Enzyme shielding by mesoporous organosilica shell on Fe3O4@silica yolk-shell nanospheres

Accepted Manuscript Enzyme shielding by mesoporous organosilica shell on Fe3O4@silica yolk-shell nanospheres

Jiandong Cui, Baoting Sun, Tao Lin, Yuxiao Feng, Shiru Jia PII: DOI: Reference:

S0141-8130(18)31273-X doi:10.1016/j.ijbiomac.2018.05.227 BIOMAC 9832

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

16 March 2018 30 May 2018 30 May 2018

Please cite this article as: Jiandong Cui, Baoting Sun, Tao Lin, Yuxiao Feng, Shiru Jia , Enzyme shielding by mesoporous organosilica shell on Fe3O4@silica yolk-shell nanospheres. Biomac (2017), doi:10.1016/j.ijbiomac.2018.05.227

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ACCEPTED MANUSCRIPT Enzyme shielding by mesoporous organosilica shell on Fe3O4@silica yolk-shell nanospheres Jiandong Cui1, * Baoting Sun1 1

Tao Lin2

Yuxiao Feng2

Shiru Jia 1*

Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin University of

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Science and Technology, No 29, 13th, Avenue, Tianjin Economic and Technological Development

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Area (TEDA), Tianjin 300457, P R China 2

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Research Center for Fermentation Engineering of Hebei, College of Bioscience and Bioengineering,

Hebei University of Science and Technology, 26 Yuxiang Street, Shijiazhang 050000, P R China Corresponding authors:

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*

Jiandong Cui, E-mail: [email protected], Tel: +86-022-60601598

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Shiru Jia, E-mail: [email protected], Tel: +86-022-60601598

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ACCEPTED MANUSCRIPT Abstract Enzyme immobilization on the external surface of solid supports is a commonly adopted method to improve stability and reuse for continuous operations, which, however, is prone to cause the enzyme denaturation due to no carriers protection.

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Herein, we describe enzyme-shielding strategy to prepare hybrid organic/inorganic

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nanobiocatalysts; it exploits the self-assembly of silane building blocks at the surface

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of immobilized enzymes on Fe3O4/silica core-shell nanospheres to grow a protective silica layer. The silica shell around the immobilized enzyme particles provides a

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“shield” to protect from biological, thermal and chemical degradation for enzyme. As

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a result, the recycling of the immobilized catalase with a protective silica layer was improved remarkably compared with immobilized catalase without a protective silica

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layer. The immobilized catalase with a protective silica layer still remained 70% of

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their original activity after 9 cycles, whereas the immobilized catalase without a protective silica layer only retained 20% of their original activity. Moreover, the

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immobilized catalase with a protective silica layer exhibited significantly enhanced

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resistance to denaturing stresses (such as proteolytic agent, denaturants, and heat). Therefore, the enzyme-shielding strategy showed promising applications for preparing obtain stable and recycled nanobiocatalyst. Keywords: Magnetic Fe3O4/silica core-shell nanospheres; Organosilica shell; Immobilization enzyme; Enzyme shielding; Nanobiocatalysts

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ACCEPTED MANUSCRIPT 1. Introduction In recent years, enzymes are becoming very versatile catalysts for a variety of industries including chemical industry, food industry and pharmaceutical industry [1,2]. However, the use of enzymes in industrial processes is limited by their significant

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fragility and fast aging in non-physiological environments [3,4]. In the past two

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decades, various strategies were developed to improve performances of the enzyme

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including genetic engineering techniques [5], chemical modification [6], and immobilization techniques [7,8]. Among these methods, enzyme immobilization on

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solid supports has been demonstrated to be a valuable approach to address enzyme

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stability, activity and selectivity [9-14]. The improved properties of immobilized enzymes are due to prevention of subunit dissociation via multisubunit immobilization,

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prevention of aggregation, autolysis or proteolysis by proteases, rigidification of the

