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Encapsulation of spherical cross-linked phenylalanine ammonia lyase aggregates in mesoporous biosilica Jiandong Cui, Ya Min Zhao, Yuxiao Feng, Tao Lin, Cheng Zhong, Zhilei Tan, and Shiru Jia J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05003 • Publication Date (Web): 05 Jan 2017 Downloaded from http://pubs.acs.org on January 5, 2017

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Journal of Agricultural and Food Chemistry

of

spherical

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Encapsulation

cross-linked

phenylalanine

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ammonia lyase aggregates in mesoporous biosilica

3

Jiandong Cui1, 2 * Yamin zhao2 1 Yuxiao Feng2 Tao Lin2 Cheng Zhong1

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1

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of 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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2

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

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*

Zhilei Tan1 Shiru Jia1 *

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

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

Corresponding authors:

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Jiandong Cui, E-mail: [email protected], Tel: +86-311-81668486

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

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ACS Paragon Plus Environment


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Abstract

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Cross-linked enzyme aggregates (CLEAs) have recently emerged as a promising method

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for enzyme immobilization due to its simplicity and low cost. However, a lack of good

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size and morphological control over the as-prepared CLEAs has limited their practical

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applications. For example, the prepared CLEAs exhibit amorphous large clusters that

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would cause significant mass-transfer limitations, which leading to low catalytic

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efficiency. Here, inspired by biomineralized core–shell structures in nature, we develop a

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novel mesoporous spherical CLEA with biosilica shell by using phenylalanine ammonia

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lyase based on CaCO3 microtemplates and biomimetic mineralization. The resultant

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CLEAs exhibited spherical structure with good monodispersity instead of the amorphous

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clusters of conventional CLEAs, and showed higher activity than that of conventional

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CLEAs. Moreover, the thermostability, the tolerance against denaturants, and mechanical

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stability of the spherical CLEAs with biosilica shell were enhanced significantly

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compared with conventional CLEAs. Especially, the spherical CLEAs with biosilica

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shell still retained 70% of its original activity after 13 cycles. Whereas, the conventional

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CLEAs only retained 35% of its original activity. This approach could be an efficient

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strategy to improve catalytic properties of CLEAs.

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Keywords: Biomimetic mineralization; CaCO3 microtemplates; Spherical CLEAs;

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Immobilization enzyme

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Introduction

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Currently enzymes are widely used in diverse sectors owing to their ease of production,

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substrate specificity and selectivity. However, for commercial viability, these biocatalysts

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display low operational stability and difficulty in recovery, leading to high cost and the

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contamination of the products.1, 2 Immobilization of enzymes is now providing powerful

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tools and devices to develop novel alternative strategies for increasing the performance,

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stability, and activities of enzymes for industrial application.3,4 The improvement of

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operational stabilization of immobilized enzymes can be due to prevention of subunit

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dissociation via multisubunit immobilization,5 prevention of aggregation, autolysis or

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proteolysis by proteases,6 rigidification of the enzyme structure via multipoint covalent

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attachment7,8 and generation of favorable microenvironments.9 The selectivity

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improvement via immobilization can be due to alteration of the geometry of the active

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center via immobilization,7,8 stabilizing enzyme conformation

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diffusion limitation.11 Among such immobilization methods, carrier-free immobilization

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has attracted a great deal of attention due to clear advantages: great volumetric

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productivity, high stability and the low production cost due to the exclusion of an

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additional carrier.12,13 Especially, cross-linked enzyme aggregates (CLEAs) seem to be a

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promising method for enzyme immobilization.14,15 The enzyme is immobilized with high

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stability and high volume activity by cross-linking the physical enzyme aggregates.16,17

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Compared with free enzymes, CLEAs proved significantly more stable to denaturation

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by heat, or organic solvents and proteolysis than the corresponding soluble enzyme.18,19

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and elimination of


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Moreover, CLEAs possess high space time yields, volumetric and catalyst

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productivities.20 However, CLEAs technology has also some disadvantages.21 For

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example, the CLEAs have wide particle distribution which affects enzyme activity and

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reaction kinetics. Amorphous large clusters would result in significant mass-transfer

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limitations, whereas the small particles cannot be filtered or recycled.22,23 Moreover,

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CLEAs may still be considered too soft for many industrial applications. Therefore, it is

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important to control the particle size of CLEA and improve their mechanical stability.24,25

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In order to overcome these problems, CLEAs of papain in commercial macroporous

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silica gel were prepared, and showed excellent operation stability.26 The formation of

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CLEAs of phenylalanine ammonia lyase (PAL) from Rhodotorula glutinis into

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macroporous silica gel exhibited good reusability.27 The magnetic CLEAs of α-amylase

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were prepared on amino functionalized magnetite nanoparticles, and exhibited excellent

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thermostability, storage stability and reusability.28 However, the noncatalytic mass of the

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carrier causes decrease of the volumetric activity and production of the biocatalyst.

