Enzyme Shielding in a Large Mesoporous Hollow Silica Shell for

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Enzyme shielding in large mesoporous hollow silica shell for improved the recycling and stability based on CaCO3 microtemplates and biomimetic silicification Jiandong Cui, Zhilei Tan, Peipei Han, Cheng Zhong, and Shiru Jia J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 28 Apr 2017 Downloaded from http://pubs.acs.org on April 29, 2017

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Journal of Agricultural and Food Chemistry

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Enzyme shielding in large mesoporous hollow silica shell for

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improved the recycling and stability based on CaCO3

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microtemplates and biomimetic silicification Jiandong Cui*†‡

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Zhilei Tan†

Peipei Han†

Cheng Zhong †

Shiru Jia† *

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†

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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

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‡

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

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

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

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*

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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

Corresponding authors:

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ACS Paragon Plus Environment


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Abstract

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We report a novel “anchor-shield” approach for synthesizing a yolk-shell structured

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biocatalytic system that consists of phenylalanine ammonia lyase (PAL) protein particles

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core and a hollow silica shell with large mesoporous by combination of CaCO3

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microtemplates and biomimetic silicification. The method is established on filling porous

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CaCO3 cores with the PAL via co-precipitation, and controlled self-assembly and

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polyondensation of silanes, and cross-link of the PAL molecules and subsequent CaCO3

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dissolution. During this process, the self-assembled layer of cetyltrimethylammonium

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bromide served as a structure directing agent of the mesostructure, and it directed the

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overgrowth of the mesostructured silica on the external surface of PAL/CaCO3 hybrid

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microspheres, after CaCO3 dissolution, the cross-linked PAL particles were encapsulated

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in the hollow silica shell. The hollow silica shell around the enzyme particles provided a

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

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result, the recycling of PAL enzyme were improved remarkably compared with adsorbed

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PAL on CaCO3. PAL particles with hollow silica shell still remained 60% of their

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original activity after 13 cycles, whereas the adsorbed PAL on CaCO3 microparticles lost

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activity after 7 cycles. Moreover, the immobilized PAL exhibited higher stability against

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proteolytic agent, denaturants, heat, and extremes pH than adsorbed PAL on CaCO3

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microparticles. These results demonstrated that the “anchor-shield” approach is an

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efficient method to obtain stable and recycled biocatalyst with yolk-shell structure.

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Keywords: Hollow silica shell; Enzyme immobilization; CaCO3 microtemplates;

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Biomimetic silicification; Stability

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Introduction

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Enzymes are highly efficient biocatalysts with high substrate specificity, selectivity, and

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mild conditions. Consequently, they have been used in an environmentally friendly

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chemical industry.

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low stability and are difficult to recover, causing high costs and the low production

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efficiency.

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produces insolubilize the enzymes, leading to easy recovery and reusability.

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improved stability are due to preventing subunit dissociation,7 decreasing aggregation,

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autolysis or proteolysis,8 enhancing enzyme rigidification9,10 and producing favorable

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microenvironments.11 The methods used for immobilization include physical adsorption,

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covalent bonding, entrapment, and cross-linking.12-15 In these methods, physical

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adsorption by porous materials exhibits some advantages, such as easy to handle, low

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cost, mild immobilization conditions and excellent applicability for various enzymes.16-19

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However, enzyme desorption from supports is a problem.20 Therefore, a suitable support

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for enzyme immobilization is important. Mesoporous materials have become a common

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choice for immobilization support because they can stabilize the enzyme like an “anchor”.

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For example, spherical mesoporous CaCO3 microparticle has been developed as efficient

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carriers due to low cost and lack of both toxicity and chemical activity with the enzyme.

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Recent years, lysozyme, horseradish peroxidase, trypsin, and guanylate kinase have been

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successfully immobilized into the CaCO3 microparticles by adsorption.21-23 However,

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leaching of enzyme from mesoporous CaCO3 microparticles is almost unavoidable during

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However, for large extent commercialization, free enzymes exhibit

As an effective strategy, immobilization improves stability of enzyme and

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The

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application. Furthermore, an enzyme immobilized on the external surface of CaCO3

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microparticles may not be protected by carriers and suffer inactivation caused by

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denaturing stresses.

