Development of Thermoregulatory Enzyme Carriers Based on

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Development of thermoregulatory enzyme carriers based on microencapsulated n-docosane phase change material for biocatalytic enhancement of amylases Jindui Li, Huan Liu, Xiaodong Wang, and Dezhen Wu ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b02200 • Publication Date (Web): 16 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017

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ACS Sustainable Chemistry & Engineering

Development of thermoregulatory enzyme carriers based on microencapsulated n-docosane phase change material for biocatalytic enhancement of amylases

Jindui Li, Huan Liu, Xiaodong Wang*, Dezhen Wu

State Key Laboratory of Organic–Inorganic Composites, Beijing University of Chemical Technology, Beijing 100029, China. * Tel: +86 10 6441 0145. Fax: +86 10 6442 1693. E-mail: [email protected] (X. Wang).

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ABSTRACT A phase change materials (PCMs)-based magnetic microcapsule system was designed as thermoregulatory enzyme carriers and then successfully constructed by microencapsulating n-docosane into an Fe3O4/SiO2 hybrid shell through interfacial polycondensation in a Pickering emulsion templating system. Scanning and transmission electron microscopic observations indicated that the as-synthesized microcapsules presented a well-defined core-shell microstructure as well as a regular spherical morphology, and their chemical structure and composition were confirmed by a series of spectroscopic characterizations. α-Amylase as a model enzyme was also successfully immobilized on the aldehyde-functionalized surface of as-synthesized microcapsules

through

covalent

bonding.

Such

covalent

immobilization

was

confirmed

by

energy-dispersive X-ray spectroscopy and X–ray photoelectron spectroscopy. Thermal analysis results indicated that the α-amylase-immobilized microcapsules obtained a high latent heat-storage capability of approximately 155 J/g and a high encapsulation efficiency of 58 %, and they also showed an effective thermoregulation capability and good thermal management performance. Most of all, the α-amylase immobilized on the microcapsules demonstrated a higher enzyme activity, thermal stability and storage stability than that on the classical SiO2 solid carriers under the fluctuation of ambient temperature. In addition, the α-amylase-immobilized microcapsules also achieved a good separability and reusability due to its superparamagnetic nature. With an enhanced biocatalytic activity, the thermoregulatory enzyme carriers developed by this work exhibit a great potential for biologic applications and can well serve for sustainable chemistry and green process. Keywords: PCMs-based microcapsules system; Thermoregulatory enzyme carriers; α-Amylases; Immobilization; Enzyme activity; Biocatalytic performance

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Introduction Sustainable development is a very hot theme in today's society, and it has become one of the largest concerns in the world due to the enormous challenges in our social and environmental resources. The goal of sustainable development is to meet the needs of the present without compromising the ability of future generations to meet their needs.1 Energy and environment are considered as two major issues concerning the sustainable development of human beings. The heavy use of fossil fuels in all over the world has brought about a series of global problems such as greenhouse gas and exhaust emissions, climate change, global warming and air pollution, which by now have become a serious threaten to the health and survival of human beings as well as the social and economic development. The exploitation of sustainable energy sources and the improvement of energy efficiency generally recognized as two effective ways to realize the sustainable development of human beings.2 Unlike fossil fuels that provide the bulk of the worldwide energy, renewable and sustainable energy can be used for a long period of time and produces far less pollution, thus making a much greater contribution to environmental and economic sustainability.3 Phase change materials (PCMs) as a class of renewable and sustainable energy materials have received considerable attention with regard to the thermal energy storage in recent years, because they can store large amounts of latent heat and then releases it depending on the thermal energy demand with only small temperature changes. With a large storage capacity and the isothermal nature of the storage process, PCMs can help bridge a gap between the availability and use of thermal energy, which avoids energy waste and improves energy efficiency effectively.4 Nowadays, PCMs have been broadly applied for energy-saving buildings, heat-pump systems, solar thermal energy storage, waste heat recovery, food and pharmaceutical refrigeration, thermoregulatory textiles used in the closing, thermal comfort in vehicles, telecom shelters in tropical regions, computer cooling and thermal protection of electronic devices.5 Moreover, the creative