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In the past decades, inorganic materials have been successfully used as hard templates

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for the synthesis of immobilized enzyme particles with control over internal structure,

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morphology, and composition.29,30 The most straightforward method towards such

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assemblies employs inorganic materials as templates such as microporous silica,31

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ploymer,32 and porous CaCO3.33 Among templates, CaCO3 microspheres have nowadays

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become one of the most popular decomposable matrices because of easy preparation

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procedure, low costs, biocompatibility, and mild decomposition conditions.34,35

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Moreover, some enzymes have been successfully entrapped into the pores of the CaCO3

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microspheres by the co-precipitation, infiltration by solvent exchange, and physical

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adsorption.30,36,37 Although the resulting immobilized enzyme particles by CaCO3

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templating exhibit regular size and monodispersity, they may still be considered too soft

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for industrial application.

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

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stresses. This biologically induced, self-assembly process, called biosilicification, is

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formed in biological environments under mild reaction conditions.38 Generally,

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biomimetic silica can be produced in vitro by mixing silicic acid with silaffin,39 or

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synthetic R5 peptide (part of the silaffin repeating unit),40 or even polymers such as

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poly(lysine)

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biosilicification have hierarchical structures, multiple morphologies with mesopores, and

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superior mechanical properties. In the study, inspired from biosilicification, we

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developed a new method to prepare a mesoporous spherical CLEA with biosilica shell

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based on CaCO3 microtemplates and biomimetic mineralization. The method mainly

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consists of a sequential reaction involving preparation of spherical CLEA particles based

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on porous CaCO3 microtemplate, and controlled self-assembly and subsequent

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polycondensation of silanes, thus resulting in the formation of biosilica coatings around

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CLEA particles (Figure 1). It is conceivable that the resultant CLEAs can form regular

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spherical particles with appropriate size under suitable conditions that would overcome

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the formation of conventional CLEAs with amorphous clusters. More importantly, the

and

polyallylamine.41 Moreover,

silica

materials

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synthesized

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biosilica shell could afford unprecedented protection from biological, thermal and

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chemical degradation. In this work, the phenylalanine ammonia-lyase (PAL, EC 4.3.1.24)

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from recombinant E. coli was selected to explore the validity of this approach. To

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prevent PAL subunit dissociation, polyethyleneimine(PEI) was used to protect PAL, and

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promote biomimetic silica formation. In addition, the structure and some properties of

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prepared spherical CLEAs with biosilica shell were investigated.

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Materials and Methods

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Chemicals

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glutaraldehyde and coomassie brilliant blue R-250 were purchased from Sigma

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Chemical Co. (St Louis, MO, USA). Trypsin (500 U/mg), tetramethoxysilane (TMOS),

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and polyethyleneimine (PEI, 1.8 kDa) were purchased from International Aladdin

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Reagent Inc (Shanghai, China), and stored at 4 °C. L-Phenylalanine was obtained from

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Beijing Chemical Reagent Company (Beijing, China). All of other chemicals were of

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analytical grade and were purchased from commercial suppliers. The recombinant BL21

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(DE3) E. coli cells (carrying recombinant expression plasmid pET28a-PAL) was

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

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medium in our lab.

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Production and purification of PAL from recombinant E. coli. BL21 (DE3) E. coli

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with pET28a-PAL was inoculated in 5 mL of LB broth with kanamycin (35 mg/L), and

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

and

microorganism.

Isopropyl-β-D-thiogalactopyranoside

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(IPTG),

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transferred to a 250 mL Erlenmeyer flask containing 100 mL of LB broth and 100 µL of

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kanamycin (35 mg/mL). The bacteria were grown at 37 °C in an incubator (200 rpm)

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until the OD600 reached approx. 0.6 and induced by adding 1 mM IPTG, and incubated at

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30 °C for 4 h. For recombinant PAL purification, E. coli cells were separated from the

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culture media by centrifugation at 4 °C. Cells pellets were resuspended in ice-cold lysis

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buffer (50 mM Tris-HCl, 500 mM NaCl, pH 8.0), and were sonicated until the solution

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became clear. The lysate was clarified by centrifugation at 4 °C, 15,000 ×g for 20 min.

