added for immobilization was fixed at 3 mg for all carriers, including GO, P-rGO, and P-rGO/ZIF-8. The Vsa and Voa were calculated by dividing the reaction time (10 min) by the amount of produced p-NP for free enzyme and immobilized enzyme, respectively.

2.5 Stabilities of Pickering Interfacial Nanobiocatalysts The pH and temperature stabilities were evaluated by measuring the residual activity of lipase after hatching the free and immobilized lipase in different pH (4.0-10.0) or temperatures (20-70 oC) for 3 h. NaAc–HAc buffer solution was chosen for pH 4.0-5.0, phosphate buffer saline (PBS) solution was chosen for pH 6.0 and Tris-HCl buffer solution was chosen for pH 7.0-10.0. The storage stability of the free and immobilized lipase was estimated by measuring the relative activity after storing in

n-hexane for a certain period of days at 4 oC. The initial activities for the free and immobilized lipase used in these stabilities evaluation were taken to be 100%. The recycling stability of immobilized lipase was assessed by measuring the enzymatic activity in each cycle. After each batch, the immobilized lipase was collected and washed with Tris-HCl (50mM, pH 7.0) and then added to the

8


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next cycle. The relative activity (%) represented the ratio of residual activity to initial activity of each sample.

2.6 Characterizations Scanning electron microscope (SEM) images of Pickering emulsions stabilized by GO nanosheets, P-rGO nanosheets and P-rGO/ZIF-8 nanosheets were recorded by using a field emission scanning electron microscope (FESEM, Nanosem 430). The water contact angles of GO nanosheets, P-rGO nanosheets, P-rGO/ZIF-8 nanosheets and P-rGO/ZIF-8/Lipase were measured using a contact angle goniometer (JC2000C Contact Angle Meter, Powereach Co., Shanghai, China). The surface chemical compositions of GO nanosheets, P-rGO nanosheets, P-rGO/ZIF-8 nanosheets, P-rGO/Lipase and ZIF-8/Lipase were analyzed by X-ray photoelectron spectroscope (XPS, PerkinElmer Phi 1600 ESCA system) using Mg Kα (1254.0 eV) as the radiation source. X-ray diffraction patterns (XRD) were recorded on a RINT2500 V X-ray diffraction-meter (Rijeka) with Cu Kα irradiation (λ=1.5406 Å). Fourier transform infrared spectroscope (FTIR) spectra were obtained on a Nicolet-6700 spectrometer with a resolution of 4 cm−1 for each spectrum.

3.

RESULTS AND DISCUSSION

3.1 Preparation and Characterizations of P-rGO/ZIF-8/Lipase Nanobiocatalysts.

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a) (1)

GO

(2)

(3)

P-rGO

P-rGO/ZIF-8

c)

b)

P-rGO/ZIF-8/Lipase

d)

Dopamine ZIF-8 Lipase

Figure 1. a) Schematic illustration of the preparation process of 2D P-rGO/ZIF-8/Lipase

nanobiocatalysts. (1) Surface coating of polydopamine on GO nanosheets, (2) Surface decoration of ZIF-8 nanoparticles on P-rGO nanosheets, (3) Surface immobilization of lipase on P-rGO/ZIF-8 nanosheets. b-d) TEM images of b) GO nanosheets, c) P-rGO nanosheets and d) P-rGO/ZIF-8 nanosheets.

The preparation of 2D P-rGO/ZIF-8/Lipase nanobiocatalysts was illustrated in Figure 1a. Oxidative self-polymerization of dopamine was firstly implemented to form polydopmaine coating on the surface of GO nanosheets. During this process, GO nanosheets were simultaneously reduced by dopamine, after which polydopamine-modified reduced graphene oxide (P-rGO) nanosheets were formed.30-31 Subsequently, the polydopamine layer served for surface decoration of ZIF-8 nanoparticles. Specifically, Zn2+ cations in ZIF-8 favorably bond to catechol groups on P-rGO nanosheets through Zn2+-catechol coordination, which led to the formation of P-rGO/ZIF-8 nanosheets. Lipase from Candida rugose was further immobilized onto P-rGO/ZIF-8 nanosheets. Briefly, such kind of microbial lipases was more commonly used than lipase derived from plants and animals mainly because of the rapid growth of microorganism, convenient production process, high catalytic activities and inexpensive costs.32-34 Lipase immobilized onto P-rGO/ZIF-8 through 10


