Surface-Functionalized Mesoporous Nanoparticles as Heterogeneous

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Surface-Functionalized Mesoporous Nanoparticles as Heterogeneous Supports to Transfer Bifunctional Catalysts into Organic Solvents for Tandem Catalysis Ningning Zhang, René Hübner, Yangxin Wang, En Zhang, Yujian Zhou, Shengyi Dong, and Changzhu Wu ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b01572 • Publication Date (Web): 30 Oct 2018 Downloaded from http://pubs.acs.org on October 31, 2018

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ACS Applied Nano Materials

Surface-Functionalized Mesoporous Nanoparticles as Heterogeneous Supports to Transfer Bifunctional Catalysts into Organic Solvents for Tandem Catalysis Ningning Zhang,a René Hübner,b Yangxin Wang,a En Zhang,c Yujian Zhou,d Shengyi Dong,e and Changzhu Wu*f a

Institute of Microbiology, Technische Universität Dresden, Zellescher Weg 20b, Dresden 01217,

Germany. b

Institute of Ion Beam Physics and Materials Research, Helmholtz-Zentrum Dresden-Rossendorf,

Bautzner Landstraße 400, Dresden 01328, Germany. c

Department of Chemistry, Technische Universität Dresden, Bergstraße 66, Dresden 01069,

Germany. d

Department of Chemistry and Food Chemistry, Technische Universität Dresden,

Mommsenstraße 4, Dresden 01062, Germany. e College

of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, Hunan,

P. R. China.

ACS Paragon Plus Environment

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f

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Danish Institute for Advanced Study (DIAS) and Department of Physics, Chemistry and

Pharmacy, University of Southern Denmark, Odense 5230, Denmark. KEYWORDS: multifunctional biocatalyst, mesoporous silica nanoparticles (MSN), palladium nanoparticles, lipase CalB, cascade reaction

ABSTRACT: The combination of chemo- and biocatalysts offers a powerful platform to address synthetic challenges in chemistry, particularly in synthetic cascades. However, transferring both of them into organic solvents remains technically difficult due to the enzyme inactivation and catalyst precipitation. Herein, we designed a facile approach using functionalized mesoporous silica nanoparticles (MSN) to transfer chemo- and biocatalysts into a variety of organic solvents. As a proof-of-concept, two distinct catalysts, palladium nanoparticles (Pd NPs) and Candida antarctica lipase B (CalB), were stepwise loaded into separate locations of the mesoporous structure, which not only provided catalysts with heterogeneous supports for the recycling but also avoided their mutual inactivation. Moreover, mesoporous particles were hydrophobized by surface alkylation, resulting in a tailor-made particle hydrophobicity, which allowed bifunctional catalysts to be dispersed in eight organic solvents. Eventually, these attractive material properties provided the MSN-based bifunctional catalysts with remarkable catalytic performance for cascade reaction synthesizing benzyl hexanoate in toluene. On a broader perspective, the success of this study opens new avenues in the field of multifunctional catalysts where a plethora of other chemo- and biocatalysts can be incorporated into surface-functionalized materials ranging from soft matters to porous networks for synthetic purpose in organic solvents.

