Biomimetic Mineralization Inducing Lipase-MOF Nanocomposite for

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Biomimetic Mineralization Inducing Lipase-MOF Nanocomposite for Pickering Interfacial Biocatalytic System Liang Qi, Zhigang Luo, and Xuanxuan Lu ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.9b00113 • Publication Date (Web): 03 Mar 2019 Downloaded from http://pubs.acs.org on March 7, 2019

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ACS Sustainable Chemistry & Engineering

Biomimetic Mineralization Inducing Lipase-MOF Nanocomposite for Pickering Interfacial Biocatalytic System Liang Qi1, Zhigang Luo1,2,3*, Xuanxuan Lu4 1. School of Food Science and Engineering , South China University of Technology, No.381 Wu Shan

Road, Guangzhou, 510640, China. 2.South China Institute of Collaborative Innovation, Xue Fu Road, Dongguan, 523808, China. 3.Overseas Expertise Introduction Center for Discipline Innovation of Food Nutrition and Human Health (111 Center), No.381 Wu Shan Road, Guangzhou 510640, China. 4.Department of Food Science, Rutgers, The State University of New Jersey, 65 Dudley Road, New Brunswick, New Jersey 08901, USA.

*Corresponding

author:

Zhigang Luo, Tel: +86-20-87113845, Fax: +86-20-87113848. E-mail address: [email protected]

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Abstract

In this study, a brand new biocatalytic system was successfully accomplished by combination of MOFs biomimetic mineralization and Pickering interfacial system for enhanced lipase catalysis and recycling in organic media. Specifically, a highly porous lipase-loaded MOF composite was synthesized via biomimetic mineralization of ZIF-8 around lipase from Candida rugosa (CRL). With increasing enzyme loading amount, the role of CRL in modulating size, crystallinity and interior or exterior structure of composite was meticulously clarified. In this process, we disclosed a gradation from crystal into amorphous form for CRL-loaded ZIF-8, and more importantly discovered an enrichment of enzymes on the surface of crystals. Combining this phenomenon with results of dynamic light scattering, zeta potential and water contact angle, as well as styrene-induced interface solidification, we confirmed the surface-embedded CRL of crystal played a role in adjusting the surface chemical characteristics of composite that directly affected the stability of emulsion and distribution rule of stabilizers on the oil-water interface. Furthermore, through the comparison of hydrolyses involving small substrate (p-nitrophenyl butyrate) and a larger one (p-nitrophenyl palmitate), a stable o/w Pickering emulsion with an optimized quantity and integrity of oil droplets was proved to achieve the highest catalytic efficiency. Notably, since size selectivity of ZIF-8 shell induced different locations of substrates in contact with enzymes, an obvious discrepancy in catalysis efficiency was observed for substrates with different sizes. Impressively, we could employ this system for transesterification by only changing the encapsulated enzyme


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(lipase B from Candida antarctica expressed in Aspergillus) and optimizing the ratio of oil/water. The excellent catalytic performance highlighted the versatility of this interfacial biocatalytic system. Key words: Biomimetic mineralization; Lipase-MOF porous composite; Oil-inwater emulsion; Interfacial biocatalysis; Size-selective catalysis. Introduction In nature, many living organisms fabricate molecular architectures specifically designed to provide exoskeletal protection and structural support for soft tissue. This biologically induced, self-assembly process, termed biomineralization, is carried out with exquisite control of crystal morphology and compositional specificity under physiological conditions1. Such natural biomineralization processes have inspired ‘biomimetic’ strategies for the synthesis of novel materials, which would significantly increase the potential for utilizing functional biomacromolecules in applications where enhanced thermal stability, tolerance to organic solvents or extended shelflifetime is required2, 3. Among

the

existing

biomimetic

mineralization

approaches,

inorganic

minerals(for example, calcium phosphate, titanate) have been demonstrated to be a shell candidate that can provide protection for bioactive macromolecules4, 5. However, these reports clearly demonstrate the fusion of organic substances and inorganic architectures is not an easy task. Indeed, to stimulate growth of a calcium phosphate shell on vaccines, genetic modification was required to incorporate a specific peptide