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chemical conjugation between amine groups of lipase and uncoordinated catechol groups of polydopamine.35 Actually, the physical adsorption of lipase by ZIF-8 may also contribute to the high loading of lipase on the P-rGO/ZIF-8 nanosheets.23, 36 2D P-rGO/ZIF-8/Lipase nanobiocatalysts were finally obtained after centrifugation followed by washing with buffer and water for several times. Transmission electron microscopy (TEM) was conducted to show the topological structure of GO nanosheets, P-rGO nanosheets and P-rGO/ZIF-8 nanosheets. As shown in Figure 1b-d, ultrathin sheet structure was well-maintained after the modification of GO nanosheets with polydopamine and further decoration with ZIF-8 nanoparticles. Moreover, ZIF-8 nanoparticles with size of 200 nm~350 nm were distributed on the P-rGO nanosheets evenly without obvious aggregation. Then, we could deduce that the two-step modification process did not destroy the 2D structure of GO nanosheets.

11



Figure 2. a) Water contact angles of GO nanosheets, P-rGO nanosheets, ZIF-8/P-rGO nanosheets

(mass ratios of ZIF-8 to P-rGO were 1:1, 1.5:1 and 2:1) and ZIF-8/P-rGO/Lipase. b) XRD patterns of GO nanosheets, P-rGO nanosheets, ZIF-8/P-rGO nanosheets (mass ratios of ZIF-8 to P-rGO were 1:1, 1.5:1 and 2:1) and ZIF-8. c) FTIR spectra of GO nanosheets, P-rGO nanosheets and ZIF-8/P-rGO nanosheets (mass ratios of ZIF-8 to P-rGO were 1:1, 1.5:1 and 2:1).

To elucidate the influence of ZIF-8 nanoparticles on the structure/property of P-rGO nanosheets, different amounts of ZIF-8 nanoparticles were added during the synthesis of P-rGO/ZIF-8 nanosheets. As one of key parameters to influence the formation of Pickering emulsions, the surface wettability of different samples were firstly measured, including GO nanosheets, P-rGO nanosheets, 12


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and P-rGO/ZIF-8 nanosheets with different mass ratios of ZIF-8 to P-rGO (1:1, 1.5:1 or 2:1) and P-rGO/ZIF-8/Lipase (mass ratio of ZIF-8 to P-rGO was 1.5:1). As shown in Figure 2a, the water contact angles for all the samples were in the range of 37-80o. This indicated GO nanosheets, P-rGO nanosheets, P-rGO/ZIF-8 nanosheets and P-rGO/ZIF-8/Lipase were relatively hydrophilic and tended to stabilize oil-in-water-type Pickering emulsions.37 As was reported,23 nanoparticles with water contact angle of slightly lower than (close to) 90o could better stabilize oil-in-water-type Pickering emulsions. Hence, Pickering emulsions composed of GO nanosheets (contact angle: 37o) and P-rGO nanosheets (contact angle: 51o) may not be quite suitable as the particulate stabilizers. The incorporation of hydrophobic ZIF-8 onto P-rGO nanosheets changed the surface wettability of rGO nanosheets.38 More amount of ZIF-8 could endow the as-prepared P-rGO/ZIF-8 nanosheets with higher hydrophobicity approaching water contact angle of 90o (Figure 2a), which would have better potential in stabilizing Pickering emulsions. Interestingly, the incorporation of lipase did not alter the surface wettability of P-rGO/ZIF-8 nanosheets. As a result, 2D P-rGO/ZIF-8/Lipase nanobiocatalysts would have similar behavior to P-rGO/ZIF-8 nanosheets in stabilizing Pickering emulsions.

The successful reduction of GO nanosheets by polydopamine and coordination of ZIF-8 nanoparticle with P-rGO nanosheets were confirmed by XRD patterns and FTIR spectra. As shown in Figure 2b, the characteristic peak of GO nanosheets appeared at 11.34o corresponded to the

interlayer d-spacing of 0.758 nm due to the formation of hydroxyl, epoxy and carboxyl groups. On the contrary, neglected peaks at 11.34o were observed on P-rGO nanosheets, indicating the chemical reduction of oxygen-containing groups on GO nanosheets.27 P-rGO/ZIF-8 nanosheets exhibited similar crystal peaks to ZIF-8 nanoparticles, suggesting minor destruction of ZIF-8 nanoparticles 13