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enzyme structure via multipoint covalent attachment, and generation of favorable microenvironments [12,15,16]. Furthermore, a large number of bioconjugation

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strategies have been also developed to enable the immobilization and protection of

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enzymes on a variety of carrier materials, such as biopolymers [17], metal-organic frameworks [18], metalloid oxides (e.g., silica) [19], and hybrid nanomaterials (e.g., hybrid nanoflower) [20,21]. These carrier materials have become a common choice for the immobilization and protection of enzymes because they can stabilize the enzyme like an “anchor”. For example, various enzymes such as catalase (CAT) [22], lipase [23], glucose oxidase (OxOx) [24], and phenylalanine ammonia lyase (PAL) [25] have been successfully immobilized on silica nanoparticles. However, the immobilization

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ACCEPTED MANUSCRIPT on the external surface of the support raises some problems. For example, leaching of enzyme from supports is almost unavoidable when enzymes are adsorbed on the surface of supports by weak interactions (such as hydrogen bonding, electrostatic interactions, etc.). An enzyme adsorbed on the external surface of material (not inside

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the materials) is not protected from carriers and deleterious interactions with

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hydrophobic interfaces like gas bubbles. Furthermore, the enzyme molecules in one

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particle may interact with the enzyme molecules in other particle [26]. In order to overcome these problems, an “anchor-shield” structure for enzyme immobilization

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was developed by encapsulating the immobilized enzyme into polymer matrix.

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Betancor et al. found that the formation of a hydrophilic shell around the enzyme (glucose oxidase, d-amino acid oxidase, and trypsin) using dextran-aldehyde could

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prevent the interaction between enzyme and hydrophobic interfaces [27]. Furthermore,

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the same group further demonstrated that the deleterious interactions can be decreased by coating immobilized support [28]. In addition, β-Glucuronidase (GUS) was

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immobilized on CaCO3 microparticles by adsorption. Subsequently, the immobilized

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GUS was entrapped again in alginate beads. The resulting immobilized GUS with “anchor-shield” structure exhibited excellent loading efficiency, reusability and storage stability [29]. OxOx was absorbed onto gold nanoparticles-CaCO3 hybrid porous microspheres and then encapsulated in silica sol. The immobilized OxOx with “anchor-shield” structure exhibited high storage stability and reusability [30]. Likewise, the encapsulated yeast alcohol dehydrogenase (YADH) in silica-coated alginate gel beads exhibited excellent reustability due to the prevention of enzyme

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ACCEPTED MANUSCRIPT leakage by silica shell [31]. Although these polymer matrices offer a degree of protection for enzyme, polymer matrix coating of the support might block off the interconnected pores and result in increased mass transfer. Furthermore, this method usually requires the use of acid or base catalysts, leading to inactivation of enzyme

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[32,33]. Therefore, it is necessary to seek for effective methods to manipulate new

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“anchor-shield” structure for enzyme immobilization.

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Recent years, silica-based mesoporous materials become choice for the encapsulation and protection of enzymes due to its low production cost, variable pore size, and high

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thermal and mechanical stability [34,35]. For example, CAT was immobilized in silica

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sol-gel film in the presence of cysteine on gold electrode. The CAT electrode showed a pair of well-defined and quasi-reversible cyclic voltammetry peaks, and could be used

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as an electrochemical biosensor for the determination of hydrogen peroxide [36].