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sequence known to have a high affinity for calcium ions5. Furthermore, coating biopolymer oligodopa around titania nanoparticles must rely on arginine that provides bioadhesion-assistance4. In spite of this, these results highlight that enhancing the interactions between inorganic and organic components is a key to inducing biomineralization. Therefore, we posit that organic–inorganic hybrid materials would provide a more versatile and general method for encapsulating biomacromolecules, especially the enzymes, as the protein domains have a high affinity for the organic moieties1. A burgeoning class of hybrid materials termed metal organic frameworks (MOFs) just meet our requirements, because they are: constructed from organic and inorganic components1, thermally and chemically stable6, 7 and can be constructed under mild biocompatible conditions6. Furthermore, MOFs possess open architectures and large pore volumes8, 9, which facilitate the transport of suitable size molecules through the protective

porous

coating10,

enabling

the

selective

interaction

of

the

biomacromolecules with the external environment. More importantly, in contrast to inorganic minerals, MOFs biomimetic mineralization is easy to achieve due to the biomacromolecules affinity towards the imidazole-containing building block arising from intermolecular hydrogen bonding and hydrophobic interactions11. Benefited from this, biomimetic mineralization involving various MOFs has been employed for abundant enzymatic catalyses12-19. However, as yet these reports have been confined to the single-phase catalysis, in which substrates, products and catalysts are commonly in the same mechanically stirred reactors. The obvious drawback of this


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mechanically stirred system is inhomogeneous mixing, which results in resilientmass/heat-transfer limitations ascribed to a modest interfacial contact between substrates and catalysts, even under vigorous stirring20. Recently,

Pickering

interfacial

catalysis,

where

colloid

particles

can

simultaneously stabilize a Pickering emulsion and catalyze reactions at the interface of two immiscible solvents, has become an attractive issue.21-23 Our group has achieved fruitful results in this field, including elaborate modification of highly emulsifying stabilizers24, 25 and establishment of environmental-responsive interfacial catalytic system26. In the system of Pickering interfacial catalysis, the location of catalysts at interfaces of micro/nano-sized droplets maximizes the extent of the catalyst–liquid (substrates) interfacial areas, overcoming the traditional transfer limitations in the single-phase biocatalysis. Meanwhile, it has been proved to exhibit a fascinating performance in biphasic reactions involving phase-transfer behavior of substrates/products, e.g., transforming poorly water-soluble substrates into watersoluble fine chemicals, fuels, etc.27-29 Therefore, these attributes inspire us to achieve a brand new enzyme-induced biocatalysis with high efficiency, stability and diversity by combination of MOFs biomimetic mineralization with Pickering interfacial system. Zeolitic imidazolate framework-8 (ZIF-8), formed by coordination between Zn2+ ions and 2-methylimidazole (2-Melm), was selected as a candidate MOF material for this study due to its high surface area, high surface activity30,

31

and exceptional

chemical and thermal stability.32 Meanwhile, since lipase from Candida rugosa (CRL) is widely involved in hydrolysis33, it was chosen as the representative enzyme.



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Through the biomimetic mineralization, we have synthesized a highly porous CRLloaded ZIF-8 composite and the role of CRL in modulating the mineralization process, involving size, crystallinity and morphology of composite, was clarified in detail. Then CRL-loaded ZIF-8 were utilized as the surfactant stabilizer for the generation of Pickering emulsions, and their distribution rule at the oil-water interface were summarized based on the characterization of surface chemical properties and results of interface solidification. Moreover, this interfacial biocatalytic system was carried out in a comparative hydrolyses between a pair of substrates to testify that their discrepancies in catalysis efficiency had to do with different locations of substrates contacting with enzymes induced by size selectivity of ZIF-8 shell. Finally, to highlight the versatility of this enzyme-MOF based Pickering interfacial catalytic system, it was also employed for a transesterification after changing encapsulated enzyme to lipase B from Candida antarctica expressed in Aspergillus (CALB) and optimizing oil/water rate. Experimental Section Materials Zn(NO3)2·6H2O

