Pickering Emulsion as an Efficient Platform for Enzymatic Reactions

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Pickering Emulsion as an Efficient Platform for Enzymatic Reactions without Stirring Lijuan Wei, Ming Zhang, Xiaoming Zhang, Hongchuan Xin, and Hengquan Yang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b01776 • Publication Date (Web): 30 Sep 2016 Downloaded from http://pubs.acs.org on October 1, 2016

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ACS Sustainable Chemistry & Engineering

Pickering Emulsion as an Efficient Platform for Enzymatic Reactions without Stirring Lijuan Wei,† Ming Zhang,† Xiaoming Zhang,† Hongchuan Xin,‡ and Hengquan Yang*,† † ‡

School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, China

Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of

Sciences, Qingdao 266101, China

*Corresponding Author： [email protected]

ABSTRACT To address the current limitations of enzymatic reactions, we develop a novel strategy to conduct stirring-free biphasic enzymatic reactions. This strategy involves translation of a conventional biphasic enzymatic reaction to a water-in-oil (W/O) Pickering emulsion system by adding a small amount of solid particle emulsifier. In such a system, enzymes, for example a Candida Antarctica lipase B (CALB), are compartmentalized within millions of micron-sized water droplets, while organic substrates are dissolved in oil phase (outside the droplets). It was demonstrated that CALB-catalyzed hydrolysis kinetic resolution of racemic esters in the stirring-free Pickering emulsion system gave reaction efficiency and enantioselectivity favorably comparable to that in the conventional biphasic system under stirring conditions, which was due to the large reaction interfacial area and the short molecule distances created by the Pickering emulsion droplets. The specific activity was found to depend on the water droplet size, highlighting the importance of the presence of droplets in the reaction system. Moreover, the convenient and effective recycling of CALB could be achieved through simple demulsification by centrifugation. After 27 reaction cycles, the ee values of ester and alcohol were still as high as 87.5% and 99%, respectively, which significantly exceed those of the conventional biphasic reaction. The high recyclability may be attributed to avoiding stirring that often causes damage of the three-dimensional structure of enzymes. This study compellingly demonstrates that Pickering emulsion is an innovative platform to process efficiently enzymatic reactions without need for stirring and immobilization.

KEYWORDS: Pickering emulsion, enzymatic reactions, hydrolysis kinetic resolution

INTRODUCTION Enzymatic reactions are highly attractive for synthesis of chiral chemicals owing to high enantioselectivity and high activity even under mild reaction conditions.1-7 Enzymes prefer water over organic solvents as their reaction medium, just as in the scenario in nature. However, the majority of reactants are soluble only in organic solvents. Enzymatic reactions, therefore, often require an aqueous-organic biphasic system. Unfortunately, biphasic systems often suffer from low reaction efficiency due to the limited reaction interfacial area and the high mass transport resistance.8-10 To address these limitations, surfactants are usually introduced to the reaction systems to form microemulsions or emulsions, creating large reaction interface areas. Despite reaction efficiency

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enhancement achieved, surfactant-stabilized emulsion systems encounter difficulty in product purification and enzyme recycling.11-

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Alternatively, stirring or shaking is implemented to increase mass transport between two phases. Unfortunately, stirring and

shaking in some cases cause the damage of enzyme three-dimensional structures.14-16 In parallel, from the practical viewpoint, enzymes should be easily recycled due to their relatively high cost. Although immobilization of enzymes can address these issues, immobilization sometimes leads to a decrease in enzyme activity.17-24 Therefore, the development of a simple protocol to improve both the enzyme catalysis efficiency and recyclability is still highly desirable. Pickering emulsions that are stabilized by solid particles are emerging as an innovative platform for designing efficient biphasic catalysis systems due to their attractive advantages over the conventional surfactant-stabilized emulsions such as ease of product-particle separation and low toxicity of particles.25-43 The large oil-water interfacial area allows biphasic reaction systems to access high efficiency, which has been extensively demonstrated in hydrogenation, oxidation as well as enzymatic reactions.32-38 More interestingly, we have found that Pickering emulsion systems enable biphasic reactions to proceed efficiently through auto-diffusion of molecules without need for stirring due to large interface area and short molecule diffusion distance.31 Such a stirring-free process allows Pickering emulsions to emulate the reactions occurring in nature.25 These merits of Pickering emulsion systems might address the obstacles encountered currently in enzymatic reactions: deactivation caused by vigorous stirring and difficulty in enzyme recycling, but has not yet been explored.