INTRODUCTION


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The fabrication of heterogeneous biocatalysts, through entrapping or immobilizing enzymes/whole cells onto organic and inorganic supports, offers the potential to revolutionize biocatalyst performance at the industrial process scale.1 In particular, the use of heterogeneous supports simplifies biocatalyst recycling and promotes continuous process development.2-3 Today, any type of industrial enzymes can be immobilized onto a “solid” carrier whose properties can be further tuned by chemical or physical manipulation. In many examples, hydrogels4-5 and synthetic resins6-8 are used as enzyme hosts for improving catalyst stability and usability at ambient/elevated pressures and temperatures. However, catalytic efficiency using these carriers is often compromised by their macroscopic sizes and nonporous structures that give rise to a small interfacial contact during reactions.9 In contrast to traditional materials, mesoporous particles become ideal supports owing to their self-assembled porous structures that allow for high enzyme loadings with large interface contact.10-11 Moreover, the porous networks can provide enzymes a protective environment against harsh reaction conditions, such as extreme pH, elevated temperature, and organic solvents.12-13 Recently, the mesoporous silica nanoparticles (MSN) with hierarchical pores or dendritic pores were reported to be good supports for enzyme immobilization.14-20 In addition, mesoporous materials were particularly explored to accommodate two catalytic species into their hierarchical structures for cascade catalysis.21 Since there is no need to purify and isolate intermediates, such bifunctional system can reduce operation time, production cost and waste, and meanwhile, enhance overall yield.22-23 Many groups have adopted this concept by combining metal particles (e.g., Ru, Au, Pt, and Pd) with other active species on the porous surface,24-25 achieving exceptional cascade productivity towards, for instance, knoevenagel condensation26 and oxidation of cinnamyl alcohol.27 Despite these successes, enzymes are sparingly combined with metal particles or


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organocatalysts for the same principle, because the incorporation of these distinct species (e.g., different stability and size) into a size-restricted porous material remains a challenge to date. Starting in 2013, the Bäckvall group reported the first example of bifunctional biocatalysts, where they pioneered a crosslinking process to co-immobilize Candida antarctica lipase B (CalB) and palladium nanoparticles (Pd NPs) in siliceous mesocellular foams.28 Such co-immobilization offered the two catalysts to cooperatively catalyze the dynamic kinetic resolution (DKR) of a primary amine with improved yield and enantioselectivity. Very recently, Zhang et al. further improved the fabrication approach by positional loading of Pd NPs and CalB into the inner core and outer shell of mesoporous silica, respectively.29 The spatial separation of two active sites thereby resulted in an even better yield. These two pioneering examples successfully illustrate mesoporous materials to be suitable to support bifunctional metal-enzyme composition for cooperative reactions. However, from a practical point of view, the current preparation protocols are based on as-synthesized porous scaffolds that are neither tailor-made to specific reaction conditions nor provide an optimal enzyme microenvironment, thus leaving much room for further improvement. Herein, we report a stepwise approach that allows loading Pd NPs and biocatalysts into mesoporous silica nanoparticles (MSN) whose surface hydrophobicity is finely tuned via postmodification with alkyl chains. The tailored material surface not only provides an interfacially active hydrophobic microenvironment to Candida antarctica lipase B (CalB) but also enables to transfer the bifunctional MSN from water into a wide range of organic solvents, making the system suitable in a reaction medium of interest. Moreover, their hydrophobized surface allows high protein loading because of the hydrophobic interactions and possible hydrogen bonding,30 and facilitates nonpolar substance diffusion across the hierarchical mesoporous structure. As a result


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of these properties, the combination of lipase and Pd NPs in our system allows to catalyze a cascade reaction with high efficiency.

RESULTS AND DISCUSSION The synthesis route of the bifunctional biocatalyst is illustrated in Scheme 1. It started with preparing mesoporous silica nanoparticles (MSN) according to the previously reported method.31 Pd(0) NPs were then loaded into these as-synthesized MSN particles by in-situ reduction of Pd(AcO)2 in the presence of NaBH4 solution, obtaining a hybrid Pd@MSN. The original MSN display a white color, while after the Pd NPs encapsulation, the MSN-based nanocomposites show a sharp contrast color of black, which is a straightforward proof of successful Pd loading in MSN (Figure S1). In order to change the particles from hydrophilic to hydrophobic, long-chain alkanes were introduced to the surface of Pd@MSN by reacting with trimethoxy(octadecyl)silane (TMODS), obtaining [email protected]

Scheme 1. Schematic illustration of the construction of CalB@Pd@mMSN.