(99.0%),

2-methylimidazole

(2-Melm,

99.0%),

sodium dodecyl sulfate (SDS), bicinchoninic acid (BCA), p-nitrophenyl butyrate (pNPB), p-nitrophenyl palmitate, (p-NPP), 1-butanol, vinyl acetate, 3-(4-hydroxyphenyl) propan-1-ol, vinyl laurate, lipase from Candida rugosa (CRL, lyophilized powder) and lipase B from Candida antarctica expressed in Aspergillus (CALB, liquid form )


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were purchased from Sigma-Aldrich (Shanghai, China). All other chemicals, such as n-heptane, hydrochloric acid, hydrofluoric acid were of analytical grade and obtained from commercial sources. Methods Synthesis of CRL-loaded ZIF-8 by biomimetic mineralization Various amounts (20, 40, 80, 160 mg) of CRL were added into a solution of 2methylimidazole (1.25 M, 40 ml) in deionized water. A separate solution of Zn(NO3)2·6H2O (0.31 M, 4 ml) was also prepared. These two solutions were combined and then agitated at 600 rpm for 0.5h at room temperature. The obtained products were firstly dispersed in SDS solution (10 wt%) at 60 C for 10 min to wash off the free enzymes on the crystal surface, followed by three centrifugation/wash cycles (10000rpm, 6min) with deionized water. The resulting CRL-loaded ZIF-8 with various CRL addition was named ZIF-8@CRL-20, ZIF-8@CRL-40, ZIF-8@CRL-80 and ZIF-8@CRL-160, respectively. The synthesis of pure ZIF-8 followed the same procedure to the preparation of CRL-loaded ZIF-8 but in the absence of enzyme solution. Preparation of Pickering emulsion 1.0 mL of n-heptane and 1.0 mL of CRL-loaded ZIF-8 suspensions (1 wt%) were charged in 10 mL plastic vessels at room temperature. Pickering emulsions were readily formed using a homogenizer (IKA Ultraturrax T25, Germany) at a stirring rate of 13,000 rpm for 30s.



Pickering interfacial biocatalysis 1.0 mL of n-heptane containing 2.5 mg mL-1 of p-NPB or p-NPP and 1.0 mL of PBS solution (10 mM, pH 7.4) containing 10 mg of CRL-loaded ZIF-8 were homogenized at 13000 rpm to form Pickering emulsion, then the catalytic reaction was conducted with a gentle stirring (200 rpm). After a defined interval, the reaction was terminated by adding 1 mL of 0.5 M Na2CO3, followed by centrifugation for 5 min at 6000 rpm. The absorbance of the water phase filtered through a 0.22 μm filter was measured at 410 nm using a UV-vis spectrophotometer (Hitachi U-3010, Japan) and the amount of liberated p-nitrophenol (p-NP) was calculated according to the standard curve (UV-vis vs. concentration of p-NP, Fig.S1). To minimize potential errors, pure ZIF-8 replaced CRL-loaded ZIF-8 for the blank. One activity unit (U) was defined as the enzyme amount needed for converting 1 μmol substrate per minute. The specific activities (U·mg-1) of encapsulated lipase were determined under the same condition within 5 min. All reactions were repeated at least three times. The catalytic recyclability was assessed by measuring the conversion rate in each cycle. After each batch, the CRL-loaded ZIF-8 was collected through centrifugation (4000 rpm) and then washed with PBS solution (10 mM, pH 7.4) and utilized for the next cycle. The maximum conversion rate was taken to be 100%, and the relative conversion rate (%) represented the ratio of residual to maximum conversion rate of each sample. Characterization of CRL-loaded ZIF-8


