A Mild Strategy To Encapsulate Enzyme into Hydrogel Layer Grafted

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A Mild Strategy to Encapsulate Enzyme into Hydrogels Layer Grafted on Polymeric Substrate Xing Zhu, Yuhong Ma, Changwen Zhao, Zhifeng Lin, Lihua Zhang, Ruichao Chen, and Wantai Yang Langmuir, Just Accepted Manuscript • Publication Date (Web): 25 Nov 2014 Downloaded from http://pubs.acs.org on November 25, 2014

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A Mild Strategy to Encapsulate Enzyme into Hydrogels Layer Grafted on Polymeric Substrate Xing Zhu,† Yuhong Ma,‡ Changwen Zhao,*,† Zhifeng Lin,† Lihua Zhang,† Ruichao Chen,† Wantai Yang*, †,‡

†

State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical

Technology, Beijing 100029, China ‡

Key Laboratory of Carbon Fiber and Functional Polymers, Ministry of Education, Beijing

University of Chemical Technology, Beijing 100029, China

ABSTRACT: Although hydrogel network has been widely investigated as carrier for enzyme immobilization, to in situ encapsulate enzymes into hydrogel network in an efficient, practical and active way is still one of great challenges in the field of biochemical engineering. Here we reported a new protocol to address this issue by encapsulating enzyme into poly(ethylene glycol) (PEG) hydrogel network grafted on polymeric substrates. In our strategy, isopropyl thioxanthone semi-pinacol (ITXSP) dormant groups were firstly planted onto the surface of a plastic matrix with low density polyethylene (LDPE) film as model by a UV-induced abstracting hydrogencoupling reaction. Then as a proof of concept, lipase which could catalyze esterification of

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glucose with palmitic acid, was in situ net-immobilized into a PEG based hydrogel network layer through a visible light induced surface controlled/living graft cross-linking polymerization. This strategy demonstrates the following novel significant merits: 1) in comparison with the UV irradiation or high temperature, the visible light and room temperature used provide a friendly condition to maintain activity of enzyme during immobilization; 2) the uniqueness of controlled/living cross-linking polymerization not only makes it easy to form uniform PEG hydrogel network which is benefit to avoid the leakage of net-immobilizing enzyme, but also to tune the net-thickness or capacity to accommodate enzyme; 3) compared to systems of nanoparticles and porous matrixes, the flexible/robust end-products of the surface netimmobilizing enzyme with polymer film are more suitable to be applied in bioreactor due to their features of easier separation and reuse. We confirmed that this catalytic film could retain almost full of its initial activity after 7 batches of 24 h esterifications. The proposed strategy provides an extremely simple, effective and flexible method for enzyme immobilization. KEYWORDS: Visible light, enzyme immobilization, polymer films, cross-link

INTRODUCTION

Immobilization of the enzyme on or within a support is of particular importance to avoid contamination of the product by the enzyme, permit their reusability without the loss in activity. Presently a variety of inorganic (silica, zeolites, alumina, etc.) or organic materials (hydrogel, porous acrylic resins, polymeric membrane, etc.) have been explored as support for enzyme


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immobilization.1-3 As a unique soft material, the cross-link network structure of hydrogel provides enough capacity which is suitable to accommodate various size of matters including small molecules, nanoparticles, enzymes and cells.4-7 Hydrogels are water swollen 3D polymer networks with utilities in diverse areas of biotechnology and biomedicine.8 These materials have significant advantages over solid matrices in that a well-hydrated environment favors stability and function of immobilized enzymes. 9-13 However, most hydrogel networks suffer from a lack of sufficient mechanical strength due to its heterogeneous network structure thus are easy to be damaged during practical use.14-17 This disadvantage will become more severe when the hydrogel network was fabricated to thin film. One feasible solution to overcome this limitation is to graft the hydrogel networks onto supporting substrate which have a superior mechanical strength. Compared to inorganic materials, polymeric material is considered to be appropriate substrate due to its similar flexibility to the hydrogel networks18 which allows this enzyme-loaded bilayer film rolled up to save volume in catalytic reaction. Moreover, attaching thin hydrogel networks on flexible support will greatly eliminate the diffusion restriction encountered by bulk hydrogel networks9 and shorten the access time of encapsulated enzyme and substrate. Beyond that, polymeric matrix meets the demands by its low prices and ease of processing which might contribute to the industrial application (e.g., affinity membranes for bio-separations, and membrane reactors for catalytic conversions) in the future.19 However, several key issues must be taken into account to achieve the above mentioned design. First, the surface graft polymerization should be able to perform effectively because most


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of polymeric materials have low surface energy and inert C−H surfaces.20 Second, the mesh size of hydrogel network must be uniform and match the size of enzymes to make sure no leaching of enzymes during catalytic process.21 Third and most important, synthesis condition of hydrogel network should be mild and not adverse to activity of enzyme. To encapsulate enzyme into hydrogel network usually requires enzyme being present during polymerization, which generally involves in utilizing of oxidizing initiator like ammonium persulfate5 or UV irradiation.22 These negative factors caused the enzyme subjects to suboptimal conditions (high temperature, high energy radiation or toxic chemicals) and can result in a decrease or loss of its catalytic activity.2325

Therefore, to immobilize the enzyme into a hydrogel network in situ under mild conditions

(without heating, UV radiation or strong oxidizing agent) is of significant importance. Up to now, many surface initiated techniques have been developed to fabricate surface graft network or polymer brushes, including atom transfer radical polymerization (ATRP),26 reversible addition

fragmentation

chain

transfer

polymerization

(RAFT),27

nitroxide-mediated

polymerization (NMP)28 and photoiniferter polymerization.29,30 However, all of the methods mentioned above need tedious pretreatment process to introduce initiator onto substrate, and even worse, extreme reaction conditions such as high temperature (for example, most lipases exhibit activity between 30 and 50 °C),31 toxic catalysts or UV radiation could deactivate enzymes by destroying their structures.23-25 Therefore, to develop a mild surface-initiated graft polymerization approach friendly to enzyme is still a great challenge.