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In addition to Pd NPs, CalB (Figure S2) was subsequently immobilized to the hydrophobized Pd@mMSN via hydrophobic interactions, forming a bifunctional biocatalyst, denoted as CalB@Pd@mMSN. In order to optimize the protein loading, Pd@mMSN particles were mixed with different concentrations of CalB (5, 20, and 40 mg mL-1), achieving CalB-5@Pd@mMSN, CalB-20@Pd@mMSN, and CalB-40@Pd@mMSN, respectively. The exact protein loading on particles was determined by Bradford assay, showing that CalB-5@Pd@mMSN, CalB20@Pd@mMSN, and CalB-40@Pd@mMSN contain 3.65, 7.41, and 10.76 µg mg-1 CalB, respectively (Figure S3). This finding illustrates the enzyme loading depending on the initial amount of added enzymes and the adsorption capacity could reach 15 µg mg-1. Such dependence could be also proved by thermogravimetric analysis (TGA) (Figure S4). In contrast, the unfunctionalized Pd@MSN can only immobilize a little CalB (2.23 µg mg-1) even mixed with a higher initial concentration of CalB, which further indicates the importance of surfacemodification. Besides, the Pd loading was determined by inductively coupled plasma optical emission spectrometry (ICP-OES), showing 5.3% and 4.6% Pd content on Pd@MSN and Pd@mMSN, respectively. Interestingly, after CalB adsorption on the CalB-5@Pd@mMSN, CalB20@Pd@mMSN, and CalB-40@Pd@mMSN, Pd content slightly decreased to about 4.2% which is assumed to be due to the loss of unencapsulated Pd NPs from the MSN surface and the weight increase of the particle by loading CalB. This assumption is proved by the fact that Pd loading remains constant in the successive reaction process. The mesoporous nanoparticles were initially characterized by powder X-ray diffraction (XRD). The broad peak from 20 o < 2 < 23 o in the wide-angle XRD pattern can be ascribed to amorphous silica (Figure 1a).34 The peaks at around 1.5 o and 2.4 o in the small-angle XRD pattern could be assigned to the typical plane of MSN, thus confirming the successful preparation of mesoporous


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MSN (Figure S5).35 For the Pd-containing samples, the peaks centered at 2 = 39.4 o, 46.1 o, 68.3 o,

and 81.1

o

in the wide-angle XRD pattern belong to the 111, 200, 220, and 311 diffraction

maxima of metallic Pd characterized by its face-centered cubic structure, respectively.36-37 The disappearance of the characteristic diffraction peaks at 1.5 o and 2.4 o in small-angle XRD is related to the destruction of regular mesoporous structure of MSN caused by the incorporation of Pd, which indicates the filling of the MSN pore channels by Pd NPs.31, 38 The specific surface areas and porosities of the resultant materials were measured by the N2 adsorption/desorption experiments. The curve of MSN represents a type-IV isotherm pattern, indicating mesoporosity. 39

In comparison with the parent MSN, the remarkable decrement in N2 adsorption capacity and

significant pore size change of Pd@MSN indicate that pores of the MSN are occupied by the encapsulated Pd nanoparticles (Figure S6).34,38 The similar trends go to the Brunauer-EmmettTeller (BET) surface and pore volumes, which further confirms the loading of Pd inside pores blocking the N2 adsorption (Table 1). The resultant composites after immobilizing CalB exhibit the less reduced BET surface areas, which is due to the attachment of CalB on the surface of MSN blocking some pore openings since the pores of MSN are too small to be accessible for CalB.40-41 To investigate whether the surface modification worked out, Pd@mMSN was analyzed by Fouriertransform infrared spectroscopy (FTIR) (Figure 1b). The characteristic bands (2780 - 2900 cm-1) for Pd@mMSN correspond to the C-H stretching mode, thus clearly confirming the presence of alkane groups on MSN particles.42 Not only these surface-modified particles but also CalB@Pd@mMSN were subjected to FTIR analysis. The intensified bands at 1640 and 1560 cm-1 (Figure S7), which belong to the stretching absorption of amide I group and amide II, are found on CalB@Pd@mMSN, suggest the presence of this protein on MSN particles after enzyme immobilization.43