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technology offered by PCMs provides a new horizon for the medical services like the transportation of blood and organs, surgery-operating tables, hot-cold therapies and the treatment of neonates with birth asphyxia.6 The most commonly used PCMs are salt hydrates, fatty acids and esters and various paraffin waxes, and they have been used for thermal energy storage in the low to medium temperature ranges for many years. New developments into higher temperature ranges are making use of metallic materials and inorganic salts which can operate in the temperature range of > 500 °C.7 These PCMs can be operated either in bulk or in the microencapsulated state. Since PCMs generally perform a phase transformation between solid and liquid in thermal cycling, microencapsulation naturally becomes an obvious choice for the use of PCMs. Microencapsulation cannot only make PCMs a form stability to offer the ease of handling, but also supplies a large specific surface area for PCMs to enhance their thermal conduction and heat transfer.8 The microencapsulation of PCMs can facially be conducted by a physical or chemical method. Both inorganic chemicals and organic polymers can be adopted as shell materials to encapsulate PCMs into core-shell structural microcapsules,9 and these shell materials include SiO2,10,11 CaCO3,12,13 Al(OH)3,14 TiO2,15 melamine-formaldehyde copolymers,16 polyurea-formaldehyde copolymers,17 poly(methyl methacrylate),18 polystyrene,19 polyurethane,20 and even water-soluble materials.21 The newest trend in development of microencasulated PCMs are making use of inorganic functional materials to encapsulate PCMs. A number of successful cases have been reproted, indicating that some of paraffins could be feasibly encapsulated into the brookite TiO2,22,23 ZrO2,24,25 ZnO,26 Cu2O,27 Fe3O4/SiO2,28 and Ag/SiO2 hybrid shells.29 These functional shell materials endow the microencapusalted paraffins with a variety of accessory functions like sterilization, photocatalysis, photoluminescence, magnetism, electrical conduction and gas sensitivity in addition to a thermal energy-storage function. This actually offers promise in the field of PCMs-based

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multifuntional microcapsules. It is highly anticipated that the development of PCMs-based multifuntional microcapsules for bioapplicatons will be the next major trend in the field of thermal management and thermoregulation technologies. Enzymes as a type of important biocatalysts serve a wide variety of biological functions for various industrial applications and have been extensively used for brewing industry, food processing, dairy industry, molecular biological engineering, starch industry and paper industry as well as for the productions of pharmaceuticals, fine chemicals and biofuels.30 It is generally accepted that enzymes play an important role in sustainable and green chemistry due to their high catalytic efficiency, mild conditions, and less pollution to the environment.31 High activity and high thermal stability are generally expected for enzymes when performing a biological catalysis. Therefore, to enhance the biocatalytic performance as well as the economy for the industrial applications, enzymes are needed to be chemically or physically immobilized on appropriate solid carriers so as to stabilize their active forms and also achieve an ease separation from the reaction medium.32 Moreover, mesoporous silica and porous carbon materials were also used as supports for the immobilization of enzymes due to their large specific surface areas and high immobilization amount.33,34 However, most of the enzymes are thermally sensitive and only exhibit a high catalytic activity in the optimal temperature range because of the kinetic and thermodynamic features of biochemical reactions.35 Considering the fact that the phase change takes place at constant temperature, it is possible for enzymes to smooth out the ambient temperature variations by using PCMs-based microcapsules as enzyme carriers. With this idea in mind, we successfully designed a magnetic microcapsule system based on an n-eicosane core and Fe3O4/TiO2 hybrid shell for the immoblization of lipases in our previosu study,36 and it was found that, compared to those inertial solid or porous supporters, such a type of enzyme carriers not only provided high thermal stability, long storage durability and good reusability but also effectively

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improved the biocatalytic activity under the fluctuation of ambient temperature due to the thermoregulation of their PCM core. In the current work, we attemped to design a type of thermoregulatory enzyme carries based on PCMs-based microcapsules for biocatalytic enhancement of amylases. Amylases are one of the most members in the family of enzymes, and they exhibit a wide spectrum of application in brewing industry and food processing.37,38 The aim of this work is to develop a new type of enzyme carriers with the characteristics of high immobilization, automatic thermoregulation and easy separation from the reaction media and also to open up a new application in the fields of biologic technology and engineering.

Experimental Section Materials.

Tetraethyl

orthosilicate

(TEOS),

cetyltrimethyl

ammonium

bromide

(CTAB)

and

3-Aminopropyltriethoxysilane (APTES) were purchased from Tianjin Fuchen Chemical Reagent Co., Ltd., China. n-Docosane was purchased from Acros Organics Company, USA. Fe3O4 nanoparticles were prepared according to the method as described in the literature

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and sealed in a vacuum bag before use.

α-Amylase and starch soluble were commercial supplied by AoBoxing Bio-Tech Co., Ltd., China. Sodium hydroxide (NaOH), potassium iodide, iodine, citric acid monohydrate, sodium citrate, gultaraldehyde and formamide were commercially provided by Sigma-Aldrich and used as received. Synthesis of magnetic microcapsules. The magnetic microcapsules consisting of n-docosane core and Fe3O4/SiO2 hybrid shell were synthesized by using TEOS as a silica source through interfacial polycondensation in a Pickering emulsion templating system, where Fe3O4 nanoparticles were used as a Pickering stabilizer. The synthetic method and typical process were circumstantially described in Support Information in accordance with the methodology reported by the literature.28 Immobilization of α-amylase onto microcapsules. First of all, the as-synthesized magnetic microcapsules were surface-functionalized with amino groups before conducting the immobilization of α-amylase. In a

