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Oriented Enzyme Immobilization at the Oil/Water Interface Enhances Catalytic Activity and Recyclability in a Pickering Emulsion Jinghui Wang, Renliang Huang, Wei Qi, Rongxin Su, and Zhimin He Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02862 • Publication Date (Web): 02 Oct 2017 Downloaded from http://pubs.acs.org on October 9, 2017

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Oriented Enzyme Immobilization at the Oil/Water Interface Enhances Catalytic Activity and Recyclability in a Pickering Emulsion

Jinghui Wang,† Renliang Huang,*,‡, Wei Qi,*, †, § Rongxin Su†, § and Zhimin He†

†

State Key Laboratory of Chemical Engineering, School of Chemical Engineering and

Technology, Tianjin University, Tianjin 300072, P. R. China ‡

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

China §

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin 300072, P. R. China

* Author to whom any correspondence should be addressed E-mail: [email protected] (R. H.), [email protected] (W. Q.) Tel: +86 22 27407799. Fax: +86 22 27407599.

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ABSTRACT: Enzyme-loaded water-in-oil Pickering emulsion is a promising system for biphasic catalytic reactions. In this paper, we report on oriented enzyme immobilization at the oil/water interface in a Pickering emulsion, in which CHO-Janus silica nanoparticles (CHO-JNPs) are utilized as a stabilizer of the emulsion and support for the enzyme to enhance both catalytic activity and recyclability. The catalytic performance of this immobilized enzyme (lipase from Candida sp.) was evaluated by esterification of hexanoic acid and 1-hexanol in a water/heptane biphasic medium. The results show that the specific catalytic activity of the immobilized enzyme (33.2 U mL-1) was 6.5 and 1.4 times higher than that of free enzyme (5.1 U mL-1) and encapsulated enzyme in the liquid core (23.3 U mL-1), respectively. Moreover, the immobilized enzyme demonstrated good stability and recyclability, retaining 75% of its activity after 9 cycles. We expect that oriented enzyme immobilization at the oil/water interface will be an important strategy for enhancing catalytic performance in Pickering emulsions.

Key words: Janus particle, Pickering emulsion, enzyme immobilization, oil/water interface, colloidosomes

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INTRODUCTION Enzymes have extensively been used as biocatalysts for the synthesis of green chemicals and medicine due to their high chemo-, stereo-, and regioselectivity in catalytic reactions under mild conditions.1-4 However, free enzyme is sensitive to temperature, pH and organic solvent resulting in poor stability and low recyclability.5-6 For instance, organic media, extensively used as a reactant solvent, are adverse to the hydration layer of the enzyme.5 It is generally believed that immobilization of an enzyme is a convenient and appropriate way to promote stability and recyclability of the enzyme in practical catalysis.3, 5, 7-12 For instance, Kuwahara et al 13-14 immobilized lipase within silica nanoparticles with oil-filled core-shell structure for highly efficient biocatalysis. Generally, encapsulation of enzymes is a simple way that provides an aqueous micro-environment to address this problem.15-17 Many works so far have concentrated on utilizing nanoparticles to stabilize emulsions, known as Pickering emulsions, for enzyme encapsulation.6, 17-25 Pickering emulsions have been proved to be an efficient carrier of enzyme encapsulation for catalysis in organic media. Hydrophobic silica nanoparticles have generally been used as a stabilizer in Pickering emulsions to reduce surface tension of water-in-oil emulsions.17, 20, 22, 24, 26 For example, Wu et al 20 first utilized hydrophobic silica nanoparticles to stabilize a Pickering emulsion for enzyme immobilization. Recently, Zhang et al 24 used hydrophobic silica nanoparticles to stabilize a continuous flow Pickering emulsion for confining water-soluble catalysts in organic-aqueous biphasic catalysis. To enhance the stability and recyclability of Pickering emulsions, chemical cross-linking agents have been employed to crosslink nanoparticles at