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Yang and Rånby firstly proposed a surface-initiated controlled radical polymerization based on photoinduced initiator immobilization technique (aromatic ketones abstracting hydrogen reaction) in 1996,32 and this method was further developed by Bowman et al.33 In this system, UV light were utilized as triggering source. In our recent work, visible light was used to take place of the UV irradiation to eliminate the adverse effect of UV irradiation on biomolecules. In those work, isopropyl thioxanthone (ITX) was photoreduced under UV light and sequentially coupled onto the surface of polymeric substrates to produce an isopropyl thioxanthonesemipinacol (ITXSP) “dormant” group, which can serve as reactive site to initiate surface grafting polymerization under visible light.34,35 In this work, we further developed this method into a chemical strategy of “visible light induced surface graft cross-linking polymerization”, and especially its bio-application for in situ enzyme net-immobilization. This design has two advantages: 1) energy of visible light irradiation is low enough not to affect the activity of delicate enzymes; 2) surface-initiated controlled graft cross-linking polymerization enables to form uniform PEG hydrogel network and precise control of thickness of hydrogel layer, which can avoid the leakage of netimmobilizing enzyme and readily adjust the amount of enzyme carried by hydrogel layer. We chose poly(ethylene glycol) diacrylate (PEGDA) as bifunctional monomer and lipase as model enzymes to evaluate the feasibility of our design. The results showed that lipase can be immobilized uniformed into P(PEGDA) hydrogel networks successfully with retention of catalytic activity and this enzyme encapsulated polymer film shows a good active stability after


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repeatedly conducting catalytic reaction. Although we used LDPE film as model substrate here, this strategy can also be applied on other polymeric substrates such as polymer film, fiber and non-woven fabric, to produce various kinds of robust enzyme immobilization products. These low-cost and new forms of products will provide an alternative separation and recycling methods for particular application which is not suitable to particle carriers. MATERIALS AND METHODS. Low density polyethylene (LDPE) films with a thickness of 50 µm were obtained from Beijing Plastic Factory No.7. The films were extracted with acetone for 48 h and then dried in a vacuum oven at 30 °C. Isopropyl thioxanthone (ITX) was obtained from TH-UNIS Insight Co., LTD. Poly(ethylene glycol) diacrylate (PEGDA) with molecular weight of 575 was purchased from Sigma-Aldrich Chemical Co. Lipase from porcine pancreas (PPL, EC 3.1.1.3) was ordered from TCI (Tokyo, Japan). Glucose, palmitic acid, tert-Butanol, Triton X-100, and p-nitrophenyl palmitate (p-NPP) came from Alfa Aesar Chemical Co. Preparation of Free and Cross-Linked PPL Solutions. Excess amounts of PPL were added into 20 mL phosphate buffer saline (PBS, KH2PO4 = 0.2 g/L, Na2HPO4.12H2O = 2.9 g/L NaCl = 8 g/L, KCl = 0.4 g/L, 0.01M, pH = 7.4) by shaking, and the mixture was left to stir at 4 °C for 24 h. After that, centrifuge (1439 g) was used to get supernatant from the PPL solutions. A concentration of 8.6 mg mL-1 free PPL solutions were finally prepared and stored at 4 °C. To prepare for the cross-linked PPL solutions, concentrations varying from 0.05% - 1% (v/v) of glutaraldehyde were added into the free PPL solutions for PPL linkage. In order to


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reduce the biotoxicity effect of glutaraldehyde, the cross-linking time was controlled for 30 min and then used to the grafting reactions immediately. Introducing ITXSP Groups onto LDPE Films. ITXSP was immobilized on LDPE films under UV irradiation through the sandwich structure as reported method.26 Briefly, certain amount of ITX solutions (acetone as the solvent, 40 µL, 3 mmol mL-1) was droped onto the LDPE film and then placed the film between two quartz plates to spread the solutions uniformly. This sandwich system was irradiated under high-pressure mercury lamp (wavelength 254 nm, 9 mW cm-2 ) for 3 min at room temperature, and then LDPE with ITX semipinacol groups (LDPEITXSP) were obtained after the reaction. The LDPE-ITXSP films were extracted with acetone for 24 h and washed with acetone alternately three times to remove the residual ITX. Finally, the films were dried in a vacuum oven at room temperature and then the whole film was packed up with tinfoil. In Situ PPL Immobilization by Visible-Light Induced Surface Graft Cross-Linking Polymerization. Two structures (the sandwich structure and groove structure) were applied to immobilize PPL to obtain grafting layers with quite different thickness. The stock solution of PPL was mixed with PEGDA to a certain concentration via shaking for 10 min. In the sandwich system, 40 µL PEGDA/PPL solutions were cast onto a LDPE-ITXSP film, and the film was then sandwiched between two quartz plates to spread out the PEGDA/PPL solutions. The time of the polymerization was varied from 10 min to 120 min under xenon lamp (filter was added with band pass of 380-700 nm, irradiation intensity was 3 mW cm-2 at 420 nm).


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To prepare patterned film, the only difference was that the quartz plate placed onto the LDPEITXSP surface was replaced with a photomask. In the groove system, a silicone rubber with square groove of depth of 2 mm was closely placed onto the LDPE-ITXSP film, and a certain volume of PEGDA/PPL solutions were added into the groove. The system was covered by a quartz plate. Then this system was placed under the same xenon lamp for 180 min at room temperature. To prepare patterned film, the only difference was that the quartz plate was replaced with a photomask. The LDPE films loaded with PPL which was entrapped into the P(PEGDA) hydrogel networks, noted as LDPE-g-P(PEGDA)/PPL, were immersed into the PBS for 72 h and washed with PBS (0.01M, pH = 7.4) alternately three times to remove the non-immobilized PPL and homopolymer, and the films were stored at 4 °C in the presence of 5 mL PBS (0.01M, pH = 7.4). To prepare for the patterned LDPE-g-P(PEGDA)/PPL with fluorescence, the patterned LDPE-g-P(PEGDA)/PPL was immersed into the Rhodamine B solution (10-5 g/L) for 30 min at room temperature. Thorough washing with PBS solution was performed to remove the fluorescent agent without electrostatic interaction, all steps being operated in the darkroom. The Detection of PPL Immobilized on the LDPE Film. Bradford’s method was used to detect the ratio of PPL immobilized in P(PEGDA) hydrogel networks. Full details about this procedure was described elsewhere.36 PPL solutions were used as standard to construct the calibration curve in our work. All the PPLs absorbed on quartz plate and LDPE-gP(PEGDA)/PPL after graft reaction were thoroughly rinsed and collected to determine non-