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typical process: about 0.1 g of as-synthesized magnetic microcapsules was dispersed in 30 mL of deionized water, followed by the addition of ammonia water (4 mL) to form an alkaline suspension. Afterward, 3 mL of APTES was added into the suspension with stirring at 60 °C for 15 h under a nitrogen atmosphere. After the amino-functionalization reaction was completed, the resulting magnetic microcapsules were collected by a magnet and then washed with deionized water thoroughly to remove the free APTES. The amino-functionalized magnetic microcapsules were separated by the magnet for further use. In the case of immobilizing α-amylase onto the surfaces of magnetic microcapsules, approximately 0.1 g of amino-functionalized magnetic microcapsules was dispersed in 30 mL of aqueous solution containing 1.5 g of gultaraldehyde with stirring for 1 h at room temperature under the protection of nitrogen. With the completion of amino-functionalization reaction, the resultant magnetic microcapsules were collected by a magnet and then washed with deionized water thoroughly to remove the free gultaraldehyde. In succession, 0.1 g of aldehyde-functionalized magnetic microcapsules was dispersed in a citric acid-sodium citrate buffer solution (30 mL, 0.1 mol/L, pH = 6.0), and then 2.0 mL of α-amylase aqueous solution (1.0 wt %) was added. The resulting suspension was mechanically agitated at room temperature for 4 h. The α-amylase immobilized on the magnetic microcapsules was obtained after washing with the same buffer solution and magnetic separation. This immobilized α-amylase was stored in the same buffer solution for future use. Characterizations. The morphologies of various microcapsule samples were observed by scanning electron microscopy (SEM; S-4700, Hitachi, Japan), and the surface elemental analysis was also performed with an energy-dispersive X-ray (EDX) spectrometer attached to the SEM instrument. The microstructures of microcapsule samples were investigated by transmission electron microscopy (TEM; Tecnai G2 F20 S-TWIN, FEI, USA). The surface chemical distributions of microcapsule samples were detected by X–ray photoelectron spectroscopy (XPS; EscaLab 250Xi, Thermo Fisher Scientific, USA) on a XPS system, and

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their chemical compositions were confirmed by Fourier-transform infrared (FTIR) spectroscopy (Nicolet iS5, Thermo Fisher Scientific, USA). The crystalline structures of microcapsule samples were determined by X-ray powder diffraction (XRD; D-Max 2500, Rigaku, Japan) using Cu-Kα radiation (λ = 1.5418 Å), and their shell structures were also detected by Raman-scatting depth profiles using a confocal Raman microscope (inVia™, Renishaw, UK) at an excitation wavelength of 514 nm. Differential scanning calorimetry (DSC) was conducted to characterize the latent heat storage/release behaviors of various samples using a differential scanning calorimeter (Q20, TA Instruments, USA) at a scanning rate of 10 °C/min. Thermogravimetric analysis (TGA) was conducted to evaluate the thermal stabilities of microcapsule samples on a thermogravimetric analyzer (Q50, TA Instruments, USA) in nitrogen at a heating rate of 10 °C/min. A custom-designed experimental setup was used to evaluate the thermoregulation performance of microcapsule samples at different ambient temperatures. This setup could stimulate the thermoregulation behaviors of PCMs by recording the transient response to temperature during the thermal energy absorption and release and was schematically illustrated in Figure S1 (see Supporting Information). The thermoregulatory capability of microcapsule samples was also evaluated by infrared thermography using a thermal imaging camera (E40, FLIR Instruments, USA). The thermographic images were taken for the specimens on a hot plate at different heat time, and the temperature distributions were analyzed by using the FLIR Tools software. The magnetic properties of enzyme carriers were measured by a vibrating sample magnetometer (VSM-7410, Lake Shore, USA) in an applied field ranging from -20,000 to 20,000 Oe at room temperature. The magnetic retentivity, magnetization saturation and coercivity were achieved on the basis of the measured magnetization curves. The enzyme activities of immobilized α-amylase as well as the free one were assayed by measuring the concentration of residual starch after hydrolysis in the citric acid-sodium

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citrate buffer solution using an ultraviolet (UV)-visible spectrophotometer (UV-2550, Shimadzu, Japan), and the detailed assay method was described in Supporting Information.

Results and Discussion An ideal type of enzyme carriers are supposed to comply with several criteria like such as good thermal and chemical stabilities, good biocompatibility, nontoxicity, large specific surface, easy covalent immobilization with various enzymes, easy separation from reaction mixture and convenient recycling for reuse.39,40 To meet these criteria as well as the cost-effective demand, we designed an Fe3O4/SiO2 hybrid as shell material to construct a PCMs-based magnetic microcapsule system as thermoregulatory enzyme carriers. The incorporation of Fe3O4 can impart a magnetic separation function to the enzyme carriers for easy reuse. Considering the fact that α-amylases play a key role in a broad range of industrial processes, we adopted the α-amylase as a model amylase for the study of immobilization and biocatalytic activity on the microencapsulated PCMs. Meanwhile, a paraffin-type PCM, n-docosane, was selected as a core material for the microcapsule system due to its suitable phase-change temperature range in accordance with the high enzyme activity of α-amylase. Figure 1 illustrates the synthetic strategy of this microcapsule system and the subsequent immobilization procedure of α-amylases. For the purpose of fabricating an Fe3O4/SiO2 hybrid shell onto the n-docosane core, an oil-in-water Pickering emulsion containing the mixture of TEOS and n-docosane was prepared first by using Fe3O4 nanoparticles as a Pickering solid stabilizer and formamide as a dispersion medium in place of water. And then, CTAB as a templating agent was incorporated into the Pickering emulsion to construct a Pickering emulsion templating system for the further self-assembly and interfacial polycondensation of silica precursors. The polycondensation of silica precursors was initiated and promoted by trace amount of water onto the surface of n-docosane micelles to fabricate an Fe3O4/SiO2 hybrid shell. Such a synthetic mechanism was described in detail in our recently