during the incorporation process. Furthermore, the absorption bands assigned to O-H (vO-H at 3450 cm-1), C-O (vC-O at 1060 cm-1), C-O-C (vC-O-C at 1250 cm-1) and C=C (vC=C at 1619.4 cm-1) in the FTIR spectrum (Figure 2c, black curve) was originated from the several oxygen-containing groups on GO nanosheets, such as hydroxyl, epoxy and carboxyl groups.39 The intensity of the bands as mentioned above decreased sharply in P-rGO nanosheets, again confirming the chemical reduction of most oxygen-containing groups in GO nanosheets.27 After decoration with ZIF-8 nanoparticles, the stretching vibrations of Zn-N, C=N and C-N appeared at 412 cm-1, 1421 cm-1 and 1142 cm-1, respectively.40 Additionally, with the amount increase of ZIF-8 nanoparticles, the intensity of these bands were gradually increased, showing the elevated loading of ZIF-8 nanoparticles on the P-rGO nanosheets. Combined with the XRD and water contact angle results, ZIF-8 nanoparticles were proven to be decorated on the surface of P-rGO nanosheets without structure changes, which owed the ability to regulate the surface wettability.

14000

C-O-C 286.6 eV

12000 10000 8000 6000

C=O 288.2 eV

4000 2000

C-O-C 286.6 eV

6000 4000

C=O 288.2 eV

C-OH 285.6 eV

10000

O1s

C-O-Zn 531.7 eV

5000

O1s

526

f)

C=O 531.3 eV

7000

C-O 532.8 eV

6000 5000

534

536

Binding Energy (eV)

538

540

526

530

532

534

536

538

540

N1s C-N 399.7 eV

2800 2600 2400 2200

C=N 400.9 eV

2000 1800

4000 532

528

3200 3000

4000 530

4000

Binding Energy (eV)

e) 9000 Intensity (a.u.)

C-OH 532.8 eV

7000

528

6000

2000

8000

8000

8000

Binding Energy (eV)

9000

3000 526

12000

0 276 278 280 282 284 286 288 290 292 294

Binding Energy (eV)

6000

14000

8000

2000

O1s

C-OH 532.8 eV

16000

10000

C-OH 285.6 eV

c) 18000

C-C 284.6 eV

12000

0 276 278 280 282 284 286 288 290 292 294

d)

C1s

Intensity (a.u.)

Intensity (a.u.)

b) 16000

C1s C-C 284.6 eV

Intensity (a.u.)

14000

Intensity (a.u.)

a)

Intensity (a.u.)

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

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528

530

532

534

536

538

540

Binding Energy (eV)

14


1600 395

400

405

Binding Energy (eV)

410

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Figure 3. a, b) XPS C1s spectra and fitted curves of a) GO nanosheets and b) P-rGO nanosheets. c-e)

XPS O1s spectra and fitted curves of c) P-rGO nanosheets, d) P-rGO/ZIF-8 nanosheets and e) ZIF-8/Lipase. f) XPS N1s spectra and fitted curves of P-rGO/Lipase.

X-ray photoelectron spectroscopy (XPS) were further employed to evaluate the alternation of surface chemical compositions of GO nanosheets before and after polydopamine modification, ZIF-8 decoration, and enzyme loading. In Figure S1, after modification with polydopamine, a new N1s peak emerged on GO nanosheets, indicating the formation of polydopamine coating on the surface of GO nanosheets.38 The C1s signals in GO and polydopamine-modified GO nanosheets showed several typical peaks that were assigned to C-C/C=C (284.6 eV), C-OH (285.6 eV), C-O-C (286.6 eV) and C=O (288.2 eV). The chemical reduction of GO nanosheets by polydopamine was evidenced by the significantly decreased intensity of the epoxide (C-O-C) groups on surface of GO nanosheets (Figure 3a and d).41-43 The increased intensity of C-OH groups may be ascribed to the introduction of

catechol groups in polydopamine. Compared to the O1s peaks of P-rGO nanosheets (Figure 3b), an emerged peak of C-O-Zn at 531.7 eV was observed in P-rGO/ZIF-8 nanosheets (Figure 3e), which confirmed the occurrence of coordination reaction between Zn2+ in ZIF-8 nanoparticles and catechol groups in polydopamine.44-45 As was well known, P-rGO nanosheets possessed chemical reactivity towards enzymes, whereas ZIF-8 nanoparticles had strong physical affinity towards enzymes. The multiple interactions between lipase and P-rGO/ZIF-8 nanosheets may contribute the high loading of enzymes, which were then studied and clarified in an indirect way. In brief, P-rGO/Lipase and ZIF-8/Lipase were, respectively, prepared and characterized by XPS for revealing the interactions. A new peak at 400.9 eV of C=N in N1s spectra of P-rGO/Lipase proved that lipase was chemically 15



conjugated onto polydopamine (Figure 3c),46-47 whereas the new peaks at 532.8 eV (C-O) and 531.3 eV (C=O) in O1s spectra of ZIF-8/Lipase demonstrated that lipase was physically adsorbed onto ZIF-8 nanoparticles (Figure 3f).