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immobilized amount. For testing the release behavior of PPL, LDPE-g-P(PEGDA)/PPL were immersed into PBS in a shaking incubator. And the amount of PPL in PBS was analyzed at regular intervals. The PPL concentrations after immersion were measured by monitoring its absorbance at 595 nm using a UV-vis Spectrophotometer. The concentration of PPL in the buffer solution was obtained from a calibration curve, and the amount of PPL release at time t (Mt) was calculated from accumulating the total PPL release up to that time. The ratio of PPL release, Mt/M0, could then be calculated. Here M0 is the amount of initially immobilized PPL. Lipase Activity Assays: Lipase activity assay was performed using p-NPP as the substrate, and this method was described by Winkler and Stuckmann in 1979.37 The reaction mixture consisted of 4 mL solutions (1 mL of Triton X-100, 37.8 mg of p-NPP in 100 mL of 50 mM Tris-HCl buffer, pH 8.0), dispersed for 5 min using an ultrasonic bath. The mixture was prewarmed to 37 °C, and then 120 µL of enzyme solution or immobilized PPL film was added. After 15 min of incubation, the reaction was stopped by the addition of 15 mL ethanol. The optical density of the solution was measured at 405 nm. One unit (U) of lipase activity was defined as the amount of enzyme that released 1 µmol of p-nitrophenol per min under the above conditions. The Esterification Reaction Catalyzed by the LDPE-g-P(PEGDA)/PPL Film. The catalytic performance of immobilized enzyme was evaluated by a commonly used esterification reaction to synthesize palmitolyglucose ester.38 Typically, glucose (0.1 mmol) and palmitic acid (0.4 mmol) were dissolved in 2 mL tert-butanol containing 1% (v/v) of H2O. Then a 2 cm × 2 cm


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o

LDPE-g-P(PEGDA)/PPL film and 0.107 g 3A molecular sieves were added into the reaction system. The esterification was performed in water bath with shaking at 50 °C for 24 hours. The reaction was monitored by TLC (1 : 8, MeOH/CHCl3). Upon the completion of the reaction, LDPE-g-P(PEGDA)/PPL film was taken out. The solvent was removed under reduced pressure and then the resulting residue was washed with H2O to remove the glucose. Finally, the precipitate was purified by silica gel chromatography with the mixture of methanol and chloroform (11/89, v/v) as eluant. The product was obtained as a white powder. Detailed spectroscopic data of palmitolyglucose ester were provided in Supporting Information. Instruments and Characterization. GBC Cintra 20 spectrophotometer (GBC Scientific Equipment) was used to measure the UV–Vis absorption spectra of films. The X-ray photoelectron spectra (XPS, ESCALAB 250 from Thermo Fisher Scientific Co.) were performed with monochromator. Atomic force microscopy (AFM, CP- Ⅱ from VEECO Co.) and the scanning electron microscopy (SEM, SD-7400 from HITACHI Co.) were used to determine the thickness of grafting layers. The patterned LDPE-g-P(PEGDA)/PPL structure with fluorescence was characterized by confocal laser scanning microscope (CLSM, FV1000 from OLYMPUS Co.). Dynamic light scattering (DLS, Zetaplus from BRUKER Co.) was used to detect the diameter of the enzyme clusters. The chemical structure of enzymatically prepared palmitolyglucose was determined by 13C NMR and 1H NMR (Bruker AVANCE Digital 600MHz Nuclear Magnetic Resonance Spectrometer, BRUKER Co.) using DMSO-d6 as solvent at 151 and 600 MHz, respectively. The FTIR spectra of the product were recorded on a Nicolet


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NEXUS 670 Fourier-Transform Infrared Spectrometer (Thermo Nicolet Co., USA). The mass spectrometry (MS, UPLC/Premier from WATERS Co.) was used to detect the mass of palmitolyglucose. RESULTS AND DISCUSSION Fabrication of LDPE-g-P(PEGDA)/PPL Film. The process of net-immobilization of PPL into P(PEGDA) grafted onto low density polyethylene (LDPE) films was summarized in Scheme 1. The ITX was first coupled to LDPE surface under UV irradiation by abstracting hydrogen and coupling reaction (Scheme 1a). The formed ITXSP dormant groups could be photolyzed under visible light and generated surface carbon radical and ITXSP radical. The surface radical could initiate graft polymerization of PEGDA, while ITXSP radical was relatively stable and could not initiate polymerization but mediate the polymerization by reversible deactivation of propagating radicals. Compared with grafting polymerization of single-functional monomer, the surface grafting polymerization of bifunctional monomer like PEGDA could form hydrogel networks with storage function instead of polymer brush. High content of the ether bonds in the PEGDA chains made the hydrogel network hydrophilic and biocompatible, which was natively suitable as support for enzyme immobilization. When free lipases from porcine pancreas (PPL) were added to monomer solution of PEGDA, they could be in situ encapsulated into the newly formed hydrogel network during graft cross-linking polymerization. We designed two routes to prepare LDPE-g-P(PEGDA)/PPL. In Scheme 1b, PEGDA/PPL solutions were directly cast onto a LDPE-ITXSP film, and this film was then sandwiched between two quartz plates to spread out


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the PEGDA/PPL solutions. This sandwich structure could obtain a relatively thin cross-linked layer. While in Scheme 1c, a silicone groove was used to accommodate more PEGDA/PPL solutions which could prepare thicker cross-linked layer. After the grafting reaction, LDPE-gP(PEGDA)/PPL could be obtained (Scheme 1d). As a proof of concept, the catalytic film was used to catalyze the esterification reaction to synthesis palmitolyglucose ester (Scheme 1e).