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Enzyme loading capacity of CRL-loaded ZIF-8 was calculated by BCAmethod. Specifically, 5 mg of sample was destroyed with 3 ml of 0.2 M HCl aqueous solution and 20 l HF (48 % water solution), followed by centrifugation at 10000 rpm for 10 min. 0.2 ml of supernatant filtered through a 0.22 μm filter was mixed with 2 ml of BCA reagent, which was incubated at 31C for 60 min prior to UV measurement. To minimize potential errors arising from the low pH, pure ZIF-8 replaced CRL-loaded ZIF-8 for standardization. The difference of intensity at 562 nm of the sample and the standard was used to calculate the concentration of CRL within the ZIF-8 based on the curve (UV-vis vs. concentration of CRL, Fig.S1) The calculation of immobilization efficiency is based on eqn (1)

` Immobilization efficiency (%) =

(m-CV) 100% m

(1)

where m (mg) is the total amount of CRL introduced into the solution; C (mg·mL-1) and V (mL) are the CRL concentration and the volume of the supernatant, respectively. Fourier transform infrared spectroscopy (FTIR) spectra of CRL-loaded ZIF-8 were collected between 4000 and 500 cm-1 by Vector 33-MIR FTIR spectrophotometer (Brukev Optik, Germany) with 256 scans at a resolution of 4 cm-1. The powder (3 mg) was ground with spectroscopic grade KBr (200 mg) powder, and pressed into 1 mm pellets. A blank disc was used as the background.



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The changes in fractions of secondary structure of free CRL and CRL-loaded ZIF-8 were estimated by using FT-IR analysis tool according to the previous work34, 35.

The resolution of FT-IR spectra of samples was enhanced by taking its secondary

derivative of 1600-1700 cm-1 (amide I region). Then, the presented spectra were smoothed with a 20-point Savitzky-Golay by Systat PeakFit version 4.12. Furthermore, curve fitting of the amide I region was done and areas under multicomponent peak were quantified using multi-peak fitting. Thermal

gravimetric

analyses

(TGA)

in

air

were

performed

on

a

TG 209 F1 Libra Thermogravimetric Analyzer (NETZSCH, Germany). The samples were heated from room temperature to 600 C at a rate of 20 C /min under air atmosphere. X-Ray Diffraction (XRD) patterns were obtained using a D/max-IIIA full automatic XRD instrument (Rigaku, Japan). Diffractograms were collected at 40 kV and 30 mA with nickel-filtered Cu Kα radiation (λ=1.5405 Å). Powdered sample was scanned from 5 to 60°(2θ) at a scanning rate of 4°/min. The size distributions of samples were characterized by nano ZS instrument (Malvern Instrument Ltd., UK) at room temperature. The wavelength of light source was 633 nm and scattering angle was kept at 90°. Morphology observations were performed using a Merlin scanning electron microscope (Zeiss Co., Germany). An energy dispersive spectroscope (EDS) attached to SEM was conducted to analyze the elemental composition. Before the test, samples were dispersed in anhydrous alcohol (0.1 wt%) under mechanical agitation of 600 rpm for 2 min. One drop of suspension was carefully placed on a glass slide and dried


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at room temperature. After that, the samples were coated with Pt for conductivity and put under SEM for observations. Interior structures analysis was taken on a JEM-1400 plus transmission electron microscope (JEOL, Japan) with an accelerating voltage of 120 kV. Before the observation, a drop of anhydrous alcohol containing the synthesized samples (0.1 wt%) was added on a carbon grid and dried at room temperature. If the removal of enzyme was needed, the calcination of samples was conducted with muffle furnace (Zhengzhou Brother Furnace Co., Ltd, China) at 300 C for 2h. N2 adsorption/desorption isotherm was measured at 77 K using a Flowsorb Ⅲ 2310 Surface Characterization Analyzer (Micromeritics Instrument Co., USA) after the sample was first degassed at 150 °C overnight. Surface areas were determined by the Brunauer-Emmett-Teller (BET) method based on multi points in the p/p0 range of 0.04-0.32, and total pore volume was determined using the adsorption branch of N2 isotherm curve at the p/p0 = 0.99 single point. Pore size distribution was determined using the adsorption branch of N2 isotherms. Micropore size distribution analysis was carried out using the Horvath-Kawazoe method. Mesopore size distribution was calculated using the Barrett-Joyner-Halenda method.