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Then, the solution was loaded onto a 5 mL nickel Sepharose 6 Fast Flow column. After

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the protein was loaded onto the column, the column was washed with 5 column-volumes

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of washing buffer (50 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 8.0). The

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target protein was eluted with a stepwise gradient of 10, 50, 100, and 200 mM imidazole,

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using two column volumes for each step. Elution fractions of 1 mL were collected and

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analyzed by SDS-PAGE.

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Preparation of CaCO3 microtemplates. CaCO3 microtemplates were prepared by a

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modification of the procedure of Volodkin et al. 33 Briefly, the crystallization process was

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initiated by rapid mixing of 0.33 M CaCl2 and 0.33 M Na2CO3 solutions (including 30%

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ethylene glycol). The mixture was intensively agitated at 500 rpm with a magnetic stirrer

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for 2 h. After centrifugation, the white precipitate of CaCO3 particles was washed three

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times with ethanol and dried in oven at 60 °C for 1 h.

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Preparation of conventional CLEAs and spherical CLEAs with biosilica shell. The

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conventional CLEAs were prepared as the general procedure.27 For preparation of

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spherical CLEA with biosilica shell, 50 mg CaCO3 microparticles were added into

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purified recombinant PAL solution (6 mg/mL). The suspension was stirred at room

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temperature for 0.5 h. Then ammonium sulfate was added to yield a final ammonium

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sulfate saturation of 40%. After shaking the mixture at 100 rpm for 5 min, the particles

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were crosslinked via glutaraldehyde (25 % v/v) at a final concentration of 0.3% (v/v) for

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1 h at 4 °C under shaking at 100 rpm. After centrifugation, the precipitates were washed

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for three times by 25 mM Tris-HCl buffer (pH 8.8) and resuspended in deionized water.

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The suspension was slowly titrated with 0.1 M HCl till pH 5.2. After the CaCO3

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templates were dissolved, the spherical CLEA particles were obtained. Subsequently, the

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prepared spherical CLEA particles (20 mg) and 6 mg/mL PEI were mixed in 25 mM

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potassium phosphate buffer (pH 6.8) at room temperature. The resultant mixtures were

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mixed with 10 µL of 1 M hydrolyzed TMOS, and after 5 min, the mixtures were

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centrifuged for 5 min at 10,000 ×g at 4 °C. The resultant spherical CLEAs with biosilica

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shell were washed three times with deionized water and resuspended in 25 mM Tris-HCl

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buffer (pH 7.0) at 4 °C prior to use.

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Activity assay. The activities of free recombinant PAL, conventional CLEAs, spherical

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CLEAs, and spherical CLEAs with biosilica shell were measured by the modification of

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the procedure.23 A small amount of enzyme samples was added to an enzymatic reaction

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medium. The reaction mixture containing 25 mM Tris-HCl buffer (pH 8.8), 25 mM

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L-phenylalanine, and enzyme samples was incubated at 30 °C for 20 min. The reaction

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

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clear supernatant was measured at 278 nm with a 752 spectrophotometer (Shanghai

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

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the amount of enzyme required to convert one µmol of L-phenylalanine to

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trans-cinnamic acids per min under the above conditions.

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Characterization. Scanning electron microscope (SEM, JEOL S4800) and transmission

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electron microscope (TEM, JEOL JEM2100) was used for imaging morphologies of the

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CaCO3 microparticles, conventional CLEAs, spherical CLEAs, and spherical CLEAs

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with biosilica shell, respectively. The pore size distributions of spherical CLEAs with

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biosilica shell were determined following the Brunauer-Emmett-Teller (BET) method of

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nitrogen adsorption/desorption on a Beckman coulter SA3100 analyzer at 77 K. The

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chemical structures of spherical CLEAs with biosilica shell were analyzed with a Fourier

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Transform Infrared Spectroscopy (FTIR) spectrum (NEXUS870 infrared spectrometer,

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(Thermo Nicolet Corporation, Madison, WI). Energy-dispersive spectrometer (EDS) (S2

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Ranger, Bruker, Germany) was utilized for the elemental composition of spherical

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CLEAs with biosilica shell. Thermal gravimetric analysis (TGA) of spherical CLEAs

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with biosilica shell was conducted with a SDT Q600 analyzer (TA Instruments-Waters

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LLC, USA) by heating from 25 to 800 °C under nitrogen gas.