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published articles.28,36 With the formation of this microcapsule system, the covalent immobilization of α-amylase was conducted on the aldehyde-functionalized surface of as-synthesized microcapsules through a grafting reaction between the amino groups of α-amylase and the aldehyde groups on the microcapsule surface. This immobilization mechanism is also explained schematically in Figure 1. A morphological investigation indicates that the as-synthesized microcapsules exhibit spherical monodisperse particles with a diameter of 2–3 µm as observed from their SEM micrograph in Figure 2a, and a magnified SEM micrograph shows that these microcapsules have a smooth compact surface as seen in Figure 2b. A nitrogen adsorption-disoperation experiment indicates that the microcapsules only have a specific surface area of 3.94 m2/g due to their smooth surface. This value is evidently lower than the specific surface areas of porous materials (>1000 m2/g) as reported,33,34 which may be disadvantageous for the microcapsules to obtain the high immobilization loading of enzymes. Moreover, a core-shell structure for the as-synthesized microcapsules is observed from the SEM micrograph of a damaged microcapsule in Figure 2c, and such a well-defined core-shell structure can be further confirmed by the TEM micrographs in Figure 2g and 2h. It is noteworthy that, after immobilized with α-amylase, the surface of microcapsules is found to become considerably coarse and seems to be fully covered with α-amylase as seen in Figure 2d–2f, nevertheless, their core-shell structure is well maintained (see Figure 2i). The α-amylase attached on the microcapsule surface can also be clearly distinguished through a comparative observation from the TEM micrographs in Figure 2h and 2j. These results verified the formation of core-shell structural microcapsules as well as the actual existence of α-amylase on the microcapsule surface. The chemical compositions and structures of as-synthesized microcapsules and the microcapsules immobilized with α-amylase were determined by FTIR spectroscopy, XRD and Raman scatting spectroscopy with depth profiles. As observed from the FTIR spectra in Figure 3a, the as-synthesized

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microcapsules not only show most of the absorption peaks corresponding to the methyl and methylene groups of n-docosane in their infrared spectrum, but also exhibit the characteristic bands with regard to the Si–O and Fe–O bonds. The XRD pattern of as-synthesized microcapsules in Figure 3b reveal a series of diffraction peaks assigned to the crystallographic planes of n-docosane as well as a set of diffraction peaks attributed to the reflections of Fe3O4. Meanwhile, the XRD pattern of microcapsule shell only shows the characteristic diffraction peaks of Fe3O4, which is consistent with those of pristine Fe3O4 nanoparticles with an inverse spinel structure as marked in Figure 3b according to the standard card data of JCPDS card no. 29–1360. These results suggest that the as-synthesized microcapsules consist of the n-docosane core and Fe3O4/SiO2 hybrid shell. Moreover, a Raman-scatting depth profiling detection clearly gives the distribution of Fe3O4 nanoparticles within the microcapsule shell. As observed from the Raman spectra of as-synthesized microcapsules in Figure 3c, a series of characteristic bands of inverse spinel Fe3O4 corresponding to the modes of Eg, T2g and A1g can be clearly distinguished only at the scanning depth of 0.3 µm. This indicates that Fe3O4 nanoparticles were not distributed on the surface of as-synthesized microcapsules but embedded into the microcapsule shell. It is noteworthy in the infrared spectra of the amino-functionalized and α-amylase-immobilized microcapsules that the H2N–R vibration for Amide I, HN–R2 vibration for Amide II and N–R3 vibration for Amide III can be observed at 1640, 1557 and 1392 cm-1, respectively. However, the other characteristic peaks of immobilized α-amylase were overlapped by the absorption bands from the microcapsules. In order to confirm the covalent immobilization of α-amylase, XPS and EDX spectroscopy were performed to investigate the surface elemental distribution and chemical structures of microcapsule samples. It is observed in Figure 3d and 3e that the EDX patterns of the as-synthesized and α-amylase-immobilized microcapsules both show the signals from the Si, O and Fe atoms as well as the carbon background. The