Controlled digestion was carried out when ZIF-8@CRL-20 was taken as example: 150 μL HF was firstly diluted with 20 mL deionized water. ZIF-8@CRL-20 (10, 20, 40 or 80 mg) which had been dried at 60 C overnight was then immersed in above solution for 30 min. After the centrifugation (10000rpm, 5min), the supernatant was obtained through a 0.22 μm filter and the amount of diffused enzyme was determined from BCA method. To minimize potential errors arising from the low pH,



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pure ZIF-8 replaced ZIF-8@CRL-20 for standardization. All reactions were repeated at least three times. Controlled digestion of ZIF-8@CRL-40,80,160 was the same as that of ZIF-8@CRL-20, except for adjusting volume of HF to 140, 133 and 120μL, respectively, Characterization in Pickering emulsion Surface Coverage (Cc) of emulsion droplets was obtained based on the following assumption: (1) the emulsion droplets were monodisperse and spherical,(2) the nanoparticles lay flat on the surface of the dispersed droplets, and (3) all the nanoparticles were attached to the droplets in a monolayer. The Cc of the dispersion droplets was calculated according to our previous report.26 Since the oil core of Pickering emulsion possesses a lower density compared to water, the emulsion layer would float on top of water phase and corresponding droplet gradually keep steady when standing for minutes. Emulsion Index (EI) was measured according to eqn (2):

EI 

H2 100% H1

(2)

where H2 is the height of the observed emulsion and H1 is the height of all the phases. The emulsion droplets were visualized using a MB11 polarizing microscope (Shanghai optical instrument factory, China) equipped with a video camera. Emulsion droplets were placed directly onto a glass slide and captured under 50-200 magnification. The average size and size distribution of droplets was determined through statistical analysis from optical microscope images using Image-pro Plus 6.0.


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The water contact angles of samples prepared on tablet press were measured using a DCa40 MICRO contact angle goniometer (DATA physics, Germany). Zeta potential measurements were measured using a nano ZS instrument (Malvern Instrument Ltd., UK) equipped with a dip cell. The concentration of samples was maintained at 0.1 wt % and the experiments were conducted at room temperature. The n-heptane-water interface tensions were measured using Du–Noüy ring method (DIN 53915 and ASTM-971) by an automatic surface tensiometer DCAT 21 system (DATA physics，Germany).

For easy observation, Pickering emulsion was solidified by copolymerization reaction. The procedure was the same as that of traditional Pickering emulsion, except 0.56 wt% of AIBN, 1.4 wt% styrene and 2.1% g divinylbenzene (DVB) was dissolved in 1mL n-heptane prior to emulsification. Then, the resulting emulsion was poured into a 10 mL plastic vessel and deoxygenated with N2 for 20 min. The reaction mixture was heated to 65 °C and subsequently polymerized for 16 h. During this process, hollow polystyrene microcapsules were formed as the n-heptane was a poor solvent for the cross-linked polystyrene network leading to phase separation and precipitation at the oil/water interface, locking the stabilizers into the capsule surface (polystyrene layer). Therefore, we could peculate the location of stabilizers at the actual oil-water interface by the observing them at polystyrene layer in the hollow microcapsules. Results and discussion



In this study, we have synthesized a series of highly porous composite through the biomimetic mineralization of ZIF-8 around CRL. According to the BCA method, enzyme loading capacity and immobilization efficiency of samples were summarized in Table.1. Such an increased CRL content could also be verified by the EDS result (Fig.S2). To ascertain that CRL was indeed encapsulated by ZIF-8 framework in the biomimetic mineralization process, CRL-loaded ZIF-8 that had been washed with a surfactant to remove any surface bound proteins were examined by Fourier transform infrared spectroscopy (FTIR). As shown in Fig.1A, besides the characteristic absorption peaks for ZIF-8 (1581 cm-1 for C=N stretching vibration on the imidazole ring, 421 cm-1 for Zn-N stretching vibration), the stretches characteristic of CRL at 1,600~1,700 cm