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Measurement of kinetic parameters. Kinetic parameters of free PAL, conventional

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CLEAs, and spherical CLEAs with biosilica shell were estimated by measuring initial

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reaction rates using different L-Phenylalanine concentrations in the range of 1.25-50 mM

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in buffer solution (Tris-HCl, 50 mM, pH 8.8) at 30 °C. Km and Vmax for free PAL,

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conventional CLEAs, and spherical CLEAs with biosilica shell were calculated by the

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Lineweaver-Burk double-reciprocal plot method of Michaelis-Menten Equation,

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respectively.

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The stability of spherical CLEAs with biosilica shell. Thermal stability of the free

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PAL, conventional CLEAs, and spherical CLEAs with biosilica shell was evaluated by

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measuring the residual activity after incubating at 60 °C for 10-60 min, respectively. The

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stability of free PAL, conventional CLEAs, and spherical CLEAs with biosilica shell

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against different chemical denaturants was tested by measuring the residual activity after

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incubating at 30 °C for 30 min. The denaturing solutions consisted of urea (6 M), sodium

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dodecyl sulfate (SDS, 2 %, w/v) or ethanol (40 %, v/v) in 25 mM Tris-HCl buffer (pH

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8.8). Enzyme resistance to proteolysis in the presence of trypsin was also tested by

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incubating free PAL, conventional CLEAs, and spherical CLEAs with biosilica shell in

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25 mM Tris-HCl buffer (pH 8.8) containing 5 mg/mL of trypsin for a certain time at

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50 °C. The residual activities of free PAL, conventional CLEAs, and spherical CLEAs

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with biosilica shell were measured, respectively. In addition, the free PAL, conventional

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CLEAs, and spherical CLEAs with biosilica shell were immersed in 50 mM sodium

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phosphate buffer solution (pH 7.5) at 25 °C and shaken at 200 rpm for a certain time to

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detect mechanical stability. Then the enzyme samples were taken out and centrifuged at

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each time point, the residual activities in these immobilized enzymes and the supernatant

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liquid were measured, respectively. Lastly, the reusability of immobilized PAL was

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assessed by measuring the activity in each cycle. The conventional CLEAs and spherical

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CLEAs with biosilica shell were collected and washed with buffer solution (Tris-HCl, 50

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mM, pH 8.8) after each batch and then added to the next cycle, respectively.

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Results and discussion

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Preparation and characterization of spherical CLEAs with biosilica shell. The

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preparation of the spherical CLEAs with biosilica shell is shown schematically in Figure

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1. First, the CaCO3 vaterite particles was synthesized by simply mixing CaCl2 and

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Na2CO3 solutions (including ethylene glycol). Second, PALs were precipitated into the

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pores of the CaCO3 templates by addition of ammonium sulfate, and crosslinked by

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addition of glutaraldehyde. Third, the CaCO3 templates were removed by change of the

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pH to acidic region (up to pH 5.2), and spherical CLEAs were obtained. In a final step

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the resulting spherical CLEAs were encapsulated in biomimetic silica by biosilicifiation

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to form spherical CLEAs with biosilica shell. The morphologies of the prepared CaCO3

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templates, conventional CLEAs, spherical CLEAs, and spherical CLEAs with biosilica

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shell were characterized by SEM and TEM. As shown in Fig. 2, the spherical CaCO3

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microspheres with a diameter about 2 µm possessed coarse and robust surface (Fig. 2a

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and 2b), and it also can be observed clearly that the CaCO3 microspheres are composed

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from channel-like spherulitic nanocrystals of a size of nanometers, suggesting that the

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CaCO3 microspheres had a highly developed porous internal structure (Fig. 2c) (the

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channel-like structure is indicated by the arrow). To further confirm the porous internal

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structure of the CaCO3 microspheres, we conducted the TEM images. As can be seen

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from Fig. 3a, the CaCO3 microspheres exhibited well-defined channel-like structures

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with interconnected pores (the channel-like structure is indicated by the arrow). The

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porous structure of the CaCO3 microspheres are also found in previous reports.36,37 The

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channel-like structures with interconnected pores allow one to load a large amount of

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adsorbing enzyme molecules, due to the high surface area available in the porous

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structure. Fig. 2d showed that conventional CLEAs exhibited large size particles with

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amorphous clusters and no defined morphologies. Whereas, spherical CLEAs and

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spherical CLEAs with biosilica shell were relatively uniform architectures with ball

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shape (Fig 2e and 2f). Furthermore, TEM further confirmed that spherical CLEAs and

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spherical CLEAs with biosilica shell had higher monodispersity than that of

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conventional CLEAs (Fig 3c and 3d). Especially, a silica coating of higher contrast

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surrounding the spherical CLEA particles could be clearly observed by TEM (Fig. 3d),

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indicating that the spherical CLEAs were successfully encapsulated in biomemitic silica.

