Encapsulation of Laccase in Silica Colloidosomes for Catalysis in

Article pubs.acs.org/Langmuir

Encapsulation of Laccase in Silica Colloidosomes for Catalysis in Organic Media Chi Zhang,†,‡ Chunyan Hu,†,‡ Yongliang Zhao,§ Martin Möller,§ Kelu Yan,*,†,‡ and Xiaomin Zhu*,§ †

College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, 2999 North Renmin Road, Shanghai 201620, China ‡ Key Lab of Textile Science &Technology of Ministry of Education, 2999 North Renmin Road, Shanghai 201620, China § Interactive Materials Research - DWI an der RWTH Aachen e.V. and Institute for Technical and Macromolecule Chemistry of RWTH Aachen University, Forckenbeckstraße 50, D-52056 Aachen, Germany S Supporting Information *

ABSTRACT: We report on the encapsulation of a laccase solution in silica colloidosomes of different shell structures and the study of catalytic performance of the encapsulated enzyme in toluene. The encapsulation is carried out by linking silica nanoparticles at the water/oil interface using hyperbranched polyethoxysiloxane (PEOS) in an aqueous-laccase-solution-intoluene Pickering emulsion. By varying the weight ratio of silica particles to water (Rs/w), colloidosome shell can be adjusted from a particle monolayer up to a bilayer bound with a sandwiched nanoporous silica thin film formed from PEOS. The encapsulated laccase exhibits catalytic activity and reusability that are controlled by Rs/w. With the increase of Rs/w the reusability of the enzyme improves, meanwhile, its activity declines. This method allows fabricating enzyme microcapsules with tailored activity and controlled release properties.

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INTRODUCTION Enzymes are important biocatalysts for a broad variety of organic reactions, providing extraordinarily high catalytic efficiency and selectivity under mild and sustainable conditions.1−3 Although certain enzymes can be active in water-free organic media,4 the application of enzymes is limited by the factor that most enzymes only exhibit high activity in water,5 whereas substrates of organic reactions are often only soluble in organic solvents. For such systems, biphasic conditions are usually required. A common way is to create an emulsion where an aqueous enzyme solution is dispersed in an organic medium containing the substrate.6 For the emulsion stabilization, surfactant molecules are used; however, they may affect the enzymatic activity,7 and the isolation of the final product is usually a problem. In order to adapt to organic reaction media, enhance stability, and enable reusability, enzymes can be immobilized on specific carriers via physical adsorption, covalent binding, aggregation, or encapsulation.8−10 Silica has been widely used as a biocompatible and stable matrix for the enzyme © 2013 American Chemical Society

immobilization by means of different techniques including sol−gel technology,11−13 bioinspired silica formation,14 and entrapment in mesoporous silica.15−20 Enzymes encapsulated in silica by these means are meant to be used in aqueous media, and for organic solutions, new silica encapsulation methods are still to be established. However, colloid particles including silica nanoparticles can be employed instead of traditional surfactants to stabilize emulsions.21−24 These so-called Pickering emulsions have been used to fabricate microcapsules (colloidosomes) by fixing the particle assemblies at the interface.25 Because of a great degree of control on the shell permeability by varying either the particle size or degree of fusing, colloidosomes find a high potential in a number of technological applications. By using Pickering water/oil emulsions as templates, aqueous solutions of enzymes were successfully encapsulated in colloidosomes for catalysis in organic media.26,27 Not only Received: October 22, 2013 Revised: November 25, 2013 Published: November 25, 2013 15457

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laccase-loaded colloidosomes were isolated by centrifugation and redispersed in toluene for enzyme assays. Field Emission Scanning Electron Microscopy (FE-SEM). FESEM measurements were carried out on a Hitachi S-4800 FieldEmission SEM. The samples were prepared by placing a drop of the colloidosome dispersion in toluene on a silicon wafer substrate. Before being placed into the specimen holder, the samples were air-dried under ambient conditions. Laser Scanning Confocal Microscopy (LSCM). LSCM on a Carl Zeiss LSM 700 (Jena, Germany) was employed to image colloidosomes loaded with FITC-laccase. Fluorescent micrographs were taken by using the ZEN2010 software. The samples were diluted prior to measurements. The specimen was illuminated with light of 488 nm, and the emission at 525 nm was used for imaging. Catalytic Performance and Reusability Tests. The catalytic activity of laccase encapsulated in colloidosomes in toluene was determined spectrophotometrically by using 2,6-DMP35,36 as substrate by monitoring the increase in absorbance at 469 nm. All spectrophotometric assays were performed with a Unico UV-2000 spectrophotometer. The laccase activity in this work is defined as the amount of 2,6-DMP (in μmol) that is converted in an hour. For enzyme assays a dispersion of laccase-loaded colloidosomes was added to a toluene solution of the substrate to result in a dispersion containing 0.5 mM 2,6-DMP and 1 mg/mL laccase. For measuring the activity of nonencapsulated laccase, 1.0 g of a citrate-phosphate buffer solution of pH 4 containing 10.0 mg of laccase was added to 10 mL of a 5 mM toluene solution of 2,6-DMP. As for the reusability test, the laccase loaded colloidosomes were isolated by centrifugation after each activity measurement. Afterward, the precipitate was washed several times with pure toluene and redispersed in toluene for the next test cycle. The initial activity measured during the first assay was taken as 100%.