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oil/water interface.6, 17, 19, 27-29 For example, Chen et al 30 used a thiol-based cross-linking agent, which was reacted with allyl-functionalized lignin in the interface, to form stable capsules. Gao et al 31 used lipase-containing dendritic mesoporous silica nanospheres to construct biocatalytic colloidosome for enhanced enzyme catalysis. In our group, we reported the synthesis of cross-linkable colloidosomes by the selective polymerization of dopamine at oil/water interfaces in a Pickering emulsion.17 As demonstrated previously, chemical cross-linking greatly strengthens the stability and recyclability of the encapsulated enzyme. However, it inevitably leads to lower mass-transfer and catalytic efficiency in some cases. There exists a dilemma for direct encapsulation of enzymes in a Pickering emulsion: while high recyclability requires a cross-linking treatment, high catalytic efficiency demands the interparticle pores remain unblocked. Janus nanoparticles (Janus NPs) are colloid-sized particles possessing two or more regions with drastically diverse chemical or physical surface properties.32-39 Some approaches, such as phase separation in (mini)emulsions,40 Pickering emulsion41 and seeded emulsion polymerization,42 have been developed to fabricate large quantities of Janus NPs with particular characteristics. The characteristics of tunable asymmetric anisotropic structure make Janus NPs appropriate for a wide range of applications. For instance, amphiphilic Janus NPs exhibit better performance in stabilizing Pickering emulsions than isotropic particles because of their hydrophobic portion immersed in the oil phase and hydrophilic portion in the aqueous phase, which has the same surface tension reducing effect as a surfactant.18, 33, 43 Yang et al.43 synthesized dumbbell-shaped bi-component mesoporous Janus NPs to stabilize Pickering emulsion for a biphasic hydrogenation catalysis reaction that exhibits more than

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three-fold increase in catalytic efficiency compared with the traditional Pt loaded catalyst. In our group, Cao et al 18 prepared amphiphilic Janus NPs to stabilize a Pickering emulsion for enzyme encapsulation, in which the enzyme was dispersed freely in the aqueous core. As we know, the enzyme reaction occurs at the oil-water interfaces, which requires contact between enzyme and substrate. Therefore, the enzyme in the aqueous core is not a good choice for such interfacial reactions. In addition, when the particles are not cross-linked together, the removal of the organic phase during the recycling process inevitably causes the collapse of emulsion droplets and thus the leakage of enzyme. To solve these problems, we attempted to construct a novel Pickering emulsion stabilized with aldehyde-functionalized Janus NPs (CHO-JNPs), which enables us to immobilize enzymes toward the aqueous core at oil/water interfaces and simultaneously enhance the emulsion’s stability via cross-linking between enzyme and particles. Herein, we present a facile strategy for enzyme immobilization at the oil/water interface of Pickering emulsions via covalent coupling of enzyme with CHO-JNPs for organic/aqueous biphasic catalysis. Specifically, CHO-JNPs were synthesized through a wax/water emulsion method 44 for asymmetric chemical modification of silica nanoparticles.18 Subsequently, the prepared CHO-JNPs were employed to form an enzyme encapsulation Pickering emulsion. After incubation, enzyme was immobilized on the surface of the CHO-JNPs via covalent coupling. The lipase from Candida sp. was chosen as the model enzyme. The catalytic performance and recyclability of this immobilized enzyme were evaluated by esterification of hexanoic acid and 1-hexanol in water/heptane biphasic media.

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EXPERIMENTAL SECTION Materials. Lipase from Candida sp. expressed in Aspergillus niger was purchased from Sigma-Aldrich. Paraffin wax (mp 58−62 °C), Tetraethyl orthosilicate (TEOS), dodecylethyldimethylammonium bromide (DDAB), (3-aminopropyl) triethoxysilane (APTES), 1,4-Phthalaldehyde (PTA), octadecyl-tri-chlorosilane (ODTS), fluorescein isothiocyanate (FITC), and 1-hexanol, hexanoic acid were obtained from Aladdin Industrial Corp. (Shanghai, China). All other chemicals, such as anhydrous ethanol, aqueous ammonia, chloroform, aqueous hydrogen peroxide solution (H2O2, 30 wt%), concentrated sulfuric acid and heptane were commercially available and of analytical grade. Preparation of silica nanoparticles. Silica nanoparticles were prepared via the StÖber method.45 In brief, 180 mL of anhydrous ethanol, 10 mL of deionized water and 15.4 mL ammonia were mixed in a 250 mL flask under vigorous stirring at 40 °C. After that 9.5 mL of TEOS was slowly added drop-wise. The resulting mixture was incubated at 40 °C and 350 rpm for 1 h. Subsequently, the silica nanoparticles were collected by centrifugation (8000 rpm, 5 min) and washed with anhydrous ethanol and deionized water to remove the excess TEOS and ammonia. The obtained silica nanoparticles were treated with piranha solution (1:3 mixture of 30% H2O2 and concentrated H2SO4) at 80 °C 300 rpm for 1 h to graft hydroxyl on the surface of the silica nanoparticles. (CAUTION: “Piranha” solution reacts sharply with organic matter and needs to be handled with extreme care.) The hydroxylated silica nanoparticles were centrifuged (8000 rpm, 5 min), washed with deionized water and finally dried at 60 °C for