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Bovine liver CAT was immobilized in tetraethoxyorthosilicate based sol-gels. The immobilized catalase showed a significantly improved thermostability compared to the

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free enzyme [37]. D-Amino acid oxidase (DAO) and Fe3O4 magnetic nanoparticles

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were encapsulated simultaneously within biomimetic silica. The stability of DAO against thermal and hydrogen peroxide-induced inactivation was significantly improved after encapsulation [38]. However, these methods have still limitations such as poor loading efficiency and enzyme leakage [39-41]. Recently, mesoporous shell structured silica nanoparticles have been synthesized as nanoreactors for enzyme immobilization [42]. Some reports showed that the yolk-shell structures with hollow mesoporous silica shell that could make them promising candidates for enzyme

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ACCEPTED MANUSCRIPT immobilization [43]. Silica shells can prevent aggregation of catalysts, meanwhile, mesoporous shells allow small molecules access to hollow space inside for effective catalytic reactions. These recent advances of mesoporous silica shells make it possible to encapsulate enzymes into a yolk-shell structure, with the silica shell serving as a

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robust and stable encapsulation layer to protect enzymes.

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Following the above “anchor-shield” model, in this study, we report for the first time a

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enzyme-shielding strategy to grow a protective silica layer at the surface of immobilized enzymes on Fe3O4/silica core-shell nanospheres. The method consists of

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a sequential reaction involving covalent immobilization of CAT on the surface of

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Fe3O4/silica core-shell nanospheres, and controlled self-assembly and subsequent polycondensation of silanes, thus resulting in the growth of an mesoporous

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organosilica layer on the surface of the nanospheres (Figure 1). As per our expectation,

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the mesoporous organosilica layer around the immobilized enzyme particles acted as a “shield” to protect from biological, thermal and chemical degradation for enzyme.

multimeric dissociation. As a result, the recycling and stability of

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leaching and

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Furthermore, covalent bonding between enzymes and silica prevented enzyme

immobilized enzyme were remarkably improved.

2. Experimental 2.1 Materials Fluorescein isothiocyanate (FITC) and hydrogen peroxide (H2O2, 30%) were purchased

from

International

Aladdin

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Reagent

Inc.

(Shanghai,

China).

ACCEPTED MANUSCRIPT Tetramethoxysilane

(TMOS),

aminopropyltriethoxysilane

(APTES),

Cetyltrimethylammonium bromide (CTAB), and catalase (EC 1.11.1.6 from bovine liver, 1× 104 U/mg protein) and tyrpsin (EC 3.4.21.4 from bovine pancreas, 1× 104 U/mg protein) were purchased from Sigma-Aldrich. All of other chemicals were of

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analytical grade and were purchased from commercial suppliers.

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2.2 Preparation of magnetite nanoparticles

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Magnetite particles were prepared according to a previous report [44]. Briefly, 2.70 g of FeCl3. 6H2O and 7.20 g of sodium acetate were dissolved in 100 mL of ethylene

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glycol and the mixture was vigorously stirred at 200 r/m for 30 min. The obtained

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solution was transferred to a Teflon-lined stainless-steel autoclave and sealed to heat at 200 °C for 8 h. After cooling, the obtained magnetite precipitates were separated by

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a magnet, and washed several times with ethanol, and then dried in vacuum at 60 °C

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overnight.

2.3 Synthesis of Fe3O4/SiO2 particles and APTES modified Fe3O4/SiO2 particles

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The core-shell Fe3O4/SiO2 particles were prepared through a commonly used sol-gel

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method [45]. Briefly, 0.10 g of magnetite particles dispersed in the mixture of ethanol, deioned water and ammonia aqueous solution (1.0 mL, 28 wt.%), After mechanical stirred for 0.5 h at room temperature, TMOS (0.3 g) was added into the mixture for 8 h. The obtained black products were separated by a magnet, and washed with ethanol and water, and dried in vacuum at 60 °C overnight. For surface modification, the obtained Fe3O4/SiO2 powders (0.1 g) were added to isopropyl alcohol (100 mL), and 1.0 mL of APTES was then added. After bubbled with nitrogen gas for 30 min, the

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ACCEPTED MANUSCRIPT resultant dispersion was then mechanical stirring for 6 h at 70 °C. Finally, the APTES-modified Fe3O4/SiO2 particles were separated by a magnet and repeatedly washed with ethanol. 2.4 CAT immobilization on the modified Fe3O4/SiO2 particles (Fe3O4/SiO2-catalase)

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For CAT immobilization, 200 mg modified Fe3O4/SiO2 particles was mixed with 1

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mL free CAT (10 mg/mL) and incubated for 8 h with 200 rpm shaking under 25 °C.

dispersed in 0.2 M phosphate buffer (pH 7.0).