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Fourier-transform infrared (FTIR) spectra of spherical CLEAs with biosilica shell were

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shown in Fig 4a. The characteristic absorption bands observed at 1627 cm-1 and 3430

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cm-1 could be attributed to amide group and -N-H stretching vibrations, indicating the

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presence of CLEAs. Similar absorption bands were also observed in FTIR spectra of

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spherical CLEAs (Fig. 4b). The sharp absorption band observed at 1079 cm-1 is ascribed

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to Si-O-Si antisymmetric stretching, the band at 876 cm-1 is attributed to Si-OH

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stretching vibrations, indicating the formation of SiO2. In addition, the spherical CLEAs

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with biosilica shell were characterized by TG and DTG analysis under N2 atmosphere.

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Three distinct weight loss steps were observed for the spherical CLEAs with biosilica

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shell (Fig. 4c). The first weight loss step occurred at 31-115 °C due to the loss of water.

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The second weight loss step occurred in the range 200-300 °C and was attributed to the

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decomposition of CLEA particles. The third weight loss step occurred in the range

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300-500 °C, which was ascribed to the content of SiO2. EDS experiment showed that

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signals for Si, C and O were present in the spherical CLEAs with biosilica shell (Fig. 5).

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The results revealed that the spherical CLEAs with biosilica shell are composed by

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CLEA particles and SiO2. Figure 6 showed the nitrogen-adsorption isotherms for the

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spherical CLEAs with biosilica shell. The spherical CLEAs with biosilica shell exhibited

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mesoporous structure, and had the multiple level pore size distribution (a broad size

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between 2 and 40 nm). The porous structure might be critical for ensuring high catalytic

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efficiency, due to an decrease in mass transfer limitation for substrates with the interior

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enzymes of CLEAs. This hypothesis was further demonstrated by a kinetic study of the

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enzymatic activity. Although the higher value of Michaelis–Menten constant (Km) could

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be observed for spherical CLEAs with biosilica shell (Table 1) in comparison to that for

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the free PAL, Km values of the spherical CLEAs with biosilica shell were lower than that

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of conventional CLEAs, indicating that the biosilica shell did not cause the increased

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mass transfer limitation for substrates. Furthermore, it is worth noting that the maximal

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reaction rate (Vmax) of the spherical CLEAs with biosilica shell was 5-fold in comparison

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with conventional CLEAs, indicating the higher catalytic rate. In our previous study,

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conventional CLEAs was encapsulated in biomimetic silica by biosilicifiation

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(CLEAs-Si). The resultant CLEAs-Si exhibited higher stability than native enzyme and

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conventional CLEAs.23 However, it is difficult to efficiently encapsulate conventional

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CLEAs into biomimetic silica due to wide particle distribution and high polydispersity,

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which cause low activity recovery. In contrast, in this study, spherical CLEAs with high

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monodispersity were easily embedded into biomimetic silica. As a result, the spherical

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CLEAs with biosilica shell exhibited higher activity than CLEAs-Si.

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Stability of the spherical CLEAs with biosilica shell. The stability of free PAL,

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conventional CLEAs and the spherical CLEAs with biosilica shell against high

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temperature and denaturants was showed in Figure 7. The spherical CLEAs with

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biosilica shell exhibited the higher stability against high temperature than free PAL and

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conventional CLEAs after incubating at 60 °C for 60 min. Free PAL and conventional

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CLEAs only retained 19% and 25% of their initial activity, respectively, whereas the

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activity of the spherical CLEAs with biosilica shell still remained about 80% of initial

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activity (Figure 7a). Similarly, the spherical CLEAs with biosilica shell showed the high

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stability against denaturants compared to free PAL and conventional CLEAs. For

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example, in the presence of 6 M urea, both free PAL and conventional CLEAs lost most

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of activity. However, the spherical CLEAs with biosilica shell retained 85% of its initial