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subsequent experiments. Synthesis of CHO-Janus silica nanoparticles. The CHO-Janus nanoparticles were synthesized via the wax-in-water emulsion method developed by Hong et al.44 Specifically, 0.27 g of silica nanoparticles was added to 10 mL of DDAB solution (60 mg L-1) and heated to 80 °C. Afterwards, 2.7 g of paraffin wax was added to the mixture and incubated at 80 °C for 15 min to melt the wax. The obtained mixture was stirred vigorously by a modular homogenizer at 5000 rpm for 180 s and cooled to room temperature, resulting in a great amount of solid wax colloidosome with embedded silica nanoparticles. Afterwards, the obtained wax colloidosome was filtered and washed with water to remove the extra and unsolidified embedded silica nanoparticles. To modify amino groups on the exposed side of silica nanoparticles embedded in wax colloidosome, the obtained wax colloidosome was dispersed in 5 mL methanol with 0.5 mL of APTES and incubated at 37 °C, 60 rpm for 12 h. Subsequently chloroform was added to dissolve the paraffin wax, releasing the partial amino-modified silica nanoparticles. They were gathered by centrifugation (8000 rpm, 5 min) and washed with chloroform (3 times),water (3 times) and anhydrous ethanol (3 times). To couple aldehyde groups to the amino groups on the surface of silica nanoparticles, the obtained partial amino-modified silica nanoparticles were dispersed in 15 mL acetone containing 0.26 g of 1,4-Phthalaldehyde and incubated at 37 °C, 150 rpm for 24 h. The aldehyde-modified silica nanoparticles were collected by centrifugation (8000 rpm, 5 min), washed with anhydrous ethanol, and then dried at 40 °C under vacuum. To modify hydrophobic alkyl chains onto the other side of the silica nanoparticles, the

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resulting aldehyde-modified silica nanoparticles were added to 5 mL of heptane with 0.25 mL of ODTS and incubated at 25 °C, 150 rpm for 3 h, resulting in the complete synthesis of CHO-JNPs. Eventually, the CHO-JNPs were gathered by centrifugation (8000 rpm, 5 min), washed with ethanol (3 times) and dried at 50 °C under vacuum for subsequent experiments. Preparation of interfacial enzyme immobilization in Pickering emulsion constructed by CHO-JNPs. Firstly, 20 µL aqueous stock solution of lipase (purchased from Sigma without any treatment) was diluted in 380 µL of phosphate buffered saline (PBS, 10 mM potassium phosphate, pH 7.4). Subsequently, the obtained lipase solution (400 µL) was directly added to heptane solution (2 mL) containing well-dispersed CHO-JNPs (20 mg), followed by homogenization (10000 rpm) for 1 min. The obtained lipase-loaded Pickering emulsion was incubated at 37 °C for 4 h to immobilize the enzyme on the surface of the CHO-JNPs via covalent coupling. Evaluation of the catalytic properties of free, encapsulated and interfacial immobilized lipases. The catalytic properties of free, encapsulated and immobilized lipases were investigated via the esterification of hexanoic acid with 1-hexanol in heptane/water medium. Specifically, 2 mL of heptane solution containing 663 mmol L-1 hexanoic acid and 666 mmol L-1 1-hexanol was added into the enzyme-immobilized CHO-JNP emulsion (2.4 mL) prepared as above. As a comparison, free lipase and lipase encapsulated by NH2-Janus18 were also utilized to catalyze this esterification reaction. Excepting the above-mentioned factors, the other conditions including substrate concentration, solvent composition, lipase and Ro/w were the same as introduced above. All of the esterification reactions were implemented on a rotating shaker (120 rpm) at 37 °C for precise time control. Samples (150

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µL) were extracted at different time points and centrifuged (12000 rpm, 3 min) for gas chromatography (GC) analysis. The concentrations of 1-hexanol, hexanoic acid and hexylhexanoate were measured by an Agilent GC system (7890B-G3440B) equipped with a flame ionization detector (FID) on a DB-FFAP column (30 m× 0.32 mm×0.25 µm). The temperature program was as follows: start temperature is 80 °C, holding for 0.5 min. Subsequently, temperature increases according to 20 °C min-1 from 80 to 170 °C, and 5 °C min-1 from 170 °C to the end temperature 185 °C. The temperatures of the gasify room and FID are 275 °C and 250 °C, respectively. One unit of lipase activity (U) was defined as 1 µmol of product (hexylhexanoate) per minute. The specific activity (U mL-1) of free, encapsulated and interfacial immobilized lipase was measured under the same conditions within 40 min. All the measurements were repeated 3 times. Assessment of initial and re-emulsified catalytic activity of enzyme-immobilized CHO-JNP emulsion and enzyme-encapsulated NH2-JNP emulsion. We prepared the enzyme-immobilized CHO-JNP emulsion described as above and enzyme-encapsulated NH2-JNP emulsion following the method of Cao18 as a control group. Heptane solution (2 mL) containing 663 mmol L-1 hexanoic acid and 666 mmol L-1 1-hexanol was added to start the reaction and investigate the specific activity within 40 min. Subsequently, water was added into the reaction system and centrifuged to demulsify the Pickering emulsion. After demulsification, we removed the oil & water phase and washed the particles with water 3 times to remove the free enzyme completely from the system. Four hundred microliters of phosphate buffered saline (PBS, 10 mM potassium phosphate, pH 7.4) and 2 mL of heptane,