-1

were found in spectra of CRL-loaded

ZIF-8, which corresponded to amide I mainly from C = O stretching mode. These peaks provided a strong evidence for CRL encapsulation within ZIF-8, whose enhanced intensity was in accordance with increasing amount of CRL within ZIF-8 framework. As we know, the biomineralization mechanism occurring in natural processes is widely attributed to the specific ability of amino acids, peptide fragments and more complex biological entities to concentrate inorganic cations (Ca2+ and Zn2+ ) to seed biominerals36, 37. In this study, there was a slight shift of the amide I peak in the CRL-loaded ZIF-8(Fig.1B), but the same phenomenon was not observed in CRL incubated with only Zn2+ ions(Fig.1C), which confirmed a direct protein-MOF interaction due to the coordination between Zn2+ and the carbonyl


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groups on the proteins38, 39. This covalent bonding, along with hydrogen bonding and hydrophobic interactions previously reported11, 40, inevitably caused an alteration in conformational integrity of enzyme molecules, which was mainly related to the variation in their secondary structures. Amongst complete FTIR spectra, amide I band is most sensitive spectral region of protein structural components. The substantial changes after immobilization generate different frequency patterns and consequently reflect in the amide I vibration band, therefore considered for the determination of secondary structure41. The second derivative FT-IR spectra for CRL and immobilized CRL were obtained using FT-IR tool (Fig.2A). The relative contents of β-sheet structure (red, 1610– 1640 cm-1), random coil structure (green, 1640–1650 cm-1), α-helix structure (blue, 1650–1658 cm-1) and β-turn structure (yellow, 1660–1700 cm-1) were determined based on the multi-component peak areas and their corresponding fraction are summarized in Fig.2B. From the results, the slight discrepancies in secondary structural

content

among

samples

confirmed

a

well-preserved

structural

conformation of CRL after entrapment within ZIF-8. However, it was worth noting that the fraction of each secondary structural content showed a regular increase or decrease, which was strongly associated with the surface hydrophobicity of enzymes. This would be discussed in detail later in this article. Furthermore, given the biomimetic mineralization that involves the combination between organic and inorganic substance with distinct thermal properties, the variation in thermostability of composites also confirms the encapsulation of CRL.



From thermal gravimetric analysis (TGA) results (Fig.3A), in contrast to constant weight in pure ZIF-8, gradual weight losses were observed in CRL-loaded ZIF-8 up to 370°C. By comparing the weight percentage between CRL-loaded ZIF-8 and pure ZIF-8, about 5, 8, 11, 17 wt% of weight loss occurred in the composites, corresponding to the protein decomposition, as did native CRL (inset in Fig.3A). This result was approximately in agreement with that from BCA method. At around 440 ° C, the sharp weight loss corresponded to the transformation from ZIF-8 into ZnO. In this study, pure ZIF-8 formed in the aqueous solution presented a high crystalline with intensive characteristic peaks in XRD pattern (Fig.3B). Nevertheless, with increasing loaded CRL, these characteristic peaks gradually weakened and more obvious broad diffuse peaks came out (Fig.3C), presenting a typical characteristic of crystalline/amorphous hybrid structure. For visually observation, this gradual change was well presented by SEM and TEM, which showed the combination of CRL and ZIF-8 had great impact on the interior or exterior structure of CRL-loaded ZIF-8. Firstly, the size of CRL-loaded ZIF-8 could be adjusted by CRL, the average size dropped from 557nm for ZIF-8@CRL-20 to 173nm for ZIF-8@CRL-160 (Fig.4, Fig.S3 and Table.S1). Moreover, it was clear that pure ZIF-8 crystal presented rhombic dodecahedron with smooth surface. However, increasing loaded enzyme would gradually blur the crystal outline and wrinkle the surface. Initially, this morphology change only affected the local area of each crystal without greatly destructing integrity of crystal (ZIF-8@CRL-20,40 and 80). However, more irregular