Scheme 1. Schematic illustration of immobilization processes of P(PEGDA)/PPL on LDPE films and a model reaction catalyzed by using this catalytic films. (a) ITXSP was coupled on LDPE surface via UV induced photo-reduction reaction. (b) Visible light induced surface graft crosslinking polymerization of PEGDA/PPL solutions with sandwich structure to obtain a thin hydrogel layer. (c) Visible light induced surface graft cross-linking polymerization of PEGDA/PPL solutions with groove structure to obtain a thicker hydrogel layer. (d) Structure


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illustration of LDPE-g-P(PEGDA)/PPL film. (e) Repeat use of the LDPE-g-P(PEGDA)/PPL film to synthesis palmitolyglucose ester. The preparation process of LDPE-g-P(PEGDA)/PPL was characterized and confirmed by UV spectroscopy (Figure S1). To further confirm the successful preparation of LDPE-gP(PEGDA)/PPL, XPS was used to determine the chemical composition of the modified LDPE surfaces at various stages. Figure 1 shows the C 1s and S 2p core-level spectra of blank LDPE, LDPE-ITXSP, and the C 1s and N 1s core-level spectra of LDPE-g-P(PEGDA) (LDPE grafted P(PEGDA) without PPL as control) and LDPE-g-P(PEGDA)/PPL. The C 1s core-level spectrum of LDPE-ITXSP films could be curved-fitted into three peak components with binding energies at 284.6 eV for the C−H and C−C species, 286.2 eV for the C−O/C−S species, and 288.7 eV for the O=C species.39,40 Affected by the machining process, C−O (due to the thermal oxidation) and C−Si (282.6 eV, due to the silicon release agent) species appeared in the blank LDPE. Compared with the pristine LDPE, S 2p signal at binding energy of about 168.5 eV, characteristic of covalently bonded sulfur, could be found in LDPE-ITXSP films. In addition, the oxygen-tocarbon signal ratio (O/C) also increased from 0.15 to 0.22 after photoreduction of ITX. All the evidence indicates that ITXSP was successfully introduced onto LDPE surfaces. The C 1s corelevel spectrum of LDPE-g-P(PEGDA) surfaces could be curved-fitted with three peak components (C−H and C−C species, C−O species, and

O=C−O species) related to the

P(PEGDA) structures, and the XPS analysis showed that the oxygen-to-carbon signal ratio (O/C) was 0.43, which was similar to the theoretical ratio of 0.46 calculated from pure P(PEGDA).


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Thus, the results demonstrated that P(PEGDA) hydrogel network has been successfully grafted onto LDPE film. It was difficult to distinguish LDPE-g-P(PEGDA) and LDPE-gP(PEGDA)/PPL by UV spectroscopy because the thick layer decreased the light transmission of the film, which results in the UV spectrum’s baseline of this film was too high to compare with other data in Fig. S1. On the other hand, although the films with thinner layers could be characterized by UV spectroscopy, the amount of PPL was too small to detect typical peak of protein. Alternatively, XPS provided a feasible method to verify them. The C 1s core-level spectra of the LDPE-g-P(PEGDA)/PPL surfaces possessed two new peaks at about 285.9 and 287.4 eV, attributing to the C−N and O=C−N species which were associated with the amino and peptide bonds in PPL. Additionally, a N 1s signal at binding energy of about 399.5 eV, characteristic of covalently bonded nitrogen, could be found in LDPE-g-P(PEGDA)/PPL surfaces. Whereas, no N 1s signal was detected in LDPE-g-P(PEGDA) surfaces. All the above results indicate that PPL have been successfully immobilized into P(PEGDA) hydrogel networks on LDPE films. Net-immobilization of PPL and Capacity Control. To effectively net-immobilize PPL, a chemical challenge is if this surface graft cross-linking polymerization could be carried out smoothly with the saturated PPL solution (8.6 mg/mL), and at last, in situ net-immobilized them in the PEG network. After polymerization all the PPLs non-immobilized were thoroughly collected, and it was found that the amount of non-immobilized cross-linked PPL was detected to be 6% after the grafting reaction.


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Figure 1. XPS C 1s and S 2p core-level spectra of the (a, b) blank LDPE, (c, d) LDPE-ITXSP, the C 1s and N 1s core-level spectra of (e, f) LDPE-g-P(PEGDA) and (g, h) LDPE-gP(PEGDA)/PPL. To investigate the distribution of PPL in P(PEGDA) hydrogel network, we prepared patterned LDPE-g-P(PEGDA)/PPL surface with column arrays, which were stained by rhodamine B through its electrostatic interaction with PPL. Figure 2 represents an example of 3D fluorescence images of these stained microarrays characterized by CLSM. It could be clearly observed that the patterned layer had a thickness of 68 µm. More importantly, due to the fact that rhodamine B can only interact with PPL, thus the column shape and uniform chromaticity of red fluorescence indicates that PPLs were embedded uniformly into the P(PEGDA) hydrogel network both in lateral and vertical direction.


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Figure 2. CLSM images of the patterned LDPE-g-P(PEGDA)/PPL dyed by Rhodamine B. Based on the results above, a plausible mechanistic interpretation of enzyme immobilization could be deduced as follows. According to our experimental setup, the LDPE-ITXSP was placed under the PEGDA/PPL solutions. When the polymerization was triggered by the visible light irradiation, the surface radical formed from photolyzed of ITXSP dormant groups initiated graft cross-linking polymerization of PEGDA from the bottom to up slowly. During this process, although PEG did not have specific interact with PPL, because of the gravity effect and the restriction of the reaction setup, PPL could be in situ encapsulated into the PEG-network formed gradually/uniformly. Thus when the hydrogel layer grew up to contact the quartz plate and reached its maximum thickness, most of enzymes should be encapsulated into the P(PEGDA) hydrogel network. Due to solubility limitation of PPL in reaction solution, to increase the thickness of P(PEGDA) layer is main method to accommodate more PPLs. Therefore, the thickness control