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incidental elementary mappings clearly demonstrate the silica-based matrix and dispersed Fe element. Furthermore, in addition to the signals of Si, O and Fe atoms, a new signal corresponding to the N atom can be found in the EDX pattern of the microcapsules after immobilized with α-amylase (see Figure 3e), and meanwhile, a dense distribution of N element on the microcapsule surface is observed in the relevant elementary mappings. These results suggest the presence of immobilized α-amylase on the microcapsule surface. The high-resolution XPS spectra depicted in Figure 3f–3k also clearly identify the surface elemental and structural variation of the microcapsules before and after immobilized with α-amylase. The presence of Si 2p, O 1s and Fe 2p signals verifies the formation of Fe3O4/SiO2 hybrid shell as observed in the high resolution specra of as-synthesized microcapsules (see Figure 3f–3h). It is noteworthy in Figure 3g that the characteristic signal associated with the O–H bond can also be distinguished in the O 1s spectrum through an XPS peak-fitting program, indicating an abundance of hydrogen groups on the surface of as-synthesized microcapsules. This is very advantageous for the further surface functionalization of amino groups and the immobilization of α-amylase. On the other hand, the high-resolution XPS spectra of α-amylase-immobilized microcapsules not only show the Si 2p, O 1s and Fe 2p signals but also exhibit a new signal for the N 1s chemical shift as seen in Figure 3i. Moreover, the existence of N–H, C–N, O=C–OH, C–C and bands can be identified through the resolutions of N 1s, C 1s and O 1s peaks by the peak-fitting program (see Figure 3i–3k). These results confirm the successful covalent immobilization of α-amylase on the surface of as-synthesized microcapsules. The

phase-change

behaviors

and

latent

heat-storage

performance

of

as-synthesized

and

α-amylase-immobilized microcapsules were investigated by dynamic DSC scans, and the obtained results are given in Figure 4a–4c. It is interestingly observed that pure n-docosane reveals a bimodal crystallization behavior by displaying two exothermic peaks at 37.9 and 40.1 °C during the cooling process

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in its DSC curve, whereas it only shows an endothermic peak at 45.7 °C for its melting. This bimodal crystallization behavior has been widely reported for various paraffin waxes and is ascribed to the formation of a metastable rotator phase before the accomplishment of full crystallization.11,41 Pure n-docosane is also found to absorb the latent heat (∆Hc) in an amount of 269.2 J/g through crystallization and then release it as fusion heat (∆Hm) with an amount of 271.2 J/g during the melting stage. This indicates that pure n-docosane has a high thermal energy-storage capability. It is notable that the as-synthesized and α-amylase-immobilized microcapsules exhibit the same phase-change behaviors with pure n-docosane, but their crystallization peak temperatures present a decrease by 2.7 °C. This may be due to the confinement effect on the crystallization of n-docosane inside microcapsules. Owing to the introduction of inertial Fe3O4/SiO2 hybrid shell, the ∆Hc and ∆Hm of as-synthesized microcapsules were reduced to 156.3 and 157.6 J/g, respectively, and the encapsulation efficency representing the effective encapsulation of n-eicosane core by the shell material is determined as 58.1% by the equaton described in the literature.12,24 It should be metioned that the synthesis of microcapsule samples in this study were carried out using the optimum fomulation developed by our previous work,28 in which the micrcapsules achieved a good balance between the mechanical strength and encapsulation efficiency by optimizing the core/shell mass ratio. For the resultant microcapsules, the core/shell mass ratio was determined as 65/35 by a comparison for the microcapsule mass before and after the solvent extraction of core material. It is notable that the phase-change enthalpies of microcapsules are slightly lower than the values calculated in accord with the weight fraction of n-docosane core. This means that not all of the n-docosane core can perform the phase changes during the heating and cooling processes. It seems that the immoblization of α-amylase does not influence the phase-change enthalpies as seen in Figure 4a, and the encapsulation efficency of α-amylase-immobilized microcapsules still reaches 58.0 %, indicating a good latent

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heat-storage capability. The phase-change reversibility and durability of α-amylase-immobilized microcapsules were evaluated by multicycle DSC scans, and the resulting DSC curves are given in Figure 4b. It is noticeable that these thermograms keep good overlaps from the first loop to the last one during the 200-cyclic scans. Meanwhile, the ∆Hc and ∆Hm of α-amylase-immobilized microcapsules present a very slight fluctuation with the cycle number as observed in Figure 4c, suggesting an excellent reversibility and stability for the phase transitions of encapsulated n-docosane within this type of enzyme carriers. In addition, the thermal stabilities of as-synthesized and α-amylase-immobilized microcapsules were analyzed by TGA. The obtained TGA curves clearly indicate that the encapsulation of n-docosane with an Fe3O4/SiO2 hybrid shell can considerably improve the thermal decomposition temperature compared to pure n-docosane as shown in Figure 4d. It is evident that the formation of inorganic shell effectively presents the microencapsulated n-docosane from thermally decomposing, thus leading to an increase in thermal degradation temperature. Furthermore, the immobilization of α-amylase seems not to affect the thermal stability of the microcapsules. This suggests that the enzyme carriers developed by this work can meet the requirement of bioapplications in a broad temperature range. The thermoregulation and thermal management performance of α-amylase-immobilized microcapsules were investigated by infrared thermography during the heating and cooling processes, and the temperature-time diagrams obtained along with the thermographic images are illustrated in Figure 4e and 4f, in which the relevant data for the SiO2 solid enzyme carriers are also presented as a control. It is observed from these two diagrams that the temperature of SiO2 solid carriers shows a continuous elevation with an increase of heating time and then decreases without any hysteresis during the cooling stage. However, a temperature lag is detected for the α-amylase-immobilized microcapsules during the heating and cooling processes as seen in Figure 4e and 4f, respectively. Such a temperature lag only occurs in the