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which were same as before, were added followed by homogenization (10000 rpm) for 1 min to re-emulsify the Pickering emulsion. Heptane solution (2 mL) containing 666 mmol L-1 1-hexanol and 663 mmol L-1 hexanoic acid was added into the obtained re-emulsified Pickering emulsion to start the reaction again and measure the specific activity within 40 min. Determination of the concentration of enzyme in the aqueous solution. The concentration of enzyme in the aqueous solution was measured by the Bradford’s method using Coomassie Brilliant Blue reagent.46 More specifically, the enzyme aqueous solution was mixed with Coomassie Brilliant Blue G-250 (0.1 g/L) with a ratio of 1:3 (v/v) for 5 min. Subsequently, the absorbance of mixture was measured at 595 nm by UV spectrum to calculate the enzyme concentration. Characterization. Optical microscopy imaging was implemented on a polarized light microscope (Shun Yu XP, China) equipped with a video camera. The average diameter distribution of the Pickering emulsion droplets was obtained through statistical analysis of more than 100 droplets. Scanning electron microscopy (SEM) images were recorded using a field-emission scanning electron microscope (FESEM, S-4800, Hitachi High-technologies Co., Japan) at an acceleration voltage of 5 kV. All samples were sputter-coated with platinum using an E1045 Pt-coater (Hitachi High-Technologies Co., Japan) before SEM observation. Elemental analysis was conducted with an energy dispersive X-ray spectrometer (EDS) equipped in the S-4800 FESEM at an accelerating voltage of 10 kV. Transmission electron microscopy (TEM) analysis was implemented on a field-emission TEM (JEM-2100F, JEOL, Japan) at an accelerating voltage of 120 kV.

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Zeta potential and dynamic light scattering measurements were performed on a Zetasizer-NANO ZSZEN3600 apparatus (Malvern Instruments, UK). The samples were prepared via dispersing 20 mg of SiO2-OH, NH2-SiO2-OH and CHO-SiO2-OH into 9 mL of deionized water. UV spectrum measurements were performed on a Dual-beam UV Spectrophotometer (TU-1900, BPGI, China). The samples were prepared by dissolving 1 mg of PTA and dispersing 5 mg of CHO-JNPs, NH2-JNPs or SiO2-OH NPs in 10 ml anhydrous ethanol. To demonstrate that the enzyme (lipase) was immobilized at oil/water interfaces, the enzyme (lipase) was first labeled with fluorescent dye (FITC). FITC-labeled lipase was prepared by overnight incubation at 4 °C in phosphate buffer (10 mM, pH 7.4) followed by dialysis (molecular weight cutoff 5 kDa) against deionized water for 72 h. Subsequently, The FITC-labeled lipase was added into heptane solution (2 mL) with well-dispersed CHO-JNPs (20 mg) by following the process introduced before. The obtained Pickering emulsion droplets were observed by laser scanning confocal microscopy (LSCM, FV-1000, Olympus, Japan) with excitation wavelength of 488 nm.

RESULTS AND DISCUSSION Scheme 1 illustrates the synthesis of CHO-JNPs and their application in enzyme immobilization at oil/water interfaces in a Pickering emulsion for biphasic biocatalysis. The CHO-JNPs were prepared through a classic method based on wax/water emulsion as described previously.44 Specially, the silica nanoparticles were prepared by the StÖber method. As shown in Figure 1a, the average diameter of the silica nanoparticles is 250 nm. The obtained silica nanoparticles, hydroxylated by piranha solution, were added into 11