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Then the immobilized CAT was separated by a magnet, and washed by DI water, and

Mesoporous

silica

layer

was

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particles (mSiO2@Fe3O4/SiO2-catalase)

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2.5 Synthesis of CAT-encapsulated mesoporous silica layer on Fe3O4/silica yolk-shell

coated

onto

Fe3O4/SiO2-catalase

to

form

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CAT-encapsulated mesoporous silica yolk-shell sphere as following: 0.2 mL CTAB

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solution (0.1 M) and TMOS (0.3 g) were added to Fe3O4/SiO2-Catalase (10 mg/mL) in 0.2 M phosphate buffer (pH 7.0), and allowed to react for 10 h with 150 rpm shaking

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under 25 °C. The product mSiO2@Fe3O4/SiO2-catalase was separated by a magnet,

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and washed by DI water, and re-suspended in 0.2 M phosphate buffer (pH 7.0). 2.6 Characterization methods Scanning electron microscope (SEM) and Transmission electron microscope (TEM) images were obtained by JEOL JSM6700 and JEOL JEM2100, respectively. N2 adsorption isotherms were obtained on a Beckman coulter SA3100 analyzer at 77 K. Specific surface areas and pore diameter distribution were calculated using Brunauer-Emmett-Teller

(BET)

and

Barrett–Joyner–Halenda

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(BJH)

models,

ACCEPTED MANUSCRIPT respectively. Fourier transform infrared (FTIR) spectra were obtained using a NEXUS870 infrared spectrometer (Thermo Nicolet Corporation, Madison, WI) using the standard KBr disk method. FT-IR measurements were conducted in the region of 400-4000 cm-1. Powder X-ray diffraction (PXRD) patterns were recorded using a

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X-ray powder diffraction (D/Max-2500 diffractometer, Shimadzu, Japan) at 40 kV

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and 40 mA. The elemental composition was obtained by using energy-dispersive

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spectrometer (EDS) (S2 Ranger, Bruker, Germany). Magnetisation measurements were carried out using a Magnetic Property Measure System (MPMS, Quantum

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design) under magnetic fields at 300 K. Confocal laser scanning microscopy (CLSM)

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was used to investigate the distribution of CAT. Prior to observation, CAT was mixed with FITC solution (50 mg/mL, FITC in acetone) for 3 min to form a highly

2.7 Activity assay

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fluorescamine [46].

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fluorescent product by the reaction between primary amines in proteins and

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The activities of free CAT and immobilized CAT were measured by the modification

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of the procedure [47]. 50 mg enzyme samples were added to phosphate buffer solution (pH 7.0, 50 mM) and then incubated at 30 °C for 1 h. After incubation, the samples were added to 0.2% hydrogen peroxide solution and the final concentrations of hydrogen peroxide were monitored by measuring absorbance at 240 nm on a 2800H spectrophotometer (Unicoi Instrument Co., Ltd. Shanghai).One unit catalase activity is defined as the amount of enzyme that decomposes of 1 μmol hydrogen peroxide per minute.

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ACCEPTED MANUSCRIPT 2.8 Measurement of apparent kinetic parameters Kinetic parameters of free CAT and immobilized CAT were estimated with increasing hydrogen peroxide concentrations from 0.01% to 0.2% by monitoring the rate of hydrogen peroxide decomposition spectrophotometrically at 240 nm. Km and Vmax for

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free CAT and immobilized CAT were calculated by the Lineweaver-Burk

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double-reciprocal plot method of Michaelis-Menten Equation, respectively.