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In order to further prevent leaching and enzyme inactivation, an “anchor-shield” structure

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was developed by encapsulating “anchors” into polymer matrix. For example,

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β-Glucuronidase (GUS) was first adsorbed on CaCO3 microparticles. Subsequently, the

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CaCO3 microparticles with GUS were entrapped in alginate beads. The resulting

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immobilized GUS exhibited excellent loading efficiency, reusability and storage

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stability.24 Oxalate oxidase (OxOx) was absorbed onto gold nanoparticles-CaCO3 hybrid

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porous microspheres and then encapsulated in silica sol. The immobilized OxOx

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exhibited high storage stability and reusability.25 In addition, yeast alcohol dehydrogenase

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(YADH) was encapsulated in silica-coated alginate gel beads. The reustability of

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immobilized YADH was enhanced significantly due to the prevention of enzyme leakage

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by silica shell.26 Although these polymer matrices offer a degree of inhibition of enzyme

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leakage, polymer matrix coating of the support might block off the interconnected pores

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and result in increased mass transfer. Furthermore, this method requires the use of acid or

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base catalysts, leading to inactivation of enzyme.27,28 Therefore, it is necessary to seek for

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effective

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immobilization.

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In nature, many living organisms can fabricate silica shell to survive environmental

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stresses.29,30 This biologically induced, self-assembly process, called biomimetic

methods

to

manipulate

new

“anchor-shield”

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structure

for

enzyme


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silicification, occurs in biological environments under mild reaction conditions. Moreover,

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silica materials synthesized with biomimetic silicification method have hierarchical

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structures, multiple morphologies

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properties.31 Recent years, silica nanoparticles with yolk–shell structure have been

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prepared and applied in enzyme immobilization.32-34 For example, lipases were

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encapsulated within mesoporous silica yolk-shell spheres based on a two-step soft

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templating method. The resulting immobilized lipase exhibited observably increased

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thermostability and resistance to proteases.35 Amyloglucosidase and Ru-B amorphous

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alloy were co-encapsulated in yolk-shell silica for one-pot dextrin conversion to sorbitol.

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The yolk-shell structured biocatalytic system exhibited high sorbitol yields and strong

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durability.36 In addition, Cross-linked enzyme aggregates (CLEAs) were entrapped into a

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graphitized mesoporous carbon network or Ca-alginate gel particles. These entrapped

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CLEAs exhibited higher operation stability than conventional CLEAs.37, 38 However, it is

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necessary to develop more efficient immobilization systems.39,40 The novel yolk–shell

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structures with hollow mesoporous silica shell that can make them promising candidates

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for enzyme immobilization due to inherent micro voids, large surfaces (inner and outer)

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and large mesostructure of the silica shells. However, to our knowledge, there are no

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reports on the yolk–shell structures with hollow mesoporous silica shell for enzyme

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immobilization.

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Following the above “anchor-shield” model, in this study, inspired by biomineralized

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core–shell structures in nature, we utilize phenylalanine ammonia lyase (PAL) from

with mesoporous, and superior mechanical

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recombinant E. coli as a model, and report for the first time a novel “anchor-shield”

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approach for synthesizing a yolk-shell structured biocatalytic system that consists of PAL

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protein particles core and a hollow silica shell with larger mesoporous based on CaCO3

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microtemplates and biomimetic silicification. The method mainly consists of a sequential

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reaction involving the PAL protein entrapment into CaCO3 cores via co-precipitation,

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controlled self-assembly and poly-condensation of silanes, and cross-link of PAL

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molecules and subsequent CaCO3 microtemplate dissolution, thus resulting in the

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encapsulation of cross-linked PAL particles in hollow silica shell (Figure 1). As we know,

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glutaraldehyde is an important cross-link agent in the enzyme immobilization. It can react

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with amine, thiol, phenol, and imidazole of proteins. The intra- and inter-molecular

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enzyme crosslinking by glutaraldehyde can increase enzyme rigidity or prevent subunit

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dissociation in multimeric enzymes.41,42 As per our expectation, the hollow silica shell

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around the enzyme particles acted as a “shield” to protect from biological, thermal and

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chemical degradation for enzyme. Furthermore, the intra- and inter-molecular enzyme

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crosslinking obstructed enzyme leaching, and prevented multimeric dissociation. As a

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result, the recycling and stability of immobilized enzyme were remarkably improved. To

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the best of our knowledge, this report is the first demonstration of a stabilized yolk-shell

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structured biocatalytic system with the hollow silica shell by combination of CaCO3

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microtemplates and biomimetic silicification.

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Experimental section

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Materials.