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temperature ranges of melting and crystallization phase transitions of n-docosane and hence is believed to result from the thermal energy absorption of encapsulated n-docosane by melting and its latent heat release by crystallization. The thermographic images inserted in Figure 4e and 4f also exhibit a noticeable color difference between the α-amylase-immobilized microcapsules and SiO2 solid carriers during the heating and cooling processes. In these thermographic images, the target color is considered as a temperature indicator, and it clearly indicates that the temperature of α-amylase-immobilized microcapsules is lower than that of SiO2 solid carriers in the heating process but higher than that of SiO2 solid carriers during the cooling stage due to their thermoregulatory capability. Moreover, the temperature variations of these two samples with heating and cooling time were monitored through a custom-designed experimental system as described by Figure S1 in Supporting Information, and the resulting temperature-time diagrams are displayed in Figure 4g and 4h. This experimental system can well simulate the thermoregulation behavior of PCMs with a variation of ambient temperature. Similar with the infrared thermographic results, the α-amylase-immobilized microcapsules also reveal the hysteresis phenomenon of temperature in both the heating and cooling processes, whereas the SiO2 solid carriers show a continuous variation whether it is heated or cooled. The regions of temperature hysteresis are in good agreement with the phase-change temperature ranges of n-docosane. These characterization results clearly prove that the PCMs-based enzyme carriers developed by this work has a good thermoregulatory capability to perform an effective thermal management. The α-amylase was covalently immobilized on the as-synthesized microcapsules as thermoregulatory carriers as well as on the SiO2 solid carriers as a control. The immobilization yield of α-amylase was calculated to be 73.4 % for the thermoregulatory carriers and 73.7 % for the SiO2 solid carriers by the Coomassie brilliant blue method,42 and its immobilization efficiency was identified as 57.9 % for the

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thermoregulatory carriers and 58.6 % for the SiO2 solid carriers by the method described in Supporting Information. A comparative investigation was performed on the temperature and acidity dependency of enzyme activity, storage stability, thermal stability and reusability for these two types of carriers. Figure 5a depicts the effect of temperature on the relative activities of the α-amylase immobilized on the microcapsules and SiO2 solid carriers. There is a maximum enzyme activity observed at 45 °C for the α-amylase immobilized on both types of carriers as well as the free one, indicating an optimum biocatalytic temperature for α-amylase.43 On the other hand, the enzyme activity of free α-amylase is always far lower than that of the immobilized one, indicating the biocatalytic superiority of immobilized α-amylase over the free one. The relative activity of α-amylase immobilized on the microcapsules is found to be considerably higher than that on the SiO2 solid carriers at temperatures higher than 45 °C, although their relative activities are similar to each other at temperature lower than 45 °C. Such an improvement in enzyme activity is attributed to the thermoregulation of α-amylase-immobilized microcapsules, because the n-docosane encapsulated within the microcapsules can adjust the temperature around these carriers by absorbing or releasing latent heat through the phase change of melt or crystallization. In this case, the ambient temperature around the carriers is close to the optimum one for the enzyme activity of α-amylase, thus leading to an enhancement in biocatalytic activity for the α-amylase immobilized on the microcapsules. Moreover, the effect of acidity on the relative activity at 45 °C given in Figure 5b indicates that both the free and immobilized present a maximum relative activity at pH 6. This confirms an optimum acidity of buffer solution for the biocatalysis of α-amylase. However, the α-amylase immobilized on the microcapsules maintains a much higher relative activity than the free one with a variation of pH value, indicating that the immobilization of α-amylase on this type of carriers can lead to a high enzyme activity over a broader range of pH value. It is well known that the variation of acidity will damage the molecular

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structure of enzymes, and the immobilization of enzymes on appropriate carriers can enhance their structural stability, thus improving the enzyme activity.44 The storage stabilities of free and immobilized α-amylase were investigated by determining their relative activities after storage at 4 °C for different periods, and obtained data are presented in Figure 5c. It is noticeable that free α-amylase only maintains approximately 60 % of relative activity after storage at 4 °C for 16 days due to is poor storage stability. The α-amylase immobilized on the microcapsules and SiO2 solid carriers exhibits a high relative activity over 80 % after storage at 4 °C for 16 days. Furthermore, the α-amylase immobilized on the microcapsules still maintains a higher relative activity than that on the SiO2 solid carriers with an increase of storage period. It is believed that the fluctuation of ambient temperature can be inhibited by thermal energy storage or release through the phase changes of the n-docosane encapsulated within these carriers, thus enhancing the storage stability of immobilized α-amylase. Figure 5d shows the relative activities of free and immobilized α-amylase after incubation at 50 °C for different hours, which reflects their thermal stabilities. Free α-amylase was found to have a poor thermal stability, because it lost almost 50 % of its initial activity after incubation at 50 °C for 2 h. However, the α-amylase immobilized both on the microcapsules and on the SiO2 solid carriers maintains a high relative activity over 80% after incubation at 50 °C for 4 h, indicating the superiority of immobilized α-amylase in thermal stability. In this case, the α-amylase immobilized both on the solid silica carriers and on the microcapsules exhibits a similar variation trend with the incubation hour. Nevertheless, there is no doubt that the α-amylase immobilized on the microcapsules shows a noticeably higher enzyme activity than that on the SiO2 solid carriers as observed in Figure 5d. This suggests that a thermoregulatory action is conducted to emending the incubation temperature by the encapsulated n-docosane for this type of carriers when a temperature fluctuation occurs, thus effectively improving the thermal stability of the α-amylase