Figure 4. a) The appearance of Pickering emulsions stabilized by GO nanosheets, P-rGO nanosheets

and P-rGO/ZIF-8 nanosheets (mass ratios of ZIF-8 to P-rGO were 1:1, 1.5:1 and 2:1); Optical micrographs of Pickering emulsions stabilized by b) GO nanosheets, c) P-rGO nanosheets and d) P-rGO/ZIF-8 nanosheets. Scale bar in (b-d): 100 µm.

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Figure 5. SEM images of Pickering emulsion stabilized by a, f) GO nanosheets, b, g) P-rGO

nanosheets and P-rGO/ZIF-8 nanosheets (mass ratios of ZIF-8 to P-rGO were c, h) 1:1, d, i) 1.5:1 and e, j) 2:1).

Studies on the wettability of GO, P-rGO and P-rGO/ZIF-8 (water contact angles: 35~80 oC) demonstrated that all samples tended to stabilize oil-in-water Pickering emulsions. Based on this hypothesis, 2 mL aqueous dispersion of P-rGO/ZIF-8 were mixed with 1 mL n-hexane and then sonicated for 1 min to prepare Pickering emulsions. Figure 4 showed the physical appearance and optical micrographs of Pickering emulsions stabilized by GO, P-rGO and P-rGO/ZIF-8. Compared to GO and P-rGO, the P-rGO/ZIF-8 exhibited better stabilizing ability in the formation of Pickering emulsions with smaller diameters (Figure 4b-d). P-rGO/ZIF-8 with higher mass ratio of ZIF-8 to P-rGO stabilized Pickering emulsions with a much clearer aqueous phase. This indicated the incorporation of ZIF-8 nanoparticles could increase the stability of Pickering emulsions (Figure 4a), which was in line with the changes of surface wettability as shown in Figure 2a. SEM images showed the morphology of Pickering emulsions stabilized by GO nanosheets, P-rGO nanosheets and P-rGO/ZIF-8 nanosheets (mass ratios of ZIF-8 to P-rGO were 1:1, 1.5:1 and 2:1, Figure 5). Notably,

17



the SEM samples were prepared by dissolving the Pickering emulsions stabilized by different graphene-based nanosheets in ethanol to remove n-hexane followed by frozen dried. Clearly, the surface of P-rGO nanosheets become rough after decoration with ZIF-8 nanoparticles. P-rGO/ZIF-8 nanosheets with higher amount of ZIF-8 nanoparticles exhibited rougher surface, and aggregations of ZIF-8 nanoparticles were observed when the mass ratio of ZIF-8 to P-rGO reached 2:1. At such a high ratio, some impurities were also observed around the Pickering emulsions probably due to the detachment of ZIF-8 nanoparticles from P-rGO/ZIF-8 nanosheets. Moreover, Pickering emulsions stabilized by P-rGO/ZIF-8 nanosheets possessed smaller and more uniform diameters in dry state by contrast with the ones stabilized by GO nanosheets and P-rGO nanosheets (Figure S2), which further proved the better stabilization of P-rGO/ZIF-8 nanosheets at the oil/water interface. 3.2 Catalytic Activity and Stabilities of 2D P-rGO/ZIF-8/Lipase Nanobiocatalysts

p-NPP

b) Loading percents (%)

a)

p-NP

100

100 80 60 40 20 0 70

d)

60

80

Conversion (%)

c)

Activity recovery (%)

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

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60 40 20

50 40 30

Free lipase GO/Lipase P-rGO/Lipase P-rGO/ZIF-8/Lipase (1:1) P-rGO/ZIF-8/Lipase (1.5:1) P-rGO/ZIF-8/Lipase (2:1) GO/ZIF-8/Lipase

20 10 0

0

0

10

18


20

30

40

Time (min)

50

60

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Figure 6. a) The hydrolysis of p-NPP to p-NP catalyzed by 2D P-rGO/ZIF-8/Lipase

nanobiocatalysts. b) Enzyme loading percents of GO/Lipase, P-rGO/Lipase, P-rGO/ZIF-8/Lipase (mass ratios of ZIF-8 to P-rGO were 1:1, 1.5:1 and 2:1). c) Activity recovery of free lipase, GO/Lipase, P-rGO/Lipase, P-rGO/ZIF-8/Lipase (mass ratios of ZIF-8 to P-rGO were 1:1, 1.5:1 and 2:1). (d) Hydrolysis of p-NPP catalyzed by free lipase and immobilized lipase as a function of reaction time.