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2.9 Stability measurement of immobilized CAT and free CAT

For the resistance to proteolysis in the presence of trypsin, Fe3O4/SiO2-catalase,

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mSiO2@Fe3O4/SiO2-catalase, and free CAT were incubated in 50 mM phosphate

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buffer (pH 7.0) containing 5 mg/mL of trypsin for 1 h at 50 °C. The residual activities of free CAT, mSiO2@Fe3O4/SiO2-catalase, Fe3O4/SiO2-catalase were measured,

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respectively. Thermal stability was examined by incubating CAT samples at 50 mM

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phosphate buffer (pH 7.0) without substrate at 60 °C for 15-60 min before measuring the residual activity. The stability of free CAT, mSiO2@Fe3O4/SiO2-catalase,

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Fe3O4/SiO2-catalase against different chemical denaturants was tested by measuring

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the residual activity after incubating at 30 °C for 30 min. The denaturing solutions consisted of urea (6 M), sodium dodecyl sulfate (SDS, 2 %, w/v) or ethanol (40 %, v/v) in 50 mM phosphate buffer (pH 7.0). The storage stability was determined as follows: CAT samples were stored at 25 °C in 50 mM phosphate buffer (pH 7.0). The residual activities of CAT samples were determined in a certain storage time. The reusability of immobilized CAT was assessed by performing several consecutive operating

cycles

using

0.2%

H2O2

10

solution

as

the

substrate.

The

ACCEPTED MANUSCRIPT mSiO2@Fe3O4/SiO2-catalase and Fe3O4/SiO2-catalase were collected by a magnet, and washed with 50 mM phosphate buffer (pH 7.0) solution after each batch and then added to the next cycle, respectively. The reusability was defined as the ratio of the

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activity for the immobilized CAT after recycling to its initial activity.

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3. Results and Discussion

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3.1 Preparation and characterization of the mSiO2@Fe3O4/SiO2-catalase The preparation of the mSiO2@Fe3O4/SiO2-catalase particle is shown schematically in

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Figure 1. First, the Fe3O4/SiO2 particles were synthesized by mixing Fe3O4 particles

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and TMOS solutions, and the obtained Fe3O4/SiO2 particles were modified by APTES. The modified Fe3O4/SiO2 particles was activated by the glutaraldehyde as bifunctional

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cross linker agent, the activated support was collected from the solution using an

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external magnetic field, and rinsed several times with distilled water to remove the excess glutaraldehyde. Afterthat, CAT were anchored on the APTES-modified

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Fe3O4/SiO2 particles. In a final step, CTAB was utilized to direct the overgrowth of a organosilica

layer

(mSiO2)

on

the

external

surface

of

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mesostructured

Fe3O4/SiO2-catalase by self-assembly and polyondensation of silanes, and the CAT-encapsulated mesoporous silica layer on Fe3O4/silica yolk-shell particles was obtained, which consists of an enzyme-immobilized Fe3O4/SiO2 core and mesoporous silica shell. The SEM images showed that these particles exhibited spherical shape with rough surfaces (Fig. 2A, 2B, 2C). In contrast to Fe3O4 nanoparticles, a thin silica shell around Fe3O4 nanoparticles was observed clearly for the Fe3O4/SiO2 nanospheres

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ACCEPTED MANUSCRIPT core from TEM images (Fig. 2D, 2E). The porous silica shell offered high surface area for the derivation of numerous functional groups, which was beneficial to immobilizing enzyme by chemical bond. Furthermore, the nitrogen-adsorption isotherms analysis exhibited that the average pore sizes of mesoporous silica shell was

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about 3.2 nm (data not shown). Besides, TEM images showed that a mesoporous

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protective silica shell with 20-30 nm thickness was formed around the immobilized

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CAT nanosphere (Fig. 2F), suggesting that the CAT-encapsulated mesoporous silica layer on Fe3O4/silica yolk-shell particles were successfully prepared.