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immobilized on this type of carriers. To confirm the application of magnetic separation for the thermoregulatory enzyme carriers developed by this work, the magnetic performance of α-amylase-immobilized microcapsules was characterized by VSM, and resulting magnetic hysteresis curve is given in Figure 5e. It is interestingly noted that the α-amylase-immobilized microcapsules demonstrate an extrinsic hysteresis behavior when an external magnetic field is applied, which is attributed to the presence of Fe3O4 nanoparticles in their shell. These carriers achieved the magnetization saturation of 8.71 emu/g and also revealed extremely low extremely magnetic retentivity and coercivity as obtained from the VSM characterization. These results clearly verify that

the

α-amylase-immobilized

microcapsules

have

a

superparamagnetic

45

nature.

Such

a

superparamagnetic nature makes the α-amylase-immobilized microcapsules collected easily by an external magnetic field. On the other hand, there is no residual magnetism retained in this type of carriers in the absence of an external magnetic field, which is very advantageous for the collection, separation and reuse of the α-amylase immobilized on these carriers. Furthermore, the digital photos inserted in Figure 5e vividly illustrate a good separability for this type of carriers. It is clearly observed that the α-amylase-immobilized microcapsules suspended in buffer solution have been completely attracted onto the wall of a glass bottle by a magnet, and these collected microcapsules can be easily resuspended in the solution after the removal of this magnet. The reusability of α-amylase-immobilized microcapsules was further evaluated by assaying the enzyme activity of immobilized α-amylase as a function of reusing times, and the obtained data are summarized in Figure 5f. As shown in Figure 5f, the α-amylase immobilized on the microcapsules reveal a gradual decrease in its relative activity with an increase of reuse time, and their residual activity still maintain over 70 % after 12 times of the cyclic reuse. The decrease in enzyme activity during the cyclic processing of reuse may be due to the exfoliation of some of the α-amylase immobilized

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on the microcapsules through physical bonding, and however the retention of biocatalytic activity is still significant for the carriers due to the immobilization of α-amylase mostly by covalent bonds.

46

These

results suggest that the α-amylase immobilized on this type of carriers have good separability and reusability for future industrial reutilization.

Conclusions We designed a PCMs-based magnetic microcapsule system as thermoregulatory enzyme carriers and then successfully constructed it by microencapsulating n-docosane into the Fe3O4/SiO2 hybrid shell through interfacial polycondensation in a Pickering emulsion templating system. The well-defined core-shell microstructure and regular spherical morphology of as-synthesized microcapsules were identified by SEM and TEM, and their chemical composition, surface elemental distribution and crystalline structure were determined by FTIR, XPS, EDX and Raman spectroscopy as well as the XRD patterns. α-Amylase as a model enzyme was also successfully immobilized on the aldehyde-functionalized surface of as-synthesized microcapsules through covalent bonding. Such covalent immobilization was vividly observed by SEM and TEM and then confirmed by XPS and EDX spectroscopy. The α-amylase-immobilized microcapsules gained a high latent heat-storage capability of 155 J/g as well as a high encapsulation efficency of 58 %. Their effective thermoregulation capability and good thermal management performance were also proved by infrared thermography and the thermoregulation behaviors observed in the custom-designed experimental setup. Most of all, the α-amylase immobilized on the microcapsules demonstrated a higher enzyme activity, thermal stability and storage stability than that on the traditional SiO2 solid carriers under the fluctuation of ambient temperature. Moreover, this type of carriers also achieved a good separability and reusability due to its superparamagnetic nature. With an enhanced biocatalytic activity, the thermoregulatory enzyme carriers developed by this work exhibits a great potential for biologic

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applications and can well serve for sustainable chemistry and green process.

ASSOCIATED CONTENTS Supporting information Description of the synthetic method for magnetic microcapsules, description of the assay method for the enzyme activities of free and immobilized α-amylase, and schematic diagram of the custom-designed experimental setup for evaluating the thermoregulation performance of PCMs-based microcapsules through the latent heat storage and release processes.

AUTHOR INFORMATION Corresponding author * Tel: +86 10 6441 0145. Fax: +86 10 6442 1693. E-mail: [email protected] (X. Wang). ORCID Xiaodong Wang: 0000-0002-8787-1268

Notes The authors declare no competing financial interest.