Isopropyl-β-D-thiogalactopyranoside (IPTG), 7


Cetyltrimethylammonium


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bromide (CTAB), glutaraldehyde (GA), and Coomassie brilliant blue R-250 were

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obtained from Sigma Chemical Co. (St Louis, MO, USA). L-Phenylalanine, trypsin

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(2500 U/mg) and tetramethoxysilane (TMOS) were purchased from International

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Aladdin Reagent Inc (Shanghai, China). All of other chemicals were of analytical grade

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and were purchased from commercial suppliers. The recombinant BL21 (DE3) E. coli

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cells (with recombinant expression plasmid pET28a-PAL) were constructed and

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maintained as 40% (v/v) glycerol stock at -80 °C in Luria-Bertani (LB) medium in our

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lab.

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Production and purification of PAL from recombinant E. coli. E. coli with

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pET28a-PAL was picked and incubated in 5 mL of LB medium with kanamycin (35

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mg/mL) at 37 °C on a shaker at 200 rpm for 12 h. Subsequently, 1 mL of culture was

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transferred into 100 mL of LB medium with kanamycin (35 mg/mL), and incubated at

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37 °C until optical density (OD600) of 0.6 was obtained. Then, the PAL expression was

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carried out by adding 0.2 mM IPTG at 30 °C for 4 h.

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For recombinant PAL puriﬁcation, cells were harvested by centrifugation at 4 °C. Cell

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pellets were resuspended in ice-cold lysis buffer (50 mM Tris-HCl, 500 mM NaCl, pH

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8.0) and sonicated until the solution became clear. The lysate was clariﬁed by

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centrifugation at 4 °C and 15,000 g for 20 min. Subsequently, the supernate was filled

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onto a 5 mL nickel Sepharose 6 Fast Flow column, and the column was washed with 5

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column volumes of washing buffer (50 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole,

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pH 8.0). The target protein was eluted with a stepwise gradient of 10, 50, 100, and 200

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mM imidazole. 1 mL fractions were collected and analyzed by SDS-PAGE.

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Encapsulation of cross-linked PAL particles in hollow silica shell. 0.3 M Na2CO3

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solution was added into a mix solution which contained the purified recombinant PAL

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solution (final proteins concentration of 6 mg/mL) and 0.3 M CaCl2 solution. The

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mixture was stirred by a magnetic stirrer for 30 min. Then 0.2 mL CTAB solution (0.1 M)

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was added to the mix solution and the solution was put into a water bath at 40 °C and

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pre-heated for 5 min. 10 µL TMOS was injected into the solution. The solution was left

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undisturbed for 12 h in a water bath. The formed particles were separated by

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centrifugation and washed by deionized water, and re-dispersed in 25 mM Tris-HCl

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buffer (pH 8.8). Then glutaraldehyde (25 % v/v) was added to crosslink PAL molecules

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in the particles for 1 h at 4 °C under shaking at 100 rpm. Subsequently, the particles were

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centrifuged and washed with 25 mM Tris-HCl buffer (pH 8.8) and resuspended in

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deionized water. Lastly, 1 M HCl solution was introduced to the particles to decompose

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the CaCO3 templates and form PAL particles with hollow silica shell. For comparison,

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adsorption of PAL on CaCO3 microparticles was carried out. CaCO3 microparticles were

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added into the purified recombinant PAL solution (final proteins concentration of 6

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mg/mL). The suspension was agitated on a magnetic stirrer for 1.5 h to reach adsorption

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equilibrium.

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PAL activity. The activities of free recombinant PAL and immobilized PAL were

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assayed as previously described by Cui et al.41 The enzyme reaction mixture contained

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25 mM Tris-HCl buffer (pH 8.8), 25 mM L-phenylalanine, and an aliquot of the enzyme

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in a total volume of 1 mL. The reaction was carried out at 37 °C for 30 min and

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terminated by the addition of 1 M HCl. After centrifugation, the absorbance of the

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supernatant at 278 nm was determined by a model 752 spectrophotometer (Shanghai

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precision and scientific instrument Co., China). One PAL activity unit was defined as the

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amount of enzyme that produced 1 µmol trans-cinnamic acid per min at 37 °C. The PAL

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immobilization efficiency and activity recovery was calculated by the following,

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respectively:

immobilization efficiency(%) = 199

m - C1V1 × 100 m

(1)

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where m (mg), C1 (mg/mL), and V1 (mL) are the mass of PAL initially added to the

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solution, the PAL concentration of the supernatant, and the volume of the supernatant,

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respectively.