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wax/water at 70 °C and then homogenized to form a Pickering emulsion. After cooling to room temperature, the hydroxylated silica nanoparticles were partially embedded on the solid wax droplets. We use APTES to graft amino groups onto the exposed surface of the silica nanoparticles. After elimination of the wax, the NH2-SiO2-OH nanoparticles were treated with PTA to couple aldehyde groups through Schiff base reaction. Afterwards, ODTS was used to graft hydrophobic chains to the other side of the silica nanoparticles to fabricate CHO-JNPs. As shown in Figure 1c-d and Figure S1, the mean diameter of the CHO-JNPs is approximate 250 nm, and the diameter distribution ranges from 230 to 320 nm, which is very close to the diameter of the original silica NPs, suggesting that the chemical surface modification has little influence on the morphology of the nanoparticles. The polydispersity index (PDI) of the CHO-JNPs is 0.371 from dynamic light scattering (DLS) measurement. As a control, we also prepared NH2-SiO2 JPs (Figure 1b) through a method reported by our group.18 To immobilize an enzyme, the as-prepared CHO-JNPs were dispersed in heptane solution and then homogenized with an aqueous solution of enzyme, forming a water-in-oil Pickering emulsion. After incubation, the enzyme was covalently immobilized on the surface of the CHO-JNPs via a Schiff base reaction between NH2 and CHO groups.

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Scheme 1. Schematic illustration of the synthesis strategy for CHO-Janus silica nanoparticles and interfacial enzyme immobilization in a Pickering emulsion constructed by CHO-JNPs via Schiff base covalent coupling for biphasic biocatalysis. The characterization of CHO-JNPs. To confirm the successful functionalization of the CHO-JNPs, FTIR spectroscopic analysis was performed. Representative FTIR spectra of hydroxylated silica nanoparticles, NH2-JNPs prepared by the method of Cao, et al.18and CHO-JNPs are shown in Figure 2a. Prior to functionalization, the spectra exhibited a broad prominent peak in the range 3700−3000 cm−1 (centered at 3417 cm−1), clearly demonstrating the presence of hydroxyl groups (Si-OH) on the surface of silica. In all of the spectra, broad absorption in the range 1200-900 cm-1 indicates the existence of asymmetric stretching at 1099 cm-1of Si-O-Si in the silica, which also possesses Si-O-Si symmetric stretching and bending vibrations at 797 and 463 cm-1, respectively. The APTES modification was demonstrated by peaks at 1467 cm-1, corresponding to C-N stretching vibration of NH2-JNPs and CHO-JNPs. Absorption from asymmetric and symmetric C-H bond (-CH2-) vibrations was observed at 2921 and 2852 cm-1, respectively, indicating the presence of hydrophobic 13


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alkyl chains on the NH2-JNPs and CHO-JNPs. It is worth mentioning that the CHO-JNPs exhibit stronger peaks at 2921 and 2852 cm-1 ascribed to aldehyde C-H and alkyl C-H stretching vibrations, respectively. Similarly, the C=N stretching vibrations were detected at 1697 cm-1 in the CHO-JNPs, which confirmed the successful covalent coupling of PTA with amino groups on the silica nanoparticles.

Figure 1. (a) SEM image of the silica particles prepared by the StÖber method. (b) SEM image of the NH2-JNPs prepared by the Wax/Water Emulsion Adsorption method. (c,d) SEM images of the CHO-JNPs. To further verify that the CHO-JNPs were successfully modified with PTA, UV spectroscopy was performed. For this purpose, equal amounts of CHO-JNPs, NH2-JNPs and hydroxylated silica NPs were dispersed in ethanol and compared with pure PTA dissolved in ethanol. As shown in Figure 2b, UV absorption at 257 nm, identical to that of PTA, appeared

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only for CHO-JNPs among the three types of nanoparticles. It is worth mentioning that although these three types of nanoparticles have UV absorption at 198 nm without exception, the CHO-JNPs have higher UV absorption than the others, which is possibly due to the synergy of UV absorption by the nanoparticles and the PTA on the CHO-JNPs. Moreover, we measured the zeta potential to study the surface charge characteristics of the nanoparticles. Because water, as the unique dispersing agent for zeta potential measurements, is averse to dispersing Janus particles, we measured the zeta potential of SiO2-OH, NH2-SiO2-OH and CHO-SiO2-OH, precursors of the Janus NPs that could disperse in water well. As shown in Figure S3, the zeta potential of the hydroxylated silica was -40.8 mV. After asymmetric modification with APTES and PTA covalent coupling to amino groups, the value shifted to +10.3 mV and +18.0 mV, respectively, indicating the successful introduction of amino groups and aldehyde groups as well as increases in stability and positive charge.

Figure 2. (a) FTIR spectra of CHO-JNPs, NH2-JNPs and SiO2 NPs. (b) UV spectra of 1,4-Phthalaldehyde (PTA), CHO-JNPs, NH2-JNPs and SiO2-OH NPs.