Acknowledgements The work was supported by the National Natural Science Foundation of China with a grant number of 51673018.

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Figure Captions Figure 1 Schematic diagram for the synthesis of microencapsulated n-docosane with an Fe3O4/SiO2 hybrid shell and the following immobilization of α-amylase. Figure 2 SEM

micrographs

of

(a,

b,

c)

as-synthesized

microcapsules

and

(d,

e,

f)

α-amylase-immobilized microcapsules; TEM micrographs of (g, h) as-synthesized microcapsules and (i, j) α-amylase-immobilized microcapsules. Figure 3 (a) FTIR spectra of (1) pure n-docosane, (2) as-synthesized microcapsules, (3) amino-functionalized microcapsules and (4) α-amylase-immobilized microcapsules; (b) XRD patterns of as-synthesized microcapsules, Fe3O4/SiO2 hybrid shell and Fe3O4 nanoparticles; (c) Raman spectra of as-synthesized microcapsules; EDX spectra of (d) as-synthesized microcapsules and (e) α-amylase-immobilized microcapsules; (f, g, h) high-resolution XPS spectra of as-synthesized microcapsules; (i, j, k) high-resolution XPS spectra of α-amylase-immobilized microcapsules. Figure 4 (a)

DSC

curves

of


pure

n-docosane,

microcapsules;

(b)

as-synthesized multicycle

microcapsules DSC

and

curves

of

α-amylase-immobilized microcapsules; (c) plots of phase-change enthalpies as a function of cycle number obtained from multicycle DSC scans; (d) TGA curves of pure n-docosane, as-synthesized

microcapsules

and


microcapsules;

(e,

f)

temperature–time diagrams of α-amylase-immobilized microcapsules and SiO2 solid carriers obtained from infrared thermography, in which the relevant thermographic images are inserted; (g, h) temperature–time diagrams of α-amylase-immobilized microcapsules and SiO2 solid carriers obtained from the thermoregulation experiment. Figure 5 Relative activities of free and immobilized α-amylase as a function of (a) ambient temperature, (b) pH value, (c) storage period at 4 °C and (d) incubation hour at 50 °C; (e) magnetic hysteresis curve of α-amylase-immobilized microcapsules along with the inserted digital photos reflecting their magnetic separability; (f) relative activity as function of cycle number for the α-amylase immobilized on the microcapsules during the reusing process.

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For Table of Contents Use Only

Synopsis A magnetic microcapsule system based on n-docosane was designed as thermoregulatory enzyme carriers for α-amylase to enhance its biocatalytic performance.

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Figure 1 Schematic diagram for the synthesis of microencapsulated n-docosane with an Fe3O4/SiO2 hybrid shell and the following immobilization of α-amylase. 75x50mm (600 x 600 DPI)



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Figure 2 SEM micrographs of (a, b, c) as-synthesized microcapsules and (d, e, f) α-amylase-immobilized microcapsules; TEM micrographs of (g, h) as-synthesized microcapsules and (i, j) α-amylase-immobilized microcapsules. 85x88mm (600 x 600 DPI)


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Figure 3 (a) FTIR spectra of (1) pure n-docosane, (2) as-synthesized microcapsules, (3) aminofunctionalized microcapsules and (4) α-amylase-immobilized microcapsules; (b) XRD patterns of assynthesized microcapsules, Fe3O4/SiO2 hybrid shell and Fe3O4 nanoparticles; (c) Raman spectra of assynthesized microcapsules; EDX spectra of (d) as-synthesized microcapsules and (e) α-amylase-immobilized microcapsules; (f, g, h) high-resolution XPS spectra of as-synthesized microcapsules; (i, j, k) high-resolution XPS spectra of α-amylase-immobilized microcapsules. 140x154mm (600 x 600 DPI)



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Figure 4 (a) DSC curves of pure n-docosane, as-synthesized microcapsules and α-amylase-immobilized microcapsules; (b) multicycle DSC curves of α-amylase-immobilized microcapsules; (c) plots of phasechange enthalpies as a function of cycle number obtained from multicycle DSC scans; (d) TGA curves of pure n-docosane, as-synthesized microcapsules and α-amylase-immobilized microcapsules; (e, f) temperature–time diagrams of α-amylase-immobilized microcapsules and SiO2 solid carriers obtained from infrared thermography, in which the relevant thermographic images are inserted; (g, h) temperature–time diagrams of α-amylase-immobilized microcapsules and SiO2 solid carriers obtained from the thermoregulation experiment. 166x258mm (600 x 600 DPI)


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Figure 5 Relative activities of free and immobilized α-amylase as a function of (a) ambient temperature, (b) pH value, (c) storage period at 4 °C and (d) incubation hour at 50 °C; (e) Magnetic hysteresis curve of αamylase-immobilized microcapsules along with the inserted digital photos reflecting their magnetic separability; (f) relative activity as function of cycle number for the α-amylase immobilized on the microcapsules during the reusing process. 146x176mm (600 x 600 DPI)


Development of Thermoregulatory Enzyme Carriers Based on

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