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Activity recovery (%) =

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Characterization. Transmission electron microscopy (TEM, JEOL JEM2100) images

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for morphologies of the CaCO3 microparticles and PAL particles with hollow silica shell

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were obtained at an acceleration voltage of 200 kV. SEM images were obtained with a

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JEOL S4800 Scanning Electron Microscope with tungsten filament as electron source

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operated at 3 kV. The Brunauer-Emmett-Teller (BET) specific surface areas were

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determined with a Beckman Coulter SA3100 analyzer at 77 K. Prior to the measurement,

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the sample was degassed at 150 °C for 6 h in the vacuum line. The chemical structures

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of PAL particles with hollow silica shell were analyzed via Fourier Transform Infrared

Total activity of immobilized PAL (U) ×100 (2) Total free PAL activity used for immobilized PAL production (U)

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Spectroscopy (FTIR) spectrum (NEXUS870 infrared spectrometer (Thermo Nicolet

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Corporation, Madison, WI) in the 600-4000cm-1 region. An energy-dispersive

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spectrometer (EDS) (S2 Ranger, Bruker, Germany) was utilized to determine the

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elemental composition of PAL particles with hollow silica shell. Thermalgravimetric

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analysis (TGA) of PAL particles with hollow silica shell was conducted with a SDT

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Q600 analyzer (TA Instruments-Waters LLC, USA) by heating from room temperature to

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to 800 °C under nitrogen at a heating rate of 10 °C /min.

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Stability measurement of PAL particles with hollow silica shell. The thermal stability

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of free PAL and immobilized PAL was evaluated by measuring the residual activity after

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incubation at 40-80 °C for 1 h. To determine the stability against chemical denaturants,

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PAL samples incubated in urea (6 M), sodium dodecyl sulfate [SDS, 2 %, (w/v)], or

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ethanol [40 %(v/v)] at 30 °C, respectively. After 30 min, the residual PAL activity was

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measured. The resistance of the enzyme to proteolysis in the presence of trypsin was also

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tested by incubating PAL samples in 25 mM Tris-HCl buffer (pH 8.8) containing 5

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mg/mL trypsin for a defined period of time at 50 °C. The residual PAL activities were

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determined. In addition, pH stability was examined by incubating PAL samples at 50

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mM phosphate buffer (pH 3-11) for 1 h before measuring the residual activity. The

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storage stability was determined as follows: PAL samples were stored at 25 °C in 25 mM

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Tris-HCl buffer (pH 8.8). The residual activities of PAL samples were determined in a

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certain storage time. Finally, the reusability was measured by carrying out a standard

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PAL activity test. After the reaction, The immobilized PAL were washed using 50 mM

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Tris-HCl buffer solution (pH8.8), and the fresh substrate solution was added for the

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another cycle of reaction.

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Results and discussion

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Encapsulation of cross-linked PAL particles in hollow silica shell and their

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characterization. Encapsulation of cross-linked PAL particles in hollow silica shell

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involved four main steps (Figure 1). First, PAL solution with CaCl2 was mixed with

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Na2CO3 solution to form PAL/CaCO3 hybrid microspheres by co-precipitation. Second,

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CTAB was utilized to direct the overgrowth of mesostructured silica (mSiO2) on the

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external surface of PAL/CaCO3 hybrid microspheres. Third, PAL molecules in

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PAL/CaCO3 hybrid microspheres were cross-linked by addition of GA. In a final step, the

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CaCO3 microtemplate was removed when the pH was made more acidic (pH60%

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of their initial activity. This experiment indicated that the silica shell provided a

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protective layer for the enzyme that prevented the egress of the proteolytic agent trypsin.

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Meanwhile, the results demonstrated that the enzymes are fully encapsulated in hollow

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silica shell. Besides, the resistance of PAL samples to denaturants was studied. The

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results were shown in Figure 5b. In the presence of 6 M urea, free PAL almost lost

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activity after 30 min, the adsorbed PAL on CaCO3 microparticles only retained 30% of

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their initial activity. However, the PAL protein particles with hollow silica shell still

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retained 75% of their initial activity. As know that urea can act as an enzyme inhibitor by

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forming H-bonds with important residues located in the active site of the enzyme. In this

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case, the silica layer provides a protective layer for the enzyme that could preserve the

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active conformation of enzyme. In the case of 40% ethanol treatment, we showed that,

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the free PAL was almost inactive, the adsorbed PAL on CaCO3 microparticles retained

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Enzyme Shielding in a Large Mesoporous Hollow Silica Shell for

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