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of P(PEGDA)/PPL layers grafted on LDPE were investigated and the results were shown in Fig. 3. In Fig. 3a, by using the sandwich structure, the thicknesses ranging from 10-1 to 101 µm could be obtained via adjusting the grafting time. The linear increase of thickness with visible light irradiation time indicated that the growth of P(PEGDA) hydrogel network from LDPE-ITXSP film was consistent with a living and well-defined process, which confirmed that the visible induced graft cross-linking polymerization could successfully proceed in the presence of PPL. To overcome the limitation of feed volume of monomer/enzyme precursor solution in the sandwich structures, the groove structure was used to obtain thicker layers. In Fig. 3b, the feed volume was fixed to 200 µL and by changing concentration of PEGDA solutions from 20 to 50 wt%, the thickness of P(PEGDA)/PPL layer could be tuned from 10 µm to 180 µm (irradiation 180 mins). Alternatively, we also could prepare thicker P(PEGDA)/PPL layer by increasing the feed volume in the groove structure. Figure 3c showed that with increasing of feed volume from 300 µL to 1200 µL, the thickness of P(PEGDA)/PPL layer exhibited linear increase from 250 µm to about 1000 µm (irradiation 180 mins). These results show us that the thickness of P(PEGDA)/PPL layers can be precisely designed/controlled.


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Figure 3. Dependence of the thickness of P(PEGDA)/PPL layer on (a) visible light irradiation time via sandwich structures, (b) the concentration of PEGDA and (c) the feed volume via groove structures. Reaction conditions: (a) the feed volume was 40 µL, concentration of PEGDA was 30 wt%, PEGDA/(PPL solutions) = 3:7 (v/v),

(b) irradiation time was 180 min,

PEGDA/(PPL solutions) = 1:1 (v/v), water was added to fix the feed volume to 200 µL, (c) irradiation time was 180 min, concentration of PEGDA was 50 wt%, PEGDA/(PPL solutions) = 1:1 (v/v). The thickness of (a) were measured by sectional analysis of AFM of patterned layer, the thickness of (b) and (c) were measured by sectional analysis of SEM. Figure 4 shows the typical images of the LDPE-g-P(PEGDA)/PPL with thickness scale of (a) 10-1-100 µm, (b) 100-101 µm, (c) 101-102 µm and (d) 102-103 µm. To obtain precise results of thickness below 10 µm, the patterned LDPE-g-P(PEGDA)/PPL films were prepared by sandwich structure. Figure 4a and 4b show the AFM images of the patterned surface, and the column with height of 0.84 and 1.44 µm were obtained with different irradiation time (70 min and 110 min). Because AFM can’t measure the height higher than 10 µm, SEM measurement was used to observe the thicker layers. The thickness of pristine LDPE film is about 50 µm, and after grafting cross-linking polymerization through the groove structure, it could be observed that the grafted P(PEGDA)/PPL layers with thickness of 25 and 430 µm were well formed on LDPE surfaces (Figure 4c and 4d). The distinct interface contrast between LDPE and P(PEGDA)/PPL, dense structure and uniform thickness of the P(PEGDA)/PPL layer verified the controllable nature of this strategy again.


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Figure 4. Typical AFM images for patterned LDPE-g-P(PEGDA)/PPL with thickness scale of (a) 10-1-100 µm (b) 100-101 µm and SEM images of the cross-sectional views of LDPE-gP(PEGDA)/PPL with thickness scale of (c) 101-102 µm (d) 102-103 µm. Retention Behavior of Immobilized PPLs and Activity Analysis. For a system encapsulating enzyme in PEG network, when it was used to catalyze reaction, to keep the enzymes into the network is very important during whole reaction process. With this aim, we firstly observed the release behavior of PPL from LDPE-g-P(PEGDA)/PPL film where it was incubated in PBS solution. As shown in Fig. 5a, a burst release occurred during the first 10 h and reached about 20% leaking ratio. After 72 h, the ratio reached about 29%. This considerable leaching of PPL should be attributed to the smaller size of PPL than mesh size of the hydrogel network. Based on the molecular weight of PEGDA 575, the ideal average mesh size of P(PEGDA) hydrogel network should be approximately 3 × 3 × 3 nm3, while the size of strip-like


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molecules of PPL is 4.6 × 2.6 × 1.1 nm3 as reported,41 and so a fair amount of these elongated PPL molecules were easily to escape from the hydrogel networks.

Figure 5. Release behavior of (a) free PPL immobilized into P(PEGDA) hydrogels and (b) cross-linked PPL (0.5% (v/v) of glutaraldehyde) immobilized into P(PEGDA) hydrogels. The thickness of grafting layer was 480 µm, and 2.5 mg PPL was immobilized on the film. Following this understanding and referring routine method for increasing size of enzymes, we used glutaraldehyde to make PPL moderately cross-link. When we used 0.5% (v/v) glutaraldehyde to cross-link PPL, the average diameter of the PPL clusters were 15.1 nm (detected by DLS), which means about 5 to 10 enzymes were cross-linked together. By the same grafting procedure, we found that the about 95 % of the cross-linked PPL could be netimmobilized in the PEG network. Then we measured its leaking behavior and the results are shown in Fig. 5b. It was observed that the burst release of PPL have been greatly suppressed after 48 h and finally only 11% of PPL leaked out after 72 h. These indicate that moderately cross-linking of PPL was effective to ensure retention of enzyme in network.