Characterization of the enzyme-immobilized CHO-JNP emulsion. The obtained CHO-JNPs were applied to construct a Pickering emulsion the stability of which was 15


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investigated subsequently; meanwhile NH2-JNPs prepared by the method of Cao et al18 served as the control group. Specifically, the obtained CHO-JNPs and NH2-JNPs were dispersed in heptane before adding into lipase dilution to form lipase-loaded Pickering emulsion after homogenization. Figure 3(a, e) show images of Pickering emulsions stabilized by CHO-JNPs (yellow) and NH2-JNPs (white), respectively, proving the successful introduction of aldehyde groups from another perspective. Figure 3(b,f) show microscopic images of droplets acquired from the enzyme-immobilized CHO-JNP emulsion and enzyme-encapsulated NH2-JNP emulsion, respectively. The droplets are spherical. It is worth noting that the droplet diameter of the enzyme-immobilized CHO-JNP emulsion (Figure 3c) is smaller than that of the enzyme-encapsulated NH2-JNP emulsion (Figure 3g) obviously on the premise of other equal conditions, probably owing to the introduction of aldehyde groups and enzyme immobilized at the water/oil interface as shown in Figure 3(d,h) changing the intensity of interfacial interaction. Figure S4a shows SEM images of enzyme-immobilized CHO-JNP emulsion droplets after the freeze-drying process. The CHO-JNPs were cross-linked together by something proved to be enzyme. The enzyme being immobilized at water/oil interfaces has an effect similar to a cross-linker on stabilizing the Pickering emulsion, which will be further demonstrated in the follow-up investigation of emulsion stability. We tilted the sample vials containing enzyme-encapsulated NH2-JNP emulsion and enzyme-immobilized CHO-JNP emulsion according to the same procedure as described before to observe the morphology of droplets, the dispersity as well as mobility of the Pickering emulsions changing over time clearly, which could reflect the stability of Pickering

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emulsions. The resulting Pickering emulsions were placed at room temperature without any disturbance. In contrast, the droplets of the enzyme-encapsulated NH2-JNP emulsion shown in Figure S5 (column 1) adhered together reducing the dispersity and mobility of the Pickering emulsion from 7 days to 28 days probably due to the breakdown of some Pickering emulsion droplets and releasing of the enzyme dilution that could adhere the droplets and particles. As shown in Figure S5 (column 2), for the enzyme-immobilized CHO-JNP emulsion, no obvious change in morphological appearance was observed after 7 days and some small emulsion droplets aggregated into bigger ones from 14 days to 28 days while no obvious change in dispersity or mobility of the Pickering emulsion was observed, indicating the high stability of the enzyme-immobilized CHO-JNP emulsion. It was mainly the immobilization of adhesive enzyme at water/oil interfaces, which has the same effect as cross-linking agents on stabilizing a Pickering emulsion, that provided the higher stability of the enzyme-immobilized CHO-JNP emulsion compared with the enzyme-encapsulated NH2-JNP emulsion.

Figure 3. (a, e) Pickering emulsion prepared via water in heptane (v/v=1/10) with enzyme

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(lipase) stabilized by CHO-JNPs and NH2-JNPs, respectively (b,f) Optical micrographs of CHO-JNP Pickering emulsion and NH2-JNP Pickering emulsion, respectively (c,g) Size distribution of CHO-JNP emulsion droplets and NH2-JNP emulsion droplets derived from the optical micrographs. (d,h) Confocal laser scanning microscopy images of CHO-JNP emulsion droplet containing FITC-labeled lipase and NH2-JNPs, respectively. The inset in (d, h) is fluorescence intensity of (d, h).

The catalytic performance of enzyme-immobilized CHO-JNP emulsion in organic-aqueous biphasic biocatalysis. To investigate the catalytic performance of enzyme-immobilized CHO-JNP emulsion in organic-aqueous biphasic biocatalysis, lipase (water soluble) from Candida sp. and the esterification of hexanoic acid and 1-hexanol (oil soluble) were chosen as the model enzyme and target reaction, respectively. There is no denying that the Ro/w, the amount of particle loading and thermal stability are critical parameters of organic-aqueous biphasic biocatalysis. We optimized these critical parameters to make the esterification react under the optimum conditions. We took enzyme-encapsulated NH2-JNP emulsion and free lipase as control groups denoted as NH2-JNP+Lipase and Lipase, respectively. As shown in Figure 4a, the interfacial enzyme immobilization of the Pickering emulsion constructed by CHO-JNPs has the highest specific activity at Ro/w=10:1; furthermore the enzyme-immobilized CHO-JNP emulsion shows higher specific activity than the enzyme-encapsulation Pickering emulsion stabilized by NH2-JNPs at all Ro/w, demonstrating that enzyme was immobilized at the oil/water interface successfully. Since in organic-aqueous biphasic biocatalysis systems, substrate in the organic phase interacts with 18