To generate Pickering emulsions, the interfacial nanobiocatalysts should possess a suitable surface wettability. In Figure 2a, we have confirmed the conjugation of lipase on P-rGO/ZIF-8 would not alter its surface wettability.47 The resultant P-rGO/ZIF-8/Lipase could well stabilize Pickering emulsions (inset of Figure 6a), suggesting the potential of P-rGO/ZIF-8/Lipase as Pickering interfacial nanobiocatalysts for biphasic catalytic reactions. Specifically, P-rGO/ZIF-8/Lipase were dispersed in the water phase and then mixed with oil phase containing p-NPP. Oil-in-water-type Pickering emulsions could be formed through vigorous shaking. The substrate p-NPP would diffuse from the internal to the interface and contact with the nanobiocatalysts, which was then catalytically converted into product p-NP. The as-produced p-NP would finally diffuse across the interface to the external aqueous phase (Figure 6a). The lipase loading percents for GO, P-rGO and P-rGO/ZIF-8 (mass ratio of ZIF-8 to P-rGO was 1.5:1) were 104.06, 64.25 and 40.33%, respectively (Figure 6b). We further compared our 2D nanobiocatalysts with other catalysts, such as membranes48,49, CRGO50 and Fe3O4 decorated RGO51. When utilized for enzyme immobilization, 2D polymer membranes usually possessed relatively lower enzyme loading percent, which was owing to the low surface area. By contrast, graphene-based carriers, including P-rGO/ZIF-8 reported in our work, exhibited much 19



higher enzyme loading percents, about 10-folds higher than the 2D polymer membranes. The immobilization efficiency increased with the mass ratio of ZIF-8 to P-rGO from 1:1 to 2:1 (Figure S3). Pickering interfacial nanobiocatalysts enabled by P-rGO/ZIF-8/Lipase exhibited similar specific

activity to those stabilized by GO/Lipase and P-rGO/Lipase. All immobilized enzymes showed a bit lower specific activity than free lipase (Figure 6c). The higher specific activity of free lipase would be caused by its intrinsic amphoteric feature which ensured lipase to stably locate at oil/water interface. The resultant emulsions would have a much higher exposed surface area, where free lipase provided more active sites for interacting with the substrate. To intensively study the catalytic process, the bioconversion of p-NPP catalyzed by Pickering interfacial nanobiocatalysts was measured with the reaction time. The reaction catalyzed by free lipase reached equilibrium in 20 min. In comparison, GO/Lipase, P-rGO/Lipase and P-rGO/ZIF-8/Lipase required 30, 40 and 50 min to reach the reaction equilibrium. The equilibrium conversion of free lipase was 62%, which was a little higher than GO/Lipase (57%), P-rGO/Lipase (60%) and P-rGO/ZIF-8/Lipase (55%, mass ratio of ZIF-8 to P-rGO was 1.5:1) (Figure 6d). The minor decrease in equilibrium conversion for the immobilized lipase was mainly attributed to the slight conformation changes of lipase during immobilization.52 For further comparison, GO/ZIF-8/Lipase was prepared by directly mixing GO with ZIF-8 followed by immobilization of lipase. Although Pickering emulsions could also be formed by this nanobiocatalysts, the p-NP conversion could only reach 40% after 40 min (the lowest value among all six immobilized lipase), indicating the essential role of polydopamine in retaining the activity of lipase.

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a)

Ⅰ

Ⅱ

Ⅲ

b)

GO/Lipase P-rGO/Lipase

P-rGO/ZIF-8/Lipase (1:1) P-rGO/ZIF-8/Lipase (1.5:1) P-rGO/ZIF-8/Lipase (2:1) GO/ZIF-8/Lipase

100

Relative activity(%)

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


80 60 40 20 0

1

2

3

4

Recycling number

5

6

Figure 7. a) Recycling process of P-rGO/ZIF-8/Lipase nanobiocatalysts including (I) In reaction, (II)

After centrifugation and (III) Re-dispersion. b) Recycling stabilities of different graphene-based nanobiocatalysts.

One of the significant advantages of Pickering interfacial nanobiocatalysts was their ease of recovery for multiple reuses. Then, we investigated the recycling stability of different types of Pickering interfacial nanobiocatalysts prepared in our study. At the end of each reaction, the catalysts could be recycled simply by centrifugation and washing for next batch (Figure 7a). The activity of GO/Lipase, P-rGO/Lipase and GO/ZIF-8/Lipase could only maintain

Robust and Recyclable Two-Dimensional Nanobiocatalysts for

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