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Figure 1. (a) Wide-angle XRD of MSN, Pd@MSN, Pd@mMSN, and CalB-20@Pd@mMSN. (b) FTIR spectra of CalB, Pd@MSN, Pd@mMSN, and CalB-20@Pd@mMSN. (c) Water contact angle images of Pd@MSN, Pd@mMSN, CalB-5@Pd@mMSN, CalB-20@Pd@mMSN, and CalB-40@Pd@mMSN; photos of Pd@MSN and Pd@mMSN dispersed in toluene (100 mg mL1).

After characterizing the materials composition, we next investigated their hydrophobicity by contact angle measurement. As shown in Figure 1c, the high contact angle (131.2 ± 1.3 o) for Pd@mMSN is observed after surface alkylation, which starkly contrasts to the low contact angle of 22.6 ± 1.0 o for unmodified Pd@MSN. This remarkable change of contact angle discloses the successful alkylation on MSN. Such hydrophobicity change could affect the particle dispersibility in solvents. For example, modified Pd@mMSN could be well dispersed in toluene while


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unmodified samples quickly aggregated. Interestingly, CalB-loaded Pd@mMSN resulted in a decrease of particle hydrophobicity. For instance, CalB-5@Pd@mMSN has a contact angle of 115.9 ± 1.4 o, which is lower than that for original Pd@mMSN. The contact angle further decreases with higher CalB loading on Pd@mMSN. This decreased contact angle indicates the hydrophilic nature of enzymes that can be further used to tune the particle hydrophobicity. Nevertheless, CalB@Pd@mMSN remains hydrophobic after enzyme immobilization, thus qualified for their following use in organic solvents.

Table 1. Structural Properties of the Tested Catalysts SBET a

VTotalb

(m2 g-1)

(cm3 g-1)

MSN

649

0.80

Pd@MSN

63

0.27

Pd@mMSN

32

0.43

CalB-5@Pd@mMSN

28.6

0.40

CalB-20@Pd@mMSN

27

0.22

CalB-40@Pd@mMSN

25.8

0.32

Sample name

a

SBET = BET Surface area, based on the adsorption data in the partial pressure of 0.05 < P/P0 < 0.20. b VTotal = Total pore volume, estimated from the adsorbed nitrogen volume at a relative pressure of about 0.95.

For organic synthesis, the choice of solvent media plays a crucial role in reaction efficiency. As such, CalB-20@Pd@mMSN was dispersed into eight different organic solvents with a wide range of polarity index. Figure 2 shows that the hybrid catalysts could be well-dispersed in all listed


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solvents. This finding suggests that surface-modified MSN can be adapted in many reaction media of interest.44-45

Figure 2. Photos of CalB-20@Pd@mMSN well-dispersed in different organic solvents (100 mg mL-1; numbers on vials mean polarity index; DCM – dichloromethane, EA – ethyl acetate, DMF – N, N-dimethylformamide, DMSO – dimethyl sulfoxide). Next, the morphology and structure of catalysts were investigated using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The SEM images of MSN and Pd@mMSN show almost identical morphology and particle diameters (Figure S8), which implies that the Pd encapsulation process has no effect to MSN surface. Interestingly, after CalB adsorption, the MSN surface becomes rather rough, presumably due to random protein distribution on MSN (Figure 3a). Inspired by this morphological observation, further TEM analysis was done to analyze the localization and composition of the hybrid catalysts. The bright-field TEM images (Figure S9) prove the highly ordered mesostructure of the parent material and the destructed mesostructures of CalB@Pd@mMSN, which further testifies the pore filling by Pd NPs. From the bright-field TEM micrograph (Figure 3b), the Pd NPs with narrow size distribution (ca. 1.80 ± 0.60 nm in diameter) are clearly observed on the mesoporous silica particles of CalB-