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enzyme in the aqueous phase to generate product, a heterogeneous catalytic reaction is generated at the organic-aqueous biphasic interface. Therefore, it is only at the organic-aqueous biphasic interface that the enzyme has catalytic activity. Under the premise of equal enzyme addition amounts, enzyme immobilized at the organic-aqueous biphasic interface has higher catalytic activity than that dispersed freely in the aqueous phase. After determination of Ro/w, we varied the amount of CHO-JNP loading to determine the optimum CHO-JNP loading. As shown in Figure 4b, when the CHO-JNP loading is 0.5% (20 mg), the enzyme-immobilized CHO-JNP emulsion has the highest specific activity. When CHO-JNP loading is less than 0.5% (0.25% 10 mg), the stability of the Pickering emulsion is decreased leading to lower specific activity. While CHO-JNP loading is more than 0.5% (0.75%, 1%), the redundant CHO-JNP aggregate at the surface of the Pickering emulsion thickens the layer of CHO-JNPs and decreases mass transfer efficiency, which resulted in lower specific activity as well. Figure 4c shows the thermal stability of enzyme-immobilized CHO-JNP emulsion, enzyme-encapsulated NH2-JNP emulsion and free lipase, which is a significant parameter in the value of potential industrial applications. The specific catalytic activity of each of these three enzyme catalytic systems decreases with the increase of incubation temperature. In regard to free lipase, owing to no addition of particles as stabilizer, the organic-aqueous biphasic interface is little leading to low specific activity (5.1 U mL-1). As the incubation temperature reaches 67 and 77 °C, there is little specific activity of free lipase because of inactivation of the enzyme at high temperature. Comparing the specific activity of enzyme-immobilized CHO-JNP emulsion with that of enzyme-encapsulated NH2-JNP

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emulsion, there is a significant decline in the enzyme-encapsulated NH2-JNP emulsion from 57 °C to 67 °C while none was found in the enzyme-immobilized CHO-JNP emulsion, the thermal inactivation point of which is similar to that of free lipase suggesting that the enzyme is dispersed freely in the micro-water droplets of the enzyme-encapsulated NH2-JNP emulsion. Until the incubation temperature ranged from 67 °C to 77 °C, there was no decline in the enzyme-immobilized CHO-JNP emulsion, the thermal inactivation point (higher than 77 °C) of which is much higher than the enzyme-encapsulated NH2-JNP emulsion and free lipase, indicating that the covalently coupled enzyme structure is more stable than free enzyme and maintains high catalytic activity in extreme temperatures, showing broad application prospects for high-temperature biocatalysis. After the determination of optimum Ro/w (10:1), amount of CHO-JNP loading (0.5%) and reaction temperature (37 °C), we studied the time course of hexanoic acid conversion as shown in Figure 4d. The enzyme-immobilized CHO-JNP emulsion shows the highest catalytic performance among the enzyme-encapsulated NH2-JNP emulsion and free lipase mainly attributed to the high stability of the Pickering emulsion creating a larger organic-aqueous biphasic interfacial area and interfacial enzyme immobilization as discussed in detail above. It should be noted that although our target reaction is conventional, our interfacial enzyme immobilization system could be applied to lots of heterogeneous catalytic reactions, more than in laboratory and even in recent research on droplets for continuous flow liquid-liquid interface catalysis.24

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NH2-JNPs+Lipase CHO-JNPs+Lipase

b) Specific activity (U/mL)

Specific activity (U/mL)

a)

30 25 20 15 10 5 0

20:1

15:1

10:1

35 30 25 20 15 10 5 0

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0.2

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CHO-JNPs+Lipase NH2-JNPs+Lipase

30

Lipase

d) 100 Conversion (%)

c) Specific activity (U/mL)

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25 20 15 10 5 0

80 CHO-JNPs+Lipase NH2-JNPs+Lipase

60

Lipase

40 20 0

30

40

50

60

70

80

0

20

40

o

60

80

100

120

140

Time (min)

Temperature ( C)