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To ascertain that CAT are indeed immobilized on Fe3O4/silica yolk-shell nanospheres,

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the mSiO2@Fe3O4/SiO2-catalase nanospheres were examined by EDS, CLSM, and FTIR. Fluorescence micrograph showed immobilization of CAT on Fe3O4/SiO2

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nanospheres by using fluorescein isothiocyanate (FITC) labeled CAT protein because

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the labeled CAT protein particles exhibited green fluorescence (Fig. 3A). Especially, after mesoporous silica layer on Fe3O4/silica yolk-shell nanospheres was formed,

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green fluorescence could still be observed, indicating that CAT was shielded by

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mesoporous silica layer (Fig. 3B). FTIR spectrum revealed that stretches characteristic at 1640-1660 cm-1 (1653 cm-1) in the mSiO2@Fe3O4/SiO2-catalase particles was ascribed to amide I in proteins (Fig. 3C), indicating the presence of CAT in the composites [48]. Likewise, the same band was also observed in free CAT. However, the band was not observed in the Fe3O4/SiO2 and Fe3O4 particles. The absorption band observed at 1090 cm-1 is ascribed to Si-O-Si antisymmetric stretching, indicating the formation of SiO2 in the composites [49]. The characteristic adsorptions

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ACCEPTED MANUSCRIPT of FeO at 591 cm-1, demonstrating the presence of the Fe3O4. Furthermore, XRD patterns (Fig. 4) show that both mSiO2@Fe3O4/SiO2-catalase and Fe3O4/SiO2 have diffraction peaks similar to that of the parent Fe3O4 particles, suggesting that the Fe3O4 particles were well retained in the silica matrix. EDS experiment showed that

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signals for C, N and O were present in the mSiO2@Fe3O4/SiO2-catalase nanospheres

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(Fig. 5C). However, C and N element was not observed in the pure Fe3O4 particles

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and Fe3O4/SiO2 nanospheres (Fig. 5A, 5B). The results showed that CAT was immobilized on Fe3O4/silica yolk-shell nanospheres. Magnetic characterization using

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a magnetometer at 300 K indicated that the Fe3O4, Fe3O4/SiO2-catalase, and

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mSiO2@Fe3O4/SiO2-catalase nanospheres have magnetization saturation values of 80.4, 77.6, and 61.2 emu/g (Fig. 6), respectively. The results confirmed the

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superparamagnetism of the particles. N2 sorption-desorption isotherms exhibited

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IV-type curves for the nanospheres (Fig. 7). The pore size distribution (Fig. 7, inset) displayed a sharp peak centered at the mean value of 17.6 nm, indicating a structure.

The

enzymatic

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mesoporous

apparent

Km

values

for

both

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mSiO2@Fe3O4/SiO2-catalase and Fe3O4/SiO2-catalase were higher than that of free CAT (Table 1), suggesting a lower substrate affinity of immobilized CAT compared to free CAT. This phenomenon was also observed by previous reports [50,51]. The apparent Vmax values of both mSiO2@Fe3O4/SiO2-catalase and Fe3O4/SiO2-catalase composites were found to be lower than that of native forms. It indicated that Kcat values of immobilized CAT suffer a progressive decrease due to the modification of the enzyme structure caused by the chemical modification. Furthermore, compared to

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ACCEPTED MANUSCRIPT free CAT, activites of both mSiO2@Fe3O4/SiO2-catalase and Fe3O4/SiO2-catalase composites decreased. This might be due to steric hindrance created by rigidification of silica shell around CAT resulting into diffusion restrictions for substrate to access the inner side of immobilized enzyme. Furthermore, multipoint attachment between

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3.2 Protective properties of silica shell for immobilized CAT

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enzyme and carriers increase enzyme rigidity.