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20@Pd@mMSN. A similar Pd NPs density and size distribution for Pd@MSN and Pd@mMSN (Figure S10) suggest that the morphological variations caused by enzyme immobilization are negligible. The high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and spectrum imaging based on energy-dispersive X-ray spectroscopy (EDXS) further confirm the homogeneous distribution of Pd NPs in Pd@mMSN (Figure S11) and CalB20@Pd@mMSN (Figure 3c). Since CalB is a multiple element-composed macromolecule, it is difficult to directly map its existence on MSN if the density is quite low. However, with OsO4 staining,46 EDXS analysis could indirectly prove the presence of OsO4-bound CalB on CalB20@Pd@mMSN (Figure S12).

Figure 3. (a) SEM image of CalB-20@Pd@mMSN; (b) bright-field TEM image of CalB20@Pd@mMSN (inset: Pd NPs size distribution); (c) HAADF-STEM image and corresponding Si, O and Pd element maps for CalB-20@Pd@mMSN.


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To directly observe proteins on MSN, CalB was labelled with fluorescein isothiocyanate (FITC) to prepare CalB-FITC@Pd@mMSN. Fluorescence microscopy images show the green particles of CalB-FITC@Pd@mMSN are dispersed in acetone, which, however, was not observed by light microscopy (Figure 4b), thus proving the presence of CalB-FITC on MSN. In addition to CalB, green fluorescent protein (GFP) and FITC-labelled glucose oxidase (FITC-GOD) were also adsorbed onto Pd@mMSN and observed with optical and fluorescence microscopy. Figure 4c and 4d show that both GFP and GOD-FITC could readily reside on silica particles. The success of immobilization of three distinct proteins illustrates that surface-modified mesoporous silica can be used as a versatile platform for preparing bifunctional catalysts from diverse biocatalyst sources.

Figure 4. (a) Scheme illustrates that diverse proteins/enzymes can be immobilized to Pd@mMSN. Optical (top) and fluorescence (bottom) microscopy images of Pd@mMSN after adsorbing (b) FITC-CalB, (c) GFP, and (d) FITC-GOD.


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After stepwise catalysts loading, we first evaluated the catalytic performance of CalB in CalB20@Pd@mMSN by a typical esterification reaction where 1-octanol and octanoic acid were converted to octyl octanoate in toluene (Figure 5a). The yield of the product octyl octanoate was measured by gas chromatography (GC) using pure octyl octanoate as the standard (Figure S13). The catalytic data in Figure 5b and 5c reveal that the lipase-catalyzed conversion by CalB20@Pd@mMSN is very fast, achieving almost 100% yield in only 20 min. In contrast, with the same duration and reaction conditions, there was only 25% yield using the identical amount of free CalB. In addition, CalB-20@Pd@mMSN shows a superior catalytic performance to the equal amount of CalB immobilized on Novozym 435 (175 mg g-1 CalB loading),47 a commercial CalB immobilisate. Besides, the control experiment with Pd@mMSN as catalyst produces no octyl octanoate without CalB loading. The superior catalytic performance by CalB-20@Pd@mMSN is attributed to the large surface contact of mesoporous carriers as well as their good dispersity in toluene. Apart from the catalytic efficiency, protein thermal stability was also evaluated in the reaction medium (Figure 5d). At 80 °C, immobilized CalB (both CalB-20@Pd@mMSN and Novozym 435) displays no activity decrease in 24 h; however, without the protective carriers, free CalB loses 70% activity after 24 h. The improved thermal stability of enzymes in CalB20@Pd@mMSN, therefore, suggests their potential as robust biocatalysts for industry-relevant application in organic solvents.


