Separated Immobilization of Incompatible Enzymes on Polymer

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Separated Immobilization of Incompatible Enzymes on Polymer Substrate via Visible Light Induced Living Photografting Polymerization Xing Zhu, Bin He, Changwen Zhao, Yuhong Ma, and Wantai Yang Langmuir, Just Accepted Manuscript • Publication Date (Web): 17 May 2017 Downloaded from http://pubs.acs.org on May 20, 2017

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Separated Immobilization of Incompatible Enzymes on Polymer Substrate via Visible Light Induced Living Photografting Polymerization Xing Zhu, †, § Bin He, † Changwen Zhao,*, † Yuhong Ma,‡ Wantai Yang*, † †

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

Materials, Beijing University of Chemical Technology (BUCT), P.O. Box 37, Beijing 100029, China ‡

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

University of Chemical Technology (BUCT), P.O. Box 37, Beijing 100029, China KEYWORDS graft polymerization, enzyme immobilization, anti-tumor drug

ABSTRACT The use of the mixed catalytic system with several enzymes can provide multiple benefits in terms of the costs, simplification of a multi-steps reaction, and the effectiveness of the complex chemical reactions. Although study of different enzymes co-immobilization system has attracted increasing attention in recent years, separately immobilizing enzymes which can not coexist on one support is still one of the great challenges. In this paper, a simple and effective strategy was introduced to separately encapsulate incompatible trypsin and transglutaminase (TGase) into different poly(ethylene glycol) (PEG) network layer grafted on low-density polyethylene (LDPE) film via visible light induced living photografting polymerization. As a proof of concept, this dual-enzymes separately loaded film was used to catalyze the synthesis of

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a new target anti-tumor drug LTV-azacytidine. The final results demonstrated that this strategy could maintain higher activities of both enzymes than the mixed co-immobilization method. And the mass spectra analysis results demonstrated that LTV-azacytidine was successfully synthesized. We believe that this facile and mild separately immobilizing incompatible enzymes strategy has a great application potential in the field of biocatalysis.

Introduction Compared with the relatively mature methods for single enzyme immobilization, immobilization of different kinds of enzymes on one substrate is more complicated and has aroused considerable interests.1-4 The simplest method to immobilize various enzymes was to mix these enzymes and fix them like a single enzyme system, which required that enzymes can work well together, or at least do not affect each other.5-8 Recently, inspired by natural metabolic pathways that catalyzed by spatially organized multi-enzymes in cells, tremendous efforts have been made to rationally design and construct separately co-localized multiple enzymes systems.912

Many substrates such as inorganic particles, protein, nucleic acid and polymer materials have

been utilized to separately position multiple enzymes to enhance the efficiency of a cascade reaction.13-15 For example, Jiang et al.16 developed a new strategy to separately immobilized three enzymes on the hybrid microcapsules to perform the cascade reaction of converting CO2 to methanol. Compared with the free enzymes’ system, the immobilized enzymes’ system showed higher activity and selectivity. Moreover, the yield of methanol was still more than 50% after 9 cycles of reusing or storage for 20 days. Palazzo et al.17 immobilized three glycol-enzymes (trehalase, glucose oxidase (GOD) and horseradish peroxidase (HRP)) via a “layer-by-layer” deposition strategy. The driving force of this assembly is the bio-specific interactions between


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sugar residues in the enzymes and Con A. After the assembly, this catalytic system has been used for the optical determination of trehalose. The final results showed that the content of trehalose in eyewash was successfully determined and all the enzymes still remained more than 50% of their activities after 11 weeks’ storage. Gustafsson et al.18 co-immobilized GOD and HRP on silica supports in a separately way by using mesoporous silica nanoparticles and denpol. The silica support was firstly coated with the dendronized polymer denpol, and then GOD was adsorbed on the silica nanoparticles. Finally, the nanoparticles loaded with GOD were coated with a denpol–HRP conjugate (HRP were covalently bound to the denpol via bis-aryl hydrazone bonds). The results demonstrated that this catalytic system remained stable for at least 2 weeks if stored in the aqueous solution of pH = 7 at 4 °C and it may find applications in bio-electrode fabrications. Despite the significant progresses have been achieved, there are still some limitations that have not been solved well, e.g., the weakness force to bound the adsorbed enzymes, the complicated operation process and the relatively low enzyme loading capacity, etc. Moreover, designing robust and easy-to-use positional immobilization techniques to preserve enzymatic activity and high catalytic efficiency is still a challenging.


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Scheme 1. Schematic route of (a) introducing ITX semipinacol (ITXSP) on LDPE film via UVinduced photo-reduction reaction; (b) preparation of patterned trypsin loaded film via visible light induced living photografting polymerization; (c) preparation of patterned TGase-trypsin loaded film (Type A) via visible light induced living photografting polymerization and (d) synthesizing LTV-azacytidine.

In our previous work, a facile strategy was developed to immobilize enzymes on polymer substrate by visible light-induced surface initiated living graft crosslinking polymerization.19,20 In that strategy, isopropyl thioxanthone (ITX) was selected as the photo-initiator and the lowdensity polyethylene film (LDPE) was used as the substrate. This technique composed of two chemical steps. Firstly, isopropyl thioxanthone semipinacol (ITXSP) dormant groups were planted on the LDPE surface through a UV induced abstracting hydrogen-coupling reaction (Scheme 1a).21 Then under visible light irradiation, the ITXSP dormant groups generated surface radicals to initiate graft polymerization of poly(ethylene glycol) diacrylate (PEGDA) to form a


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densely cross-linked layer we called PEG molecular net-cloth. When free enzymes were added to the monomer solution, they could be in situ encapsulated into the newly formed network during the graft polymerization without losing their activities. Following this strategy, HRP and GOD have been successfully co-immobilized into the PEG molecular net-cloth grafted on LDPE film and showed higher activities than enzymes immobilized under UV irradiation20. But this strategy is only suitable for the co-immobilization of enzymes which can work well together, or at least do not interfere with each other. When enzymes from different sources are incompatible and cannot be mixed together, it is not appropriate to immobilize them in one matrix. Based on this consideration, we further developed this strategy to separately immobilize incompatible enzymes on one polymeric substrate to avoid interference between them. The key point of the new strategy is the “living” characteristic of this graft polymerization, which allows sequential graft polymerization on one substrate. Therefore, there were still plenty of ITXSP dormant groups on the surface of first PEG molecular net-cloth. When another polymerization was performed, a new PEG molecular net-cloth layer can be grafted onto the former layers. Thus different kinds of enzymes can be encapsulated into different layers, in which they did not affect with each other but they can catalyze one multi-steps reaction together. Compared with other co-immobilization methods, this design has the following advantages: 1) unlike UV induced photografting, the irradiation of visible light is mild enough