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adhesive clusters was present in the formation of ZIF-8@CRL-160. For quantitatively analyzing this exterior roughness change, external specific surface area of CRL-loaded ZIF-8 was measured by nitrogen sorption study. The results confirmed that each CRL-loaded ZIF-8 indeed exhibited a growth in external specific surface area compared with that of non-CRL-loaded counterpart ( pure ZIF-8 with a similar size) (Table.2 and Figure.S4). ZIF-8@CRL-20,40 and 80 with relatively intact particles exhibited a limited increase in external specific surface area, in contrast, ZIF@CRL-160 presented a greater increase, which was in line with its more deteriorated morphology. To vividly present the possible final results of morphology change, we further increased CRL addition to 640 mg. As expected, the rhombic dodecahedron feature was totally lost in ZIF-8@CRL-640. Instead, products were full of deformed tiny particle clusters with losing most crystalline degree but entrapping more

enzyme

molecules

(Figure.S5).

As

a

result,

we

attributes

this

continuous morphology deterioration to the great interference in crystal growth induced by excessive introduction of CRL, which agrees well with the biomineralization process in which the degree of crystallinity is carefully tuned by living organisms due to complex biological regulation42. From the observation of interior structure by TEM image in Fig.4, it seemed that CRL-loaded ZIF-8 showed more light spots (from 5 to 25 nm) as loaded CRL increased, which was not observed in pure ZIF-8. These light spots resulted from the low electron-scattering density of CRL molecules and their aggregates filled in the framework of ZIF-8. With help of nitrogen adsorption/desorption study, we gave a



clear picture of pore distribution in composite after various CRL loading (Fig.5). Before the test, a calcination process at 300 °C for 2 h was carried out to remove protein molecules from the CRL-loaded ZIF-8 (the temperature was chosen based on the thermal gravimetric analysis). The distribution of micropore calculated by the Horvath-Kawazoe method revealed all composites had a similar multistage micropore structure that mainly consisted of 0.4 ~ 0.65 and 0.7 ~ 0.75 nm size, which belonged to the ZIF-8 crystal itself(Fig.5B). This confirmed a well-formed ZIF-8 framework wrapping around enzyme molecules during the mineralization. Besides, learned from the micropore structure of ZIF-8@CRL-160, we proposed that although excessive introduction of CRL brought about a macroscopic change of crystals (morphology deterioration), it had little impact on the microstructure, which ensured that ZIF8@CRL-160 still possessed the size-selective shielding effect. When it came to the mesopore size distribution using the Barrett-Joyner-Halenda adsorption method (Fig.5C), the calcinated ZIF-8@CRL-20,40 and 80 showed an increase in the pores around 5~10 and 20~30 nm size corresponding to more protein molecules and aggregates contained in the framework, which was exactly consistent with gradually decreased specific surface area (Fig.5D). Nevertheless, instead of narrow size distribution presented by other samples, there was a great increase in both size and quantity of mesopore in ZIF-8@CRL-160, indicating excessive CRL induced more serious agglomeration during the mineralization. In view of the flexible distribution of CRL within ZIF-8 framework, it was necessary to figure out whether there were any enzyme molecules embedded on the


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surface of crystal. After the same calcination process, we could clearly identify small cavities on the surface region of each CRL-loaded ZIF-8 (Fig.4). The same as TEM, this change was not seen in pure ZIF-8, which verified these cavities were due to the removal of surface-embedded CRL molecules and their aggregates. Furthermore, from ZIF-8@CRL-20 to 160, because of increased irregularity of particles, the distribution of surface-embedded enzymes seemed to be heterogeneous by finding more dense area of cavity, especially in deformed particles. For quantificationally clarifying the distribution rule of surface-embedded CRL in each sample, we designed an experiment for controlled digesting CRL-loaded ZIF8 inspired by Hong43. Specifically, a fixed amount of hydrofluoric acid (HF) in aqueous solution was added to digest various amounts of CRL-loaded ZIF-8, and the amount of diffused enzyme was determined from BCA method. According to Hong, when a very small amount of HF was added, only the surface of the MOF crystal would be digested, but as the amount of HF was increased, the inner part of the crystal would be digested accordingly. This gradual change was illustrated by the controlled digestion of ZIF-8@CRL-160 in Fig.S6. In this study, the minimum amount of HF required for complete digestion of CRL-loaded ZIF-8 was calculated based on the number of organic ligands (2-Melm) in the sample (according to the Zn element from EDS). This fixed amount of HF diluted with distilled water was then added to 2× , 4× and 8× excesses of CRL-loaded ZIF-8, adjusting molar ratio of HF to the number of 2-Melm in CRL-loaded ZIF-8 to 1/2, 1/4 and 1/8, respectively. The relative CRL content of digested samples (ratio of



diffused CRL to total CRL) are summarized in Fig.6. For ZIF-8@CRL-20,40 and 80, the relative CRL content were found to present an approximate linear growth with a slope of 1, which confirmed that, by and large, CRL was homogeneously distributed throughout these CRL-loaded ZIF-8 and released gradually with the layer-by-layer digestion of the shell. Therefore, if took their CRL loading amount into consideration, there would be a gradient increase in amount of surface-embedded enzyme for ZIF8@CRL-20,40 and 80. When the same digestion method was applied to ZIF-8@CRL160, it clearly found the relative CRL content grew almost 1.5 times for less digested samples, which inferred more enzymes distributed on the superficial layer. For explaining this phenomenon, we monitored the morphology change of ZIF-8@CRL160 in the controlled digestion (Fig.S6). Obviously, in addition to the regular digestion of intact crystals, there was a fast fusion of deformed tiny particle clusters in the early digestion(1/8 digested sample). Based on this, we attributed the heterogeneous distribution of enzyme to the existence of deformed tiny particle clusters, whose higher enzyme loading and external specific surface area facilitated more enzymes exposure to the environment and thus brought about more and quicker diffused CRL than intact crystals as surface digestion proceeded. Pickering emulsion

The confined oil droplet in oil-in-water (o/w) Pickering emulsions can be considered as an individual microreactor, which is suitable for interfacial catalyzing poorly water-soluble substrates by enlarging catalytic contact area and accelerating mass transfer. As a result, a stable o/w Pickering emulsion with an optimized quantity


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and integrity of oil droplets makes the basic and key precondition for achieving a high catalytic efficiency. Presently, pure ZIF-8 formed a stable n-heptane-in-water Pickering emulsion with average droplet size of 17.71 μm (Fig.S7), its corresponding emulsion index (EI) and droplet surface coverage (Cc) maintained at least 72 % and 100% during 12h without any disturbances at room temperature(Fig.7A). When CRL-loaded ZIF-8 served as stabilizers, it was clear to find ZIF-8@CRL-20, 40 and 80 still stabilized the emulsion well in spite of a slight reduction of EI and Cc. However, as loaded enzyme further increased, the emulsion began to deteriorate until an apparent demulsification existed in emulsion stabilized by ZIF-8@CRL-160, where average size increased to 21.81 μm together with EI and Cc falling to 37% and 69%, respectively.

Normally, smaller stabilizer has a better emulsifying effect due to its covering more interfacial areas24. In this study, however, we got a fact that ZIF-8@CRL-160 with the smallest size led to the greatest demulsification. This abnormal result reminded us to focus on the discrepancy in surface chemical characteristics of each sample, which could be characterized by zeta potential and water contact angle. As we know, ionizable 2-Melm residues in framework and amino acids contained in protein endow ZIF-8 and CRL with surface charges, thus pure CRL and ZIF-8 could be taken as polyelectrolytes and showed zeta potentials of -10.2 and -30.8 mV at neutral condition, respectively(Fig.7B). Generally, a decrease in surface charge occurs if a strong polyelectrolyte is replaced by a weak one. Given that increased number of enzyme molecules would occupy the position of partial 2-Melm residues when they were embedded on the surface of ZIF-8@CRL-20,40 and 80, thus it was



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inevitable to find gradually decreased zeta potentials of -28.9,-26.5 and -23.1 mV for these CRL-loaded ZIF-8, respectively. In spite of this, there were still enough electrostatic repulsions for them to steadily disperse in aqueous solution according to polydispersity index(PDI)

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