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The activities of PPL before and after immobilization were also investigated using Winkler and Stuckmann’s method.37 The activities of free and immobilized PPL were found to be 1.23 and 0.94 units, respectively. The activities of free and immobilized cross-linked PPL (0.5% (v/v) of glutaraldehyde) were found to be 1.00 and 0.71 units, respectively. Therefore, minus the nonimmobilized enzyme after polymerization, about 21% of PPL deactivated when free PPL was in situ immobilized, and about 24% of PPL deactivated after adding 0.5% (v/v) of glutaraldehyde. It should be noted that there may be some PPL bonded to the hydrogel network covalently via Michael addition between lysine or cysteine residues and acrylate groups of PEGDA, which may account for the deactivation of immobilized PPL. The behavior of PPL which immobilized on LDPE film is typical of the Ping Pong Bi-Bi mechanism in which only one substrate is bound to the enzyme at any time to form the substrate-enzyme complex.42 The kinetic parameters of the esterification were provided in Supporting Information. Besides, to demonstrate the universality of this strategy, other kinds of enzymes were also immobilized and their activities before and after immobilization were investigated in Supporting Information. Effect of Light Source on Activity of PPLs in Various Solvent. The activity of enzymes immobilized by in situ encapsulation technique is strongly depend on the reaction conditions since either high temperature or high energy irradiation can deactivate enzymes. In order to reflect the superiority of visible light as light source, we prepared two samples with UV as irradiation source (150s and 300s irradiation time), and compared their activity in four different solvents. As shown in Fig. 6, we found that the net-immobilized PPLs samples by both UV and


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visible light irradiated showed no catalytic activity when water or hexane was used as solvent. While in tert-butanol or acetone, the samples fabricated by visible light (irradiation 120 mins) exhibited almost 5 times higher activity than those underwent 150 s of UV irradiation. And under UV light almost no activity was detected after irradiation for 300 s in these four solvents. Base on these results, it is safe to conclude that this strategy provide an extremely friendly reaction condition for in situ enzyme immobilization. In addition, it should be noted that the immobilized PPLs showed higher catalytic activity in tert-butanol compared to others, which was the results of combined influence by substrate solubility and solvent polarity. According to literature, the solvent with strong polarity can rapidly deactivate the lipase but too weak polarity will lead to lower substrate solubility.43 Therefore, based on the above factors, tert-butanol was the optimal solvent for this reaction.

Figure 6. Effect of light source on activity of PPLs in various solvent. The esterification was performed in water bath with shaking at 50 °C for 24 hours. The concentration of molecular sieves was 53.3 g/L. All the films had same amount of immobilized PPL.


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The Catalytic Properties of LDPE-g-P(PEGDA)/PPL Films. The catalytic properties of LDPE-g-P(PEGDA)/PPL films have been studied with a model reaction using glucose and o

palmitic acid to synthesize palmitolyglucose. In this typical reaction, 3A molecular sieves were added to control the water content of this system. Various factors including the effect of specific surface area, temperature, hydrogel layer thickness and operational stability were systemically investigated to evaluate the catalytic activity of film. The detailed discussion about the effect of diffusion (Figure S2 and S3) and temperature (Figure S4) on catalytic properties of LDPE-gP(PEGDA)/PPL films were listed in Supporting Information.

The relationship between thickness of the hydrogel layer and esterification rate of palmitolyglucose was studied and results were listed in Table 1. At the same reaction condition, the esterification rate was proportional to the thickness up to 250 µm, showing that this esterification system was well catalyzed by LDPE-g-P(PEGDA)/PPL. When we further increased the thickness of the hydrogel layer, the esterification rate stopped increasing and finally stabilized around 60%. Thus, to the system in our work, the optimum thickeness of grafting layer was about 250 µm.

Table 1. Effect of the thickness of grafting layers on esterification rate. Thickness of P(PEGDA)/PPL (µm) 1.4 10

Amounts of PPL added in the grafting reaction (mg) 0.24 0.34


Esterifation rate (%) 0.94 2.84 23

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40 0.52 100 0.69 180 0.86 250 1.29 350 1.62 400 2.15 The esterification was performed in water bath with hours at 50 °C, using tert-butanol as the solvent.

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20.79 38.25 51.41 62.84 58.97 60.99 shaking for 24

The ultimate aim of immobilizing enzymes is to increase their operational stability to make them reuse for long time, and thus make the bioconversion process economically feasible. To further characterize its operational stability, the reuse of LDPE-g-P(PEGDA)/PPL was studied (Figure 7). Films immobilized free PPL showed better catalytic ability in first batch, but esterifications rate decreased after cycles of 24 hours usage and only 18% esterification rate remained after 7 batches. To improve the operational stability of PPL immobilized film, 1% (v/v) of glutaraldehyde was used to crosslink PPL thus increase size of enzyme. The esterification rate in the first cycle was 47%, and it was still above 45% after 7 cycles. The operational stability improved apparently by embedding cross-linked PPL into hydrogel networks, indicating a larger size of enzyme leading to a good retention of them in P(PEGDA) hydrogel network. However, it is certain that glutaraldehyde has biotoxicity which result in the partly deactivation of PPL. On the premise of the operational stability of immobilized PPL, further reducing dose of glutaraldehyde was necessary to improve the activity retention of enzyme. When the concentration of glutaraldehyde was 0.5%, the esterification rate increased to 52% and didn’t show any decrease after 7 cycles of usage. However, continuing to lower the concentration to 0.05%, we found that


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the esterification rate fell after 3 cycles. Considering of the ratio of PPL immobilized on films, 0.5% (v/v) of glutaraldehyde is best for our system.

Figure 7. Operational stability of LDPE-g-P(PEGDA)/PPL films in the esterification of glucose with palmitic acid in tert-butanol. The esterification was performed in water bath with shaking at 50 °C for 24 h. One cycle reaction corresponds to 24 h. PPL were cross-linked with different concentration of glutaraldehyde (0-1%, v/v). CONCLUSION A new protocol to in situ net-immobilize PPL into PEG hydrogel network grafted on polymeric substrate by visible light induced surface controlled graft cross-linking polymerization method was developed. Due to the favorable reaction condition of visible light irradiation and room temperature, most of entrapped PPL could retain their activity after graft cross-linking polymerization while similar reaction under 300 s of UV irradiation resulted in completely deactivation of PPL. The controlled feature of this method provide a effective way to uniformly encapsulating enzymes and regulate the thickness of graft layer in range of 10-1 to 103 µm, which