Figure 1. Schematic description of the Pickering emulsion strategy for stirring-free biphasic enzymatic reactions (A) and characterization of the water-in-oil Pickering emulsion (B). (a) Optical microscopy image，(b) fluorescence confocal microscopy image for the enzyme labelled by a fluorescence reagent, Rhodamine B (see Experimental Section in Supporting Information).

Herein, we demonstrate for the first time that Pickering emulsions can be used as an enzymatic reaction medium to access high reaction efficiency coupled with exceptionally high recyclability under stirring-free conditions. As Figure 1A illustrates, before reaction, a conventional biphasic enzymatic reaction is converted to a water-in-oil (W/O) Pickering

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emulsion by adding a small amount of emulsifier, creating numerous micron-sized water droplets. Being soluble in water, enzyme is thus compartmentalized in these water droplets, while organic substrates are dissolved in the oil phase surrounding the water droplets. Such high level of oil-water mixing and large oil-water interfacial area enable the enzymatic reaction to proceed efficiently without need for stirring during workup. At the end of reaction, the reaction system is subjected to demulsification through centrifugation, returning to a biphasic system with the emulsifier and enzyme retained in the aqueous phase. After the upper layer of organic product is removed through liquid transfer, the enzyme and emulsifier in the water phase can be directly used for the next Pickering emulsion reaction after reemulsification.

RESULTS AND DISCUSSION We used here partially hydrophobic silica nanospheres as emulsifier to stabilize water-in-oil Pickering emulsion. This emulsifier was prepared through modification of silica nanospheres (40-60 nm in diameter) with methyltrimethoxysilane [Transmission electron microscopy (TEM) images, N2 sorption isotherms and energy-dispersive X-ray spectrum (EDX) are displayed in Figure S1, in Supporting Information; X-Ray photoelectron spectrum (XPS) is displayed in Figure S2; Water contact angel of the modified silica pellet is determined as 93o in Figure S3]. Here we use a “shaking” method to formulate Pickering emulsions because it can avoid damage of the three-dimensional structure of enzyme caused by ultrasonic or high-speed stirring. The Pickering emulsion was confirmed to be of water-in-oil type through a droplet test. Its optical micrograph are shown in Figure 1B, a. The average size of emulsion droplets was ca. 56 μm. In order to clarify the distribution of enzyme in the reaction system, we determined the amount of enzyme adsorbed onto the emulsifier through UV-vis method (see Experimental Section in Supporting Information). It was found that there were about 53.3% of enzymes that are adsorbed onto the modified silica particles. Fluorescence confocal microscopy experiment with enzyme labelled by Rhodamine B further confirmed that a portion of enzymes were adsorbed onto the emulsifier or at the water-oil interface since the fluorescence signal was much stronger at the oil-water interface in comparison to that for the interior of the water droplets, as shown in Figure 1B, b. The catalytic performance of the stirring-free Pickering emulsion system was examined with the hydrolysis kinetic resolution of racemic esters that is widely used to synthesize chiral esters and alcohols.44-46 Its reaction efficiency was first assessed through comparison with the existing biphasic systems including a stirring-free biphasic system, a stirred biphasic system with stirring rate of 900 rpm, a stirred biphasic system with stirring rate of 1350 rpm, a stirring-free Pickering emulsion system, a stirred Pickering emulsion system with stirring rate of 900 rpm and a stirred Pickering emulsion system with stirring rate of 1350 rpm. The kinetics of hydrolysis kinetic resolution of racemic 1- phenylethyl acetate in different biphasic systems are plotted in Figure 2. These systems have significant difference in the reaction rate, reflecting by the enantiomeric excess (ee) value of 1-phenylethyl acetate change with time (Figure 2A). Apparently, the reaction rate of the stirring-free Pickering emulsion is much higher than that of conventional biphasic systems without stirring and with stirring rate of 900 rpm, and comparable to that of conventional biphasic systems with stirring rate of 1350 rpm. After 360 min, the ee values of 1-phenylethyl acetate and benzyl alcohol both reached as high as 96% in the stirring-free Pickering emulsion system (The control experiment showed that the ee value of 1-phenylethyl acetate in the absence of CALB is less than 2%). Based on the ee values of 1-phenylethyl acetate within first 60 min, the specific activity of CALB in these systems was estimated (Figure 2B). The specific activity of CALB in the conventional stirring-free

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biphasic systems is only 0.2 U mg-1. When the reaction system was stirred at a speed of 900 rpm, the specific activity dramatically increased up to 2.6 U mg-1 due to the mass transport intensification. Increasing the stirring rate up to 1350 rpm leads to a further increase in specific activity (4.6 U mg-1). Impressively, the specific activity in the stirring-free Pickering emulsion is as high as 4.5 U mg-1, which is comparable to the conventional biphasic system with a high stirring rate (at 1350 rpm). Notably, for the Pickering emulsion reaction systems, stirring does not lead to a significant increase in specific activity. The reason is that the Pickering emulsion per se has a high level of oil-water mixing, large reaction interfacial area and short molecule diffusion distance, making the stirring unnecessary. These comparisons convincingly demonstrate that the enzyme in Pickering emulsion system has sufficiently high activity, making the energy-consuming stirring operation obsolete during workup.

Figure 2. Kinetic plots of the hydrolysis kinetic resolution of racemic 1-phenylethyl acetate (A) and specific activities of CALB (B) in different biphasic systems. a, stirring-free biphasic system; b, biphasic system, stirring at 900 rpm (a magnetic stir bar with length of 10 mm); c, biphasic system, stirring at 1350 rpm; d, stirring-free Pickering emulsion system; e, Pickering emulsion system, stirring at 900 rpm; f, Pickering emulsion system, stirring at 1350 rpm. Reaction conditions: 0.3 mmol (R,S)-1-phenylethyl acetate, 4.9 mL PBS (phosphate buffer saline), 2 mL hexane, 0.15 g emulsifier if needed, 100 μL CALB, 35 °C. Specific activity is calculated based on the conversion within the first 60 min.

To clarify the role of the presence of droplets, the specific activity of CALB in the stirring-free Pickering emulsion systems with different droplet sizes were examined. Addition of different amounts of emulsifier was utilized to tune the droplet size because it was well established that the droplet diameter decreased as the amount of solid particles increased.31, 47 When the emulsifier amount was varied from 0.25 to 0.5, 1.0, 2.0 and 3.0 wt % (with respect to water), the

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average droplet diameter decreased from 200 to 137, 98, 75 and 56 μm (Figures 3A and C and Figure S4 in Supporting Information). These Pickering emulsion systems showed different reactivity, depending on the emulsifier amounts (Figure 3B). Their specific activity calculated from the kinetic profile is reflected in Figure 3C. The Pickering emulsion with 0.25 wt % emulsifier gave specific activity of 2.1 U mg-1. When increasing the emulsifier amount to 0.5 and 1.0 wt %, the specific activity increased to 4.0 and 4.5 U mg-1, respectively. As the emulsifier amount further increased up to 2.0 and 3.0 wt %, the activity increased very slightly and almost levelled off. The change in the specific activity with the emulsifier amount can be explained in terms of the water-oil interface area and the droplet surface coverage with the emulsifier amount (Figure S5, in Supporting Information). The increase in emulsifier amount caused a decrease in the droplet size, which not only results in an increase in the total water-oil interface area (S) but also the shorter reactant molecule diffusion distance due to the droplet packing effects (in the Pickering emulsion, numerous water droplets are closely packed together, creating a three-dimensional network reaction system. In such a network, enzymes are adsorbed at the oil-water interface or compartmentalized within the water droplets, while organic substrates are “confined” in the interspace among the droplets. The smaller the droplet sizes are, the shorter the diffusion distances taken by the substrate molecules are).31 The larger reaction interface area and shorter molecule diffusion distance benefit specific activity. In parallel, notably, increasing the emulsifier amount is also accompanied by an increase in the coverage of emulsifier particles at droplet interfaces C (The higher surface coverage means that more particles are located at interface. The coverage calculation is provided in Experimental Section of Supporting Information). The high coverage compromised the reaction efficiency because of sacrificing the possibility of the substrate (in oil) contact with enzyme. The trade-off between the droplet decrease and the coverage increase results in no significant change in the specific activity in a certain range.

Figure 3．． Results of the CALB-catalyzed hydrolysis of (R, S)-1-phenylethyl acetate in the emulsion systems with different amounts of emulsifier (from 0.25 to 0.5, 1, 2 and 3 wt %, with respect to water). The reaction conditions are the same as Figure 2, except the varied solid materials amount. (A) Optical micrographs for these Pickering emulsion systems with different amounts of emulsifier (scale bar is 100 μm). (B) The reaction kinetic plots. (C) Specific activity and droplet diameter changes with the amounts of emulsifier. Specific activity is calculated based on the conversion within first 60 min.

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CALB in the stirring-free Pickering emulsion systems also showed high activity towards other substrates such as racemic α-methylbenzyl butyrate, 4-methyl-2-pentanol acetate, 2-octanol acetate and 1-octen-3-yl acetate, as summarized in Table 1. For all the investigated substrates, the stirring-free Pickering emulsion systems gave 96% of ee values for esters and 99% of ee values for alcohols, within 6-10 h.

Table 1．． CALB-catalyzed hydrolysis kinetic resolution of racemic esters in the stirring-free Pickering emulsion system.

Time [h]

[S] Conv. [%]b

ees [%]c

[P] eep [%]c

1

6

49.2

96

>99

2

8

49.5

97

>99

3

10

49.2

96

>99

4

8

49.5

97

>99

5

10

49.5

97

>99

Entry

a

Substrates

a

Reaction conditions: 0.6 mmol substrate, 2 mL n-hexane, 0.15 g emulsifier, 4.9 mL buffer (PH 7.0, 0.1 M), 100µL crude enzyme, 35 oC. bConv. % = eeS/(eeP + eeS) ×100%. cThe ee values were measured by GC.

We further evaluate the recyclability of the Pickering emulsion catalysis system. After the first reaction, the Pickering emulsion reaction system was subjected to centrifugation. The Pickering emulsion was rapidly demulsified, leading to phase separation. The organic products, chiral ester and alcohol in the upper layer, could be easily isolated through a step of liquid transfer. The emulsifier and CALB retained in the lower layer were directly used to the next reaction cycle. In the second reaction cycle, the ee values of 1-phenylethyl acetate and benzyl alcohol were still as high as 95.4% and 99% (Figure 4A, A3 and A4). For the ninth reaction cycle, 97.3% and 99% of ee values were obtained within the reaction time prolonged from initial 4 h to 12 h. Impressively, after twenty seven reaction cycles, the emulsion droplet morphology were well kept (Figure 4A, A2). The ee values of 1-phenylethyl acetate and benzyl alcohol were still as high as 87.5% and 99%, respectively, which highlighted the exceptionally high recyclability of the stirring-free Pickering emulsion system. In contrast, for the conventional biphasic reaction (Figure 4B， B1 and B2), the ee value of 1-phenylethyl acetate decreased from the third reaction cycle although the ee value of benzyl alcohol had no decrease, reflecting the decrease in enzyme activity. In the ninth reaction cycle, the ee value of 1-phenylethyl acetate was only 73.6%, which is much lower than that for the stirring-free Pickering emulsion system. Such a striking comparison confirms the importance of the stirring-free Pickering emulsion system. The superiority of the Pickering emulsion may be due to avoiding the continuous stirring that caused the damage of the three-dimensional structure of enzyme.14

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Figure 4. Recycling results of the CALB-catalyzed kinetic resolution of (R, S)-1-phenylethyl acetate in the stirring-free Pickering emulsion system (A) and the conventional biphasic system (B). (A1) Optical micrographs of the fresh Pickering emulsion. (A2) Optical micrographs of the Pickering emulsion after 27 reaction cycles. (A3) The ee value of 1-phenylethyl acetate vs reaction cycle in the stirring-free Pickering emulsion; (A4) The ee value of benzyl alcohol vs reaction cycle in the stirring-free Pickering emulsion; (B1) The ee value of 1-phenylethyl acetate vs reaction cycle in the conventional biphasic system; (B2) The ee value of benzyl alcohol vs reaction cycles in the conventional biphasic system; For the conventional biphasic systems, stirring at 1350 rpm; Other reaction conditions are the same as those in Figure 2. The reaction time was prolonged from 4 h to 12 h during recycling.

CONCLUSION In conclusion, we have demonstrated a general strategy to conduct enzymatic reactions without need for stirring during workup, which is based on translation of a conventional biphasic reaction into a Pickering emulsion system. The case study of the enzymatic hydrolysis kinetic resolution of racemic ester shows that the enzyme compartmentalized within the droplets of the Pickering emulsion system has activity comparable to the conventional biphasic system with high speed stirring. The high activity at static conditions is due to the large oil-water interfacial area and the short molecule diffusion distance created by the Pickering emulsion. Impressively, the Pickering emulsion strategy allows for facile separation of product with enzyme and effective recycling the enzyme through centrifugation, without recourse to immobilizing enzymes. This study compellingly demonstrates that Pickering emulsion is an innovative platform to process efficiently enzymatic reactions without need for stirring and immobilization.

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EXPERIMENTAL SECTION Hydrolysis kinetic resolution of racemic esters in Pickering emulsions. Typically, 2 mL of n-hexane and 0.15 g of emulsifier were added into a 10 mL of vial and then dispersed by ultrasound. 100 μL of native CALB solution and 4.9 mL of PBS (phosphate buffer: 0.1 M Na2HPO4-0.1 M NaH2PO4, pH 7.0) were added. The mixture was vigorously shaking for 5 min, forming a Pickering emulsion. 0.6 mmol of racemic acetates was then added into this Pickering emulsion. After slightly stirring for 1 min, this reaction mixture stood at 35°C for a given time. At the end of reaction, this mixture was subjected to demulsification through centrifugation, for phase separation. The upper layer of organic phase was withdrawn for GC analysis. The fresh reactant and n-hexane were added to the lower layer of aqueous phase containing emulsifier. After emulsification, the second reaction cycle began. Every 5 cycles, the pH of the water phase was adjusted to 7.0 using an aqueous Na2HPO4 solution. The experimental procedure of the reaction without CALB is the same with the above process except the absence of CALB. Conventional biphasic reactions was conducted under stirring conditions (magnetic stir bar length: 10 mm, speed: 900, 1350 rpm) and other reaction conditions are the same as the Pickering emulsion reaction except the absence of emulsifier.

ASSOCIATED CONTENT

Supporting Information Experimental section; TEM images; N2 sorption analysis; EDX spectrum; XPS spectrum; Water contact angles; Appearance of the Pickering emulsion and its optical micrograph; Size distribution of the Pickering emulsion. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *[email protected] Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work is supported by the Natural Science Foundation of China (21173137 and 21573136), Program for New Century Excellent Talents in University (NECT-12-1030) and foundation for Young Sanjin Scholar.

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For Table of Contents Use Only Pickering Emulsion as an Efficient Platform for Enzymatic Reactions without Stirring

Lijuan Wei, Ming Zhang, Xiaoming Zhang, Hongchuan Xin, and Hengquan Yang*

Based on Pickering emulsion, we demonstrate a strategy to conduct biphasic enzymatic reactions without need for stirring but with high reaction efficiency and recyclability.

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Pickering Emulsion as an Efficient Platform for Enzymatic Reactions

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