Figure 4. Effect of (a) Ro/w and (b) CHO-JNP loading on the specific activity of esterification reaction. (c) Specific activity of enzyme-immobilized CHO-JNP emulsion, enzyme-encapsulated NH2-JNP emulsion and free enzyme (same amount of lipase) after incubation at different temperatures for 1 h (d) Time courses of free enzyme, enzyme-immobilized CHO-JNP emulsion and enzyme-encapsulated NH2-JNPs emulsion for the esterification of hexanoic acid with 1-hexanol. We designed an experimental method to determine the percentage of immobilized enzyme by removing the lipase that is not immobilized at the interface of a CHO-Janus Pickering emulsion. A schematic illustration and the specific activity result are shown in Figure 5a and b, respectively. The specific experimental method was described in the experimental section. In the case of CHO-JNPs, at an enzyme loading of 0.46 mg mL-1, the concentrations of proteins in the aqueous solution after demulsification were 0.027 mg mL-1. The surface

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coverage of Pickering emulsions is thus calculated as 8.7 mg protein/g CHO-JNPs. In this case, the immobilization efficiency is 94%. For NH2-JNPs, the protein concentration after demulsification was 0.28 mg mL-1, indicating that only 39% of enzymes were adsorbed on the surface of NH2-JNPs. Furthermore, the initial specific activity of the enzyme-immobilized CHO-JNP emulsion and enzyme-encapsulated NH2-JNP emulsion is 33.2 U/mL and 23.3 U/mL, respectively, while the re-emulsified specific activity is 21.6 U/mL and 6.5 U/mL as shown in Figure 5b.

Figure 5. (a) Schematic illustration of initial and re-emulsified enzyme distribution in enzyme-immobilized CHO-JNP emulsion and enzyme-encapsulated NH2-JNP emulsion. (b) Specific activity of initial and re-emulsified enzyme-immobilized CHO-JNP emulsion and enzyme-encapsulated NH2-JNP emulsion illustrated in (a).

Recycling and reuse of enzyme-immobilized CHO-JNP emulsion. To evaluate the recyclability of the immobilized enzyme, hexanoic acid and 1-hexanol were incubated in heptane (Ro/w=10:1) for esterification at 37 °C and 120 rpm for 40 min for each cycle. The enzyme-immobilized CHO-JNP emulsion, collected by removing the organic phase, was washed with fresh heptane solvent 3 times and subsequently reused for the next reaction

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cycle. As shown in Figure 6, the relative activity of the enzyme-immobilized CHO-JNP emulsion maintained at 75% after 9 cycles while the enzyme-encapsulated NH2-JNP emulsion declined to 49%. The recycling performance of the enzyme-immobilized CHO-JNP emulsion is better than previous research such as agarose cross-linker SiO2 JPs (66% after 8 cycles)18 and polyethoxysiloxane silica colloidosomes (70% after 6 cycles).19 Compared with enzyme-encapsulated NH2-JNP emulsion, the most significant difference of enzyme-immobilized CHO-JNP emulsion is the interfacial enzyme immobilization. Therefore, the result demonstrated that enzyme immobilized at an organic/aqueous biphasic interface improved the stability of Pickering emulsion droplets during the recycling process.

Figure 6. Relative activity of enzyme-immobilized CHO-JNP emulsion and enzyme-encapsulated NH2-JNP emulsion in a biphasic esterification reaction during the recycling and reuse process.

CONCLUSIONS In summary, we have successfully prepared a new Janus NP (CHO-JNPs) and applied it to construct Pickering emulsions, enabling us to immobilize enzymes at an oil/water interface 23


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for organic/aqueous biphasic biocatalysis. An efficient and simple enzyme immobilization method via covalent coupling of enzyme with CHO-JNPs along with formation of a Pickering emulsion was further developed. The immobilized enzyme displayed high catalytic activity and significantly enhanced stability in organic/aqueous media. What is more, the immobilized enzyme exhibited good recyclability performance since the enzyme at the interface serves as a cross-linker to stabilize the emulsion. Hence, oriented enzyme immobilization at the oil/water interface not only improved the catalytic performance but also enhanced the stability of Pickering emulsions. The new enzyme immobilization method is efficient, practical, and also better than other reported encapsulation methods.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (R. H.); [email protected] (W. Q.). Tel: +86 22 27407799. Fax: +86 22 27407599. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the 863 Program of China (No. 2013AA102204) and the Natural Science Foundation of China (Nos. 21777112, 21476165 and 21621004).

ASSOCIATED CONTENT Supporting Information Size distribution of the CHO-JNPs, SEM image and elements distribution of CHO-JNPs, Zeta potential of SiO2-OH, NH2-SiO2-OH and CHO-SiO2-OH, SEM image and cross-section image of colloidosome stabilized by CHO-JNPs, Photos of Pickering emulsion stabilized by 24


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NH2-JNPs and CHO-JNPs at different storage time. This material is available free of charge via the Internet at http://pubs.acs.org/.

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