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Figure 5. (a) Reaction scheme of esterification of 1-octanol and octanoic acid catalyzed by CalB. (b) Time-dependent yield of octyl octanoate catalyzed by (1) free CalB, (2) Novozym 435, and (3) CalB-20@Pd@mMSN at 25 °C in toluene. (c) Yield (20 min) of octyl octanoate using different catalysts. (d) Thermal stability of (1) free CalB, (2) Novozym 435, and (3) CalB-20@Pd@mMSN at 80 °C in toluene. The positive performance of CalB in CalB-20@Pd@mMSN encouraged us to further assess the catalytic efficiency of the immobilized Pd NPs. For this purpose, a Suzuki coupling reaction was performed in a mixed solution of ethanol and water using iodobenzene and 4-acetylphenylboronic acid as substrates and CalB-20@Pd@mMSN as catalyst (Scheme S1). By mass spectroscopy analysis (Figure S14), successful product formation with 100% conversion within 3 h is confirmed, indicating that Pd NPs remain catalytically active after the immobilization of two catalysts. The slight 0.16% Pd leaching during the Suzuki reaction was determined by ICP-OES analysis, which indicates the stability of the catalyst with negligible leaching.


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After activity proof of the two separate catalysts (CalB and Pd NPs) in CalB-20@Pd@mMSN, their combined use was demonstrated by a one-pot cascade reaction, as illustrated in Figure 6a. In the reaction, Pd NPs first reduce the benzaldehyde to benzyl alcohol under a hydrogen atmosphere. The immobilized CalB then converts the intermediate into benzyl hexanoate. The benefit of such design can avoid the isolation of active intermediates, thus providing a facile approach synthesizing esters based on benzaldehyde in a mild way for industrial use. For comparison, five control experiments were established to catalyze the cascade reaction under the identical procedure: CalB-40@mMSN (with 17.78 µg mg-1 CalB loading, Figure S3) performed with Pd@mMSN, free CalB performed with Pd@mMSN, CalB-40@Pd@MSN, CalB40@mMSN, and Pd@mMSN. The final product could be evaluated and identified by GC, 1HNMR, and

13C-NMR

(Figure S15), respectively. As illustrated in Figure 6b and 6c, CalB-

20@Pd@mMSN is the most efficient catalyst for the cascade reaction achieving 76% yield within 2 h, which is higher than the CalB-5@Pd@mMSN with 54% yield, CalB-40@Pd@mMSN with 62% yield, and three controls. Interestingly, after 2 h, CalB-20@Pd@mMSN and CalB40@Pd@mMSN exhibit higher yields than CalB-5@Pd@mMSN. In control experiments, the CalB-40@mMSN performed with Pd@mMSN gives rise to 51% yield (2h), which is lower comparing with CalB@Pd@mMSN, indicating the cooperative effect of Pd and CalB together immobilized on CalB@Pd@mMSN. Besides, there is only 14% (2 h) yield observed for free CalB with Pd@mMSN cascade reaction which contains the same amount of protein as CalB20@Pd@mMSN, which is caused by the poor protein solubility in organic solvents. Besides, the unfunctionalized CalB-40@Pd@MSN gives rise to the lowest conversion (< 1%) after 2 h, which is mainly due to the poor dispersibility and lower CalB loading. Moreover, neither CalB-


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40@mMSN nor Pd@mMSN (Figure S16) can lead to the final product benzyl hexanoate, which illustrates the necessity of cooperating Pd NPs and enzyme for cascade reaction. In regard to practical applicability, reusability is a crucial parameter for the heterogeneous catalyst. The recycling experiments were performed using CalB-20@Pd@mMSN as a catalyst in the same cascade reaction. After four-time reuse, reaction yield (4 h) remains higher than 80% compared to the first run (Figure 6d). This multiple reusability demonstrates that CalB20@Pd@mMSN is a robust and recycling heterogeneous catalyst that may be further applied in other solvent conditions for the synthetic purpose.