the high catalytic performance but also the recyclability have been clearly demonstrated. Recently we developed a new type of all-silica colloidosomes templated by water-in-oil (w/o) Pickering emulsions, where silica nanoparticles were linked by a silica precursor polymer, hyperbranched polyethoxysiloxane (PEOS), at the water/oil interface.28 In contrast to most colloidosome systems reported so far, all-silica colloidosomes prepared by us have a completely closed shell; nevertheless, small molecules can still penetrate through due to the nanoporosity of the thin silica layer obtained by PEOS condensation. Importantly, the shell of the colloidosomes can be fine-tuned from a particle monolayer up to a bilayer bound with a sandwiched thin silica film by varying the weight ratio of silica nanoparticles to water (Rs/w), and the shell structure was shown to affect significantly the release of encapsulated water molecules. Since the resulting colloidosomes are hydrophobic and well dispersible in nonpolar organic solvents, we attempt to use this concept to encapsulate aqueous enzyme solutions and employ these new enzyme-loaded allsilica colloidosomes for catalysis in organic media. As a model, laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2), which catalyzes the oxidation by molecular oxygen of a variety of aromatic substrates and attracts considerable attention for various industrial applications,29−31 is chosen. The substrates of laccase are generally hydrophobic and soluble only in organic solvents, so biphasic reaction conditions are usually needed.32,33 A question that rises here is whether the substrate and product molecules can diffuse across the silica shell. Our work addresses also the influence of the colloidosome shell structure on the activity and reusability of the encapsulated laccase.

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RESULTS AND DISCUSSIONS In our previous study, it was shown that in the w/o systems well-defined all-silica colloidosomes were formed when the pH value of the aqueous phase was in the range of 1−4.28 Meanwhile, Laccase from Trametes Versicolor exhibits a significant catalytic activity for the phenol oxidation over a pH range of 4−7.37 Therefore, in this work laccase from Trametes Versicolor dissolved in a citrate-phosphate buffer solution of pH 4.0 was encapsulated in the silica colloidosomes, and Rs/w was varied from 0.2 to 0.4. To confirm the encapsulation of laccase in the colloidosomes, laccase was first tagged by reacting with a reactive fluorescent dye, FITC. The FITC−laccase-loaded colloidosomes were then studied by means of LSCM. The LSCM micrographs presented in Figure 1 show that the colloidosomes are green fluorescent, indicating clearly that they are loaded with laccase. Figure 2 displays FE-SEM images of the silica colloidosomes encapsulating laccase solutions obtained at different Rs/w. It can be seen that the colloidosomes are spherical and that the outer surface of the shell is always fully covered by nanoparticles. Meanwhile, the coverage of the inner side by colloidal particles increases with the increase of Rs/w. At Rs/w = 0.2 and 0.3, the inner surface of the colloidosome shell is partially occupied by silica nanoparticles. The coverage of the inner surface becomes complete at Rs/w = 0.4 and a particle bilayer shell is formed. The colloidosome dimension at different Rs/w was determined by image analysis of the FE-SEM micrographs, and the results are summarized in Figure 3. The diameter of the colloidosomes was 1.39 ± 0.22, 0.99 ± 0.17, and 0.75 ± 0.12 μm for Rs/w = 0.2, 0.3, and 0.4, respectively, i.e., it decreases with the increase of Rs/w. The dimension and structure of the colloidosomes obtained in this work are similar to that reported previously by