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could finally determine the storage capacity of hydrogel layer for enzyme. The catalytic property of LDPE-g-P(PEGDA)/PPL showed that immobilized PPL exhibit improved stability at 50 °C than free enzyme. Proper cross-linking of PPL eliminated leaking of PPL and allowed repeat used of this enzyme immobilized film. This facile and effective enzyme immobilization strategy will establish a powerful platform for potential biomedical and industrial applications. Supporting Information. Effect of water contents, reaction time, diffusion and temperature on esterification. UV−visible spectra of the films. Structure characterization of palmitolyglucose ester. The mechanism of the esterification. Application to other Enzymes. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author E-mail: [email protected] (W. Y.) E-mail: [email protected] (C. Z.) Notes The authors declare no competing financial interest. ACKNOWLEDGMENT: This work was financially supported by the National Natural Science Foundation of China (Grant No.51033001, 51221002, 51103009, 51473015), National High Technology Research and Development Program (863 Program 2009AA03Z325) and the Fundamental Research Funds for the Central Universities (ZY1311). REFERENCES


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(1)

Dyal, A.; Loos, K.; Noto, M.; Chang, S. W.; Spagnoli, C.; Shafi, K. V. P. M.; Ulman, A.;

Cowman, M.; Gross, R. A. Activity of Candida rugosa Lipase Immobilized on γ-Fe2O3 Magnetic Nanoparticles. J. Am. Chem. Soc. 2003, 125, 1684-1685. (2)

Kumar, C. V.; Chaudhari, A. Proteins Immobilized at the Galleries of Layered α-

Zirconium Phosphate: Structure and Activity Studies. J. Am. Chem. Soc. 2000, 122, 830-837. (3)

Libertino, S.; Giannazzo, F.; Aiello, V.; Scandurra, A.; Sinatra, F.; Renis, M.; Fichera, M.

XPS and AFM Characterization of the Enzyme Glucose Oxidase Immobilized on SiO2 Surfaces. Langmuir 2008, 24, 1965-1972. (4)

Dai, X.; Knupp, S. A.; Xu, Q. Patterning Nanoparticles in a Three-Dimensional Matrix

Using an Electric-Field-Assisted Gel Transferring Technique. Langmuir 2012, 28, 2960-2964. (5)

Tibbitt, M. W.; Anseth, K. S. Hydrogels as Extracellular Matrix Mimics for 3D Cell

Culture. Biotechnol. Bioeng. 2009, 103, 655-663. (6)

Parascandola, P.; Branduardi, P.; de Alteriis, E. PVA-gel as an Effective Matrix for Yeast

Strain Immobilization Aimed at Heterologous Protein Production. Enzyme Microb. Technol. 2006, 38, 184-189. (7)

Wilson, L.; Illanes, A.; Pessela, B. C. C.; Abian, O.; Fernandez-Lafuente, R.; Guisan, J.

M. Encapsulation of Crosslinked Penicillin Gacylase Aggregates in Lentikats: Evaluation of a Novel Biocatalyst in Organic Media. Biotechnol. Bioeng. 2004, 86, 558-562. (8)

Fejerskov, B.; Smith, A. A. A.; Jensen, B. E. B.; Hussmann, T.; Zelikin, A. N.,

Bioresorbable Surface-Adhered Enzymatic Microreactors Based on Physical Hydrogels of Poly(vinyl alcohol). Langmuir 2012, 29, 344-354. (9)

Murayama, S.; Ishizuka, F.; Takagi, K.; Inoda, H.; Sano, A.; Santa, T.; Kato, M. Small

Mesh Size Hydrogel for Functional Photocontrol of Encapsulated Enzymes and Small Probe Molecules. Anal. Chem. 2011, 84, 1374-1379. (10)

Schachschal, S.; Adler, H. J.; Pich, A.; Wetzel, S.; Matura, A.; Pee, K.-H. Encapsulation

of Enzymes in Microgels by Polymerization/Cross-linking in Aqueous Droplets. Colloid Polym Sci 2011, 289, 693-698. (11)

Kiyonaka, S.; Sada, K.; Yoshimura, I.; Shinkai, S.; Kato, N.; Hamachi, I. Semi-wet

Peptide/Protein Array Using Supramolecular Hydrogel. Nat Mater 2004, 3, 58-64. (12)

Heo, J.; Crooks, R. M. Microfluidic Biosensor Based on an Array of Hydrogel-Entrapped

Enzymes. Anal.Chem 2005, 77, 6843-6851. (13)

Yuan, W.; Yang, J.; Kopečková, P.; Kopeček, J. I. Smart Hydrogels Containing

Adenylate Kinase: Translating Substrate Recognition into Macroscopic Motion. J. Am. Chem. Soc. 2008, 130, 15760-15761.


27

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(14)

Page 28 of 31

Li, W.; An, H.; Tan, Y.; Lu, C.; Liu, C.; Li, P.; Xu, K.; Wang, P., Hydrophobically

Associated Hydrogels Based on Acrylamide and Anionic Surface Active Monomer with High Mechanical Strength. Soft Matter, 2012, 8, 5078-5086. (15)

Jiang, G.; Liu, C.; Liu, X.; Chen, Q.; Zhang, G.; Yang, M.; Liu, F. Network Structure and

Compositional Effects on Tensile Mechanical Properties of Hydrophobic Association Hydrogels with High Mechanical Strength. Polymer 2010, 51, 1507-1515. (16)

Hoffman, A. S. Hydrogels for Biomedical Applications. Adv. Drug Del. Rev. 2002, 54, 3-

12. (17)

Tanaka, Y.; Gong, J. P.; Osada, Y. Novel Hydrogels with Excellent Mechanical

Performance. Prog. Polym. Sci. 2005, 30, 1-9. (18)

Karlsson, J. O.; Gatenholm, P. Surface Mobility of Grafted Hydrogels. Macromolecules

1999, 32, 7594-7598. (19)

Lopez, J. L.; Matson, S. L., A Multiphase/Extractive Enzyme Membrane Reactor for

Production of Diltiazem Chiral Intermediate. J. Membrane. Sci. 1997, 125, 189-211. (20)

Batich, C.; Yahiaoui, A. Surface modification. I. Graft Polymerization of Acrylamide

onto Low-density polyethylene by Ce4+-induced Initiation. J. Polym. Sci., Part A: Polym. Chem. 1987, 25, 3479-3488. (21)