Figure 6. (a) One-pot cascade reaction of benzaldehyde with ethyl hexanoate. (b) Time-dependent yield of benzyl hexanoate catalyzed by different catalysts (1) CalB-5@Pd@mMSN; (2) CalB20@Pd@mMSN; (3) CalB-40@Pd@mMSN; (4) CalB-40@mMSN performed with Pd@mMSN;


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(5) free CalB performed with Pd@mMSN; (6) CalB-40@Pd@MSN. (c) Yield (2 h) of benzyl hexanoate using different catalysts. (d) Reusability of CalB-20@Pd@mMSN. In order to get insight into understanding its stability, CalB-20@Pd@mMSN was re-subjected to FTIR analyses and TEM characterization after four-time reuse. It is found that the reaction caused no change of FTIR spectra, indicating the remaining presence of the modified surface and proteins on the hybrid catalysts (Figure S17). TEM characterization confirms that there are no significant morphological changes or Pd aggregation occurring between the fresh and recovered CalB-20@Pd@mMSN (Figure S18). These results imply that our bifunctional materials are robust under the critical reaction conditions like organic solvents. Furthermore, catalyst leaching experiments were carried out by analyzing the residual CalB and Pd NPs released from the cascade reaction (6 h). Bradford assay and ICP-OES analysis show that there is only 0.11% Pd leaching and no detectable protein leaching, which illustrates the high stability of the bifunctional materials.

CONCLUSIONS In conclusion, we have designed bifunctional biocatalysts based on mesoporous silica nanoparticles (MSN) where particle hydrophobicity was finely-tuned by surface alkylation, and two catalysts (Pd nanoparticles and CalB enzyme) were separately loaded into compartmentalized locations. This system, on the one hand, provides a large-surface-area platform to transfer catalysts into diverse organic solvents, thus can be developed as a versatile tool to accommodate other chemo- and biocatalysts in the reaction medium of interest. On the other hand, the combined use of metal and biocatalysts is demonstrated by the one-pot tandem reaction in the synthesis of benzyl hexanoate. This proof-of-principle example suggests the high potential using MSN-based bifunctional catalysts for advanced synthesis in the future.


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EXPERIMENTAL SECTION Materials. Unless otherwise stated, all chemicals were obtained from commercial suppliers and used without further purification. Candida antarctica lipase B (CalB) was purchased from c-LEcta GmbH (Leipzig, Germany), and the protein content was about 10 wt. % based on the Bradford test. Analytical techniques. Inductively coupled plasma-optical emission spectroscopy (ICP-OES) was carried out on a Perkin Elmer Optima 7000DV optical emission spectrometer. Bradford assay was carried out with TECAN infinite M200. Drop shape analysis system DSA 10 from Krüss was used to characterize the particle hydrophobicity by pressing particle samples into a smooth tablet for water contact angle measurements and each analysis was repeated for 5 times. Thermogravimetric analysis (TGA) was carried out with Mettler-Toledo TGA instrument using a 10 °C min-1 ramp up to 800 °C in air atmosphere. FTIR spectra were recorded on a FT-IR spectrometer Tensor II (Bruker) with an ATR unit. The fluorescence images were taken using the Olympus Provis AX70. The small-angle X-ray diffraction experiments were performed on a Bruker Nanostar diffractometer with Cu Kα1 radiation (λ = 154.06 pm) and a position sensitive Histar 2D detector. The wide-angle powder X-ray diffraction (XRD) analysis was performed on a PANalytical X’Pert Pro powder diffractometer with Debye-Scherrer geometry equipped with a Ge(111)-monochromator, a rotating sample stage, and a PIXcel detector, using Cu Kα1 radiation (λ = 154.06 pm). The data was collected in reflection mode using a divergence slit that kept the illuminated sample area constant. Nitrogen adsorptions are measured volumetrically at 77 K on a QuadraSorb with a sample mass of ca. 100 mg. The Brunauer-Emmett-Teller (BET) method was used to calculate the specific surface area based on the adsorption data in the partial pressure of


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0.05

Surface-Functionalized Mesoporous Nanoparticles as Heterogeneous

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