EXPERIMENTAL SECTION

Materials. Laccase from Trametes Versicolor, trimethoxy(octadecyl)silane (90%), citrate-phosphate buffer solution of pH 4.0, and fluorescein isothiocyanate (FITC) (≥90%) were purchased from Sigma-Aldrich. Tetraethoxysilane (for synthesis), absolute ethanol (for analysis), ammonia solution (25−28% w/w), toluene (for analysis), dimethylsulfoxide (DMSO) (for analysis), and 2,6-dimethoxyphenol (2,6-DMP) were purchased from Sinopharm Chemical Reagent Co., Ltd. All reagents were used as received. Water of Milli-Q grade was used for all experiments. PEOS was synthesized according to the method published elsewhere.34 The resulting PEOS had the following characteristics: degree of branching 0.54, SiO2 content 49.2%, Mn 1740, and Mw/Mn 1.9 (measured by gel permeation chromatography in chloroform with evaporative light scattering detector calibrated using polystyrene standards). Hydrophobic silica nanoparticles with a diameter of about 50 nm were prepared following a literature procedure,19 and they were dispersed in toluene to yield a homogeneous stock dispersion. Fluorophore Labeling of Laccase. For fluorescence microscopy investigation, laccase was labeled with a fluorophore, FITC. For labeling laccase a mixture of laccase (10.0 mg) and FITC (10.0 mg) was added to DMSO (3.5 mL) in a glass vial. The vial was protected against light by an aluminum foil, and the mixture was gently stirred on a magnetic stirrer at room temperature for 24 h. Later, dialysis was performed in water for 3 days in dark to remove the free FITC and DMSO; the resulting labeled laccase was isolated by freeze-drying. Encapsulation of Laccase in Silica Colloidosomes. A citratephosphate buffer solution of pH 4 (1.0 g) containing laccase (10.0 mg) and a dispersion of hydrophobic silica nanoparticles in toluene (9.4 g) with different weight ratios of silica to water (Rs/w = 0.2, 0.3, and 0.4) were mixed and emulsified using ultrasonic irradiation for 30 min (Branson Sonifier 450 cell disrupter, 3 mm microtip, 0.9 time circle, 247 W output). Afterward, a solution of PEOS in toluene with a concentration of 0.2 g mL−1 (0.6 mL) was added to the emulsion. The emulsion was stirred gently at room temperature for 3 days. The 15458

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Figure 2. FE-SEM images of silica colloidosomes encapsulating laccase solutions prepared at different weight ratio of silica nanoparticles to water (Rs/w). (a) Rs/w = 0.2, (b) Rs/w = 0.3, and (c) Rs/w = 0.4.

Figure 1. LSCM micrographs of colloidosomes loaded with FITClaccase solutions dispersed in toluene. (a) Fluorescence image, (b) bright field image, and (c) overlay of images a and b.

formation of the silica colloidosomes of different structures is presented in the Supporting Information. Laccase is only soluble in aqueous media, so its encapsulation efficiency in the silica colloidosomes can reach almost 100%. Therefore, this method is much more efficient than most silica-

us,28 indicating that the presence of laccase does not influence the formation of all-silica colloidosomes. The mechanism of the 15459

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Figure 3. Influence of weight ratio of silica nanoparticles to water (Rs/w) on colloidosome diameter. The insets are the corresponding FE-SEM image of typical colloidosomes with illustrations of the shell structure. The scale bar represents 1 μm.

based microencapsulation techniques.11−20 Furthermore, since the aqueous solution of the enzyme is loaded into the colloidosome cages, the enzyme conformation should not be altered, high biocatalytic activity can thus be expected. In contrast to classical emulsion-based encapsulation methods, in our approach silica nanoparticles are used instead of classical surfactants, and they are incorporated into the capsule shell to result in a stable solid formulation. The catalytic performance of laccase encapsulated in colloidosomes obtained at different Rs/w was investigated in toluene using 2,6-DMP as substrate, and the oxidation of 2,6DMP was monitored by UV−vis spectroscopy. The main oxidation product of 2,6-DMP is its dimeric colored form, 3,3′,5,5′-tetramethoxydiphenylquinone,38−40 having an absorption maximum at 469 nm, which corresponds to the π−π* transition of the aromatic fragment. 2,6-DMP is considered to be the most suitable substrate for this type of enzymatic assays using spectrometry due to a number of factors including the high oxidation efficiency, stability of the colored product, and its high absorption coefficient, weak acidic optimal pH. Figure 4a depicts the conversion of 2,6-DMP under the catalysis of free laccase and laccase encapsulated in silica colloidosomes with time. The catalytic activity of laccase determined from the kinetic study is summarized in Figure 4b. From these data one can see that although the laccase is encapsulated in the completely closed colloidosomes, it can still exhibit catalytic activity. This is most probably due to the nanoporous structure of the silica layer formed via the condensation of PEOS, which allows the substrate and product molecules to diffuse through. It is further demonstrated that the encapsulation of the laccase decreases its catalytic activity, and it declines further with the increase of Rs/w. The decrease of the catalytic activity of the lacasse after encapsulating in the silica colloidosomes is due to the retarded mass transfer through the colloidosome shell in comparison with barrier-free material exchange between the free laccase solution and toluene. It is hence confirmed that the formation of the particle bilayer improves the barrier properties of the colloidosomes. The rate of the enzymatic reaction seems to be controlled by the diffusion of the substrate and product molecules across the colloidosome shell. Thus, we demonstrated for the first time the possibility of the control over catalytic activity of the enzyme microencapsulated in colloidosomes by varying the shell structure.