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activity (Figure 7b). These results indicated that the biosilica shell had a beneficial effect

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on the resilience of the enzyme against high temperature and denaturants. The effect of

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the biosilica shell against proteolysis was studied in the presence of the higher trypsin

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concentration (5 mg/mL). The results were showed in Figure 8. Remarkably, in the

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presence of trypsin, both free PAL and conventional CLEAs lost activity after they were

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incubated in the presence of trypsin for 30 min, whereas the spherical CLEAs with

300

biosilica shell still retained more than 80% of its initial activity, confirming the shielding

301

effect of biosilica shell towards proteolysis. In addition, we evaluated the stability of free

302

PAL, conventional CLEAs and the spherical CLEAs with biosilica shell against

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mechanical damage by incubating them in aqueous solution under shaking conditions

304

(200 rpm). As shown in Figure 9, activity of free PAL and conventional CLEAs

305

decreased considerably and lost most activity after 16 days. However, the spherical

306

CLEAs with biosilica shell still retained about 90% of its initial activity after 16 days of

307

shaking. These results clearly demonstrated that the biosilica shell afforded

308

unprecedented protection from biological, thermal, chemical, and mechanical

309

degradation for CLEA particles.

310

Reusability of the spherical CLEAs with biosilica shell. Reusability of enzyme is a

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key factor for its cost-effective industrial use.42 The reusability of conventional CLEAs

312

and the spherical CLEAs with biosilica shell was investigated with 13 cycles. The results

313

were showed in Figure 10. The conventional CLEAs lost more than 65% of its original

314

activity after 13 cycles. In contrast, the spherical CLEAs with biosilica shell still retained

315

70% of its original activity after 13 cycles, demonstrating a very efficient recovery of the

316

enzyme catalyst. This result proved that the formation of silica shell around the spherical

317

CLEA particles readily facilitated the reuse of CLEAs, offering a long-term operational

318

stability and reusability. According to the results, the spherical CLEAs with biosilica

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shell have potential use in industrial applications.

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Acknowledgements

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This work is partially supported by the National Natural Science Foundation of China

322

(project no. 21676069) and the Natural Science Foundation of Hebei Province, China

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(project no. B2014208054). Dr. J. D. Cui also thanks supports from the Open Foundation

324

of Bio-manufacturing Subject at Hebei University of Science and Technology (project no.

325

SW5) and Key Foundation of Hebei University of Science and Technology, China

326

(project no. 2016ZDYY26).

327 328 329 330 331 332 333 334 335 336 337 338 339

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442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460

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461 462

Figure legends

463

Figure 1 Schematic illustration of the synthesis of spherical CLEAs with biosilica shell.

464

Figure 2 SEM images of (a, b) CaCO3 microsphere, (c) broken CaCO3 microsphere, (d)

465

conventional CLEAs, (e) spherical CLEAs (f) spherical CLEAs with biosilica

466

shell.

467 468 469 470

Figure 3 TEM images of (a) CaCO3 microsphere, (b) conventional CLEAs, (c) spherical CLEAs, (d) spherical CLEAs with biosilica shell. Figure 4 the FTIR data of spherical CLEAs with biosilica shell (a) and the conventional CLEA (b), and TGA (c) analysis of spherical CLEAs with biosilica shell.

471

Figure 5 Element mapping of the spherical CLEAs with biosilica shell via EDS.

472

Figure 6 N2 adsorption-desorption isotherms and pore size distribution curves of

473 474 475

spherical CLEAs with biosilica shell. Figure 7 Stability of free PAL, conventional CLEAs and spherical CLEAs with biosilica shell against high temperature (a) and denaturants (b).

476

Figure 8 Enzyme resistance to proteolysis in the presence of trypsin.

477

Figure 9 Mechanical stability of free PAL, conventional CLEAs and spherical CLEAs

478 479 480

with biosilica shell. Figure 10 Recyclability of conventional CLEAs and spherical CLEAs with biosilica shell.

481

23



Table 1. Comparison of kinetic parameters of free PAL, conventional CLEAs, and spherical CLEAs with biosilica shell

Enzyme

Km (mM)

Vmax (mM/min)

Free PAL

9.97

0.026

Conventional CLEAs

76.68

0.001

Spherical CLEAs with biosilica shell

71.28

0.005


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Figure 2


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Figure 4


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Figure 6


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Figure 8


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Figure 10


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TOC Graphic


Encapsulation of Spherical Cross-Linked ... - ACS Publications

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