Lin Chut, S.; Li, J. Reagentless Amperometric Determination of Hydrogen Peroxide by

Silica Sol-Gel Modified Biosensor. Analyst 1997, 122, 1431-1434. (22)

Andreopoulos, F. M.; Roberts, M. J.; Bentley, M, D.; Harris, J. M.; Beckman, E. J.;

Russell, A. J. Photoimmobilization of Organophosphorus Hydrolase within a PEG-based Hydrogel. Biotechnol. Bioeng. 1999, 65, 579-588. (23)

Ahern, T.; Klibanov, A. The Mechanism of Irreversible Enzyme Inactivation at

100.degree. C. Science 1985, 228, 1280-1284. (24)

Macomber, L.; Imlay, J. A. The Iron-sulfur Clusters of Dehydratases are Primary

Intracellular Targets of Copper Toxicity. Proc. Nat. Acad. Sci. 2009, 106, 8344-8349. (25)

Falguera, V.; Pagán, J.; Ibarz, A. Effect of UV Irradiation on Enzymatic Activities and

Physicochemical Properties of Apple Juices from Different Varieties. LWT-Food Sci Technol. 2011, 44, 115-119. (26)

Ejaz, M.; Yamamoto, S.; Ohno, K.; Tsujii, Y.; Fukuda, T., Controlled Graft

Polymerization of Methyl Methacrylate on Silicon Substrate by the Combined Use of the Langmuir−Blodgett and Atom Transfer Radical Polymerization Techniques. Macromolecules 1998, 31, 5934-5936.


28

Page 29 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(27)

Baum, M.; Brittain, W. J., Synthesis of Polymer Brushes on Silicate Substrates via

Reversible Addition Fragmentation Chain Transfer Technique. Macromolecules 2002, 35, 610615. (28)

Husseman, M.; Malmström, E. E.; McNamara, M.; Mate, M.; Mecerreyes, D.; Benoit, D.

G.; Hedrick, J. L.; Mansky, P.; Huang, E.; Russell, T. P.; Hawker, C. J., Controlled Synthesis of Polymer Brushes by “Living” Free Radical Polymerization Techniques. Macromolecules 1999, 32, 1424-1431. (29)

Matsuda, T.; Ohya, S., Photoiniferter-Based Thermoresponsive Graft Architecture with

Albumin Covalently Fixed at Growing Graft Chain End. Langmuir 2005, 21, 9660-9665. (30)

Barbey, R.; Lavanant, L.; Paripovic, D.; Schüwer, N.; Sugnaux, C.; Tugulu, S.; Klok, H.-

A., Polymer Brushes via Surface-Initiated Controlled Radical Polymerization: Synthesis, Characterization, Properties, and Applications. Chem. Rev. 2009, 109, 5437-5527. (31)

Kim, H. R.; Kim, I. H.; Hou, C. T.; Kwon, K. I.; Shin, B. S., Production of a Novel Cold-

Active Lipase from Pichia lynferdii Y-7723. J. Agric. Food. Chem. 2009, 58, 1322-1326. (32)

Yang, W. T.; Rånby, B. Radical Living Graft Polymerization on the Surface of Polymeric

Materials. Macromolecules 1996, 29, 3308-3310. (33)

Elliott, J. E.; Macdonald, M.; Nie, J.; Bowman, C. N. Structure and Swelling of

Poly(acrylic acid) Hydrogels: Effect of pH, Ionic Strength, and Dilution on the Crosslinked Polymer Structure. Polymer 2004, 45, 1503-1510. (34)

Bai, H. D.; Huang, Z. H.; Yang, W. T. Visible Light-induced Living Surface Grafting

Polymerization for the Potential Biological Applications. J. Polym. Sci., Part A: Polym. Chem. 2009, 47, 6852-6862. (35)

Li, C.; Glidle, A.; Yuan, X.; Hu, Z.; Pulleine, E.; Cooper, J.; Yang, W.; Yin, H. Creating

“Living” Polymer Surfaces to Pattern Biomolecules and Cells on Common Plastics. Biomacromolecules 2013, 14, 1278-1286. (36)

Bradford, M. M. A Rapid and Sensitive Method for the Quantitation of Microgram

Quantities of Protein Utilizing the Principle of Protein-dye Binding. Anal. Biochem. 1976, 72, 248-254. (37)

Winkler, U. K.; M. Stuckmann, M. Glycogen, Hyaluronate, and Some Other

Polysaccharides Greatly Enhance the Formation of Exolipase by Serratia Marcescens. J. Bacteriol. 1979, 138, 663-670.


29

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(38)

Page 30 of 31

Gumel, A. M.; Annuar, M. S. M.; Heidelberg, T.; Chisti, Y. Lipase Mediated Synthesis

of Sugar Fatty Acid Esters. Proc Biochem 2011, 46, 2079-2090. (39)

Li, C. Y.; Xu, F. J.; Yang, W. T. Simple Strategy to Functionalize Polymeric Substrates

via Surface-Initiated ATRP for Biomedical Applications. Langmuir 2012, 29, 1541-1550. (40)

Xu, F. J.; Kang, E. T.; Neoh, K. G. pH- and Temperature-responsive Hydrogels from

Crosslinked Triblock Copolymers Prepared via Consecutive Atom Transfer Radical Polymerizations. Biomaterials 2006, 27, 2787-2797. (41)

Li, Y.; Zhou, G.; Li, C.; Qin, D.; Qiao, W.; Chu, B. Adsorption and Catalytic Activity of

Porcine Pancreatic Lipase on Rod-like SBA-15 Mesoporous Material. Colloids Surf., A 2009, 341, 79-85. (42)

Paiva, A. L.; Balcão, V. M.; Malcata, F. X., Kinetics and Mechanisms of Reactions

Catalyzed by Immobilized Lipases☆. Enzyme Microb. Technol. 2000, 27, 187-204. (43)

Chang, S. W.; Shaw, J. F. Biocatalysis for the Production of Carbohydrate Esters. New

Biotechnol. 2009, 26, 109-116


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A Mild Strategy To Encapsulate Enzyme into Hydrogel Layer Grafted

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