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To assess whether the mesoporous silica shell could provide an protection against extreme conditions for immobilized CAT, we examined the stability of free CAT,

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Fe3O4/SiO2-catalase, and mSiO2@Fe3O4/SiO2-catalase against proteolytic agent,

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denaturants, and heat. The stability of free CAT, Fe3O4/SiO2-catalase, and mSiO2@Fe3O4/SiO2-catalase against high temperature was showed in Fig. 8A. The

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mSiO2@Fe3O4/SiO2-catalase exhibited the higher stability against high temperature

only

retained

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than free CAT and Fe3O4/SiO2-catalase after incubating at 60 °C for 50 min. Free CAT 16%

of

their

initial

activity,

whereas

the

activity

of

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mSiO2@Fe3O4/SiO2-catalase the still remained about 80% of initial activity. The

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increased tolerance towards high temperature may be due to the following reasons: (1) silica shell retards heat transfer. (2) the formation of multiple covalent bonds between the catalase and the carriers reduces conformational flexibility and prevents subunit dissociation [15,52]. Similarly, free CAT lost activity after 15 min in the presence of the trypsin (5 mg/mL), the Fe3O4/SiO2-catalase only retained 26% of their initial activity after 30 min. However, mSiO2@Fe3O4/SiO2-catalase still retained about 80% of their initial activity (Fig. 8B). These results indicated that the presence of silica

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ACCEPTED MANUSCRIPT shell could increase diffusion restrictions for trypsin to access the immobilized enzyme. Besides, the mSiO2@Fe3O4/SiO2-catalase showed the high stability against denaturants compared to free CAT and Fe3O4/SiO2-catalase. For example, in the presence of 6 M urea, free PAL and Fe3O4/SiO2-catalase only retained 22% and 65%

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of their initial activity, respectively. However, the mSiO2@Fe3O4/SiO2-catalase

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retained 90% of its initial activity (Fig. 8C). These results clearly demonstrated that

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the silica shell afforded protection from biological and chemical degradation for immobilized CAT. Furthermore, multiple covalent bonding between catalase and

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carriers prevented enzyme leaching and multimeric dissociation. As a result, the

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stability of immobilized enzymes were remarkably improved [53,54]. 3.3 The reusability and storage of the mSiO2@Fe3O4/SiO2-catalase

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In addition, storage stability of free CAT and immobilized CAT was also determined.

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As shown in Fig. 8D, free CAT lost activity after 3 days, and activity of Fe3O4/SiO2-catalase gradually decreased to 30% after 15 days. However, the

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mSiO2@Fe3O4/SiO2-catalase still retained 85% of their initial activity, demonstrating

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the mSiO2@Fe3O4/SiO2-catalase had good resistance to room temperature storage. Most importantly, the mSiO2@Fe3O4/SiO2-catalase exhibited better reusability than Fe3O4/SiO2-catalase. As shown in Fig. 9, Fe3O4/SiO2-catalase lost more than 80% of its original activity just after 9 cycles, whereas mSiO2@Fe3O4/SiO2-catalase still remained

70%

of

their

original

activity,

suggesting

that

the

mSiO2@Fe3O4/SiO2-catalase has robust operation stability. This result may be due to the fact that multisubunit covalent immobilization between catalase and carriers not

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ACCEPTED MANUSCRIPT only prevents the subunit dissociation, but also increases catalase rigidity. At the same time, this result also proved that the formation of silica shell around the Fe3O4/SiO2-catalase readily facilitated the reuse of immobilized CAT, offering a

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long-term operational stability and reusability.

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4. Conclusions

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In conclusion, we developed a novel enzyme-shielding strategy for synthesizing a magnetic yolk-shell structured biocatalytic system that consists of immobilized

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enzymes on magnetic Fe3O4/silica nanospheres core and a mesoporous silica shell.

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The silica shell around the immobilized enzyme particles provided a “shield” to protect from biological, thermal and chemical degradation for enzyme. Furthermore,

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covalent bonding between enzymes and silica prevented enzyme leaching and subunit

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dissociation. As a result, the resulting immobilized enzymes had improved the recycling and stability when compared to the native enzyme and the immobilized

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enzymes without the silica shell. The results suggested that enzyme-shielding strategy

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for synthesizing magnetic yolk-shell structured nanobiocatalysts with the protective silica layer has high potential for the biomedical, biosensor and biocatalysis fields.

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ACCEPTED MANUSCRIPT Acknowledgements This work is partially supported by the National Natural Science Foundation of China (project no. 21676069). Dr. J. D. Cui also thanks supports from the Natural Science Foundation of Hebei Province, China (project no. B2018208041), the Program for

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Hundreds of Outstanding Innovative Talents in Hebei province (III) under the grant

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number of SLRC2017036, and the Foundation (No. 2016IM001) of Key Laboratory

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of Industrial Fermentation Microbiology of Ministry of Education and Tianjin Key

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CE

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Lab of Industrial Microbiology (Tianjin University of Science & Technology).

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microspheres

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visible

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magnetically

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behavior, Biotechnology and Bioengineering, (40) 1992, 1427-1434.

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ACCEPTED MANUSCRIPT Figure legends Figure 1 Principle of magnetic Fe3O4@silica core shell nanosphere with silica coating for enzyme protection. Step 1: silanization magnetic Fe3O4 nanoparicles; step 2: enzyme immobilization at the surface of magnetic Fe3O4 nanoparicles by

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images

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(C)

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Figure

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covalent bond. Step 3 and 4: silane self-assembly and ploycondensation.

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mSiO2@Fe3O4/SiO2-catalase; TEM images of (D) Fe3O4, (E) Fe3O4/SiO2, and (F) Fe3O4/SiO2-catalase@SiO2.

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Figure 3 Confocal microscope images of (A) Fe3O4/SiO2-catalase and (B)

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mSiO2@Fe3O4/SiO2-catalase; FT-IR spectra analysis of (C) free CAT, Fe3O4, Fe3O4/SiO2, and mSiO2@Fe3O4/SiO2-catalase. 4

Element

mapping

of

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Fe3O4,

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Fe3O4/SiO2,

and

(C)

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mSiO2@Fe3O4/SiO2-catalase.

Figure 5 PXRD of (A) Fe3O4, (B) Fe3O4/SiO2, (C) mSiO2@Fe3O4/SiO2-catalase, and

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curves

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Fe3O4,

(B)

Fe3O4/SiO2,

(C)

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(D) standard Fe3O4.

mSiO2@Fe3O4/SiO2-catalase. Figure 7 N2 adsorption-desorption isotherms and pore size distribution curves of mSiO2@Fe3O4/SiO2-catalase. Figure 8 Stability of free CAT, Fe3O4/SiO2-catalase, and mSiO2@Fe3O4/SiO2-catalase (A) thermostability, (B) resistance to proteolysis in the presence of trypsin, (C) stability against chemical denaturants, (D) mechanical stability.

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Figure 9 Reusability of Fe3O4/SiO2-catalase and mSiO2@Fe3O4/SiO2-catalase.

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Comparison

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kinetic

parameters

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free

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Km (mM)

Vmax (mM/min)

Free CAT

15.29 ± 0.074

4.02 ± 0.021

Fe3O4/SiO2-catalase

16.61 ± 0.079

3.47 ± 0.017

21.17 ± 0.01

2.36 ± 0.011

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Highlights  The immobilized catalase in MOF with a protective organosilica shell were prepared

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(silica@CAT/ZIF-8).

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The silica shell around the immobilized enzyme particles provides a “shield” to

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enhance resistance to denaturing stresses.

he micrometer-sized silica@CAT/ZIF-8 can be easily repeatedly used without

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silica@CAT/ZIF-8

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obvious activity loss.

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CAT/ZIF-8under acid environment.

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exhibited

higher

tolerance

than