Figure 4. (a) Conversion of 2,6-DMP catalyzed by free laccase and laccase encapsulated in silica colloidosomes with different weight ratio of silica nanopartcles to water (Rs/w). (b) Catalytic activity of different laccase samples.

The silica colloidosomes consist fully of silica, so the incorporated ingredient is expected to be released under strong mechanical force due to the brittleness. Free laccase exhibits much higher activity, e.g., than the laccase encapsulated in colloidosomes with a bilayer shell. On demand the laccaseloaded colloidosomes can be broken, and then the highly active laccase solution can be released. Therefore, they can be considered as promising controlled release systems. As shown before, the free laccase shows the highest activity, however, its isolation from the reaction mixture is certainly a severe problem. One of the most important features of immobilized enzymes is their reusability. In this work, the enzyme-loaded colloidosomes were separated from the reaction mixture by centrifugation and subsequent rinsing with toluene. They were then redispersed in toluene, and the catalytic activity of the recovered encapsulated enzyme was measured. Figure 5 presents the activity of encapsulated laccase during six regeneration cycles. It shows that the reusability of the encapsulated laccase depends on Rs/w and consequently on the shell structure of the colloidsomes. For Rs/w = 0.4, where the colloidosome shell consists of a complete particle bilayer, after six recovery cycles the activity was maintained at more than 70%. At lower Rs/w, where the inner surface of the shell is not covered completely by the silica particles, the reusability of the encapsulated enzyme is a bit worse. After six consecutive recycling runs, 60% and 31% of the initial activity remained for laccase-loaded colloidosomes of Rs/w = 0.3 and 0.2, respectively. The reason of the activity loss during the recovery is still not 15460

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS C.Z. thanks the financial support of Donghua University and Garg Foundation during the research stay at DWI an der RWTH Aachen.

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ABBREVIATIONS w/o, water-in-oil; PEOS, polyethoxysiloxane; Rs/w, weight ratio of silica nanoparticles to water; FITC, isothiocyanate; DMSO, dimethylsulfoxide; 2,6-DMP, 2,6-dimethoxyphenol; FE-SEM, field emission scanning electron microscopy; LSCM, laser scanning confocal microscopy

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Figure 5. Catalytic activity of laccase encapsulated in silica colloidosomes with different weight ratio of silica nanopartcles to water (Rs/w) during six regeneration cycles.

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clear. We speculate that it might be connected with the breakage of the colloidosomes or water loss during the regeneration process. As we demonstrated previously,19 the mechanical strength and the barrier properties of the colloidosomes increases with the transition from a monolayer to a bilayer shell structure during the increase of Rs/w, as a result, the reusability of the laccase-loaded colloidosomes also improves.

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CONCLUSIONS In this work we have presented a new approach for microencapsulation of enzymes for catalysis in organic media. An aqueous solution of laccase was successfully encapsulated in silica colloidosomes by linking nanoparticles at the water/oil interface using PEOS in an aqueous-laccase-solution-in-toluene Pickering emulsion, and the shell structure was varied from a noncomplete to a completely particle bilayer by changing Rs/w. The catalytic performance of the encapsulated laccase in toluene was demonstrated by using 2,6-DMP as substrate, and the recyclability of laccase was effectively realized. At Rs/w = 0.4, where the colloidosome shell comprised the complete particle bilayer, the laccase exhibited the highest reusability, but the lowest activity. The decrease of Rs/w led to the increased enzyme activity but worsened reusability. Furthermore, because of the brittleness of the shell, the enzyme-loaded silica colloidosomes can be considered as promising controlled release systems.

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ASSOCIATED CONTENT

S Supporting Information *

Mechanism of the formation of silica colloidosomes of different structures. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*(K.Y.) Tel: +86-21-67792604. Fax: +86-21-67792608. E-mail: [email protected]. *(X.Z.) Tel: +49-241-8023341. Fax: +49-241-8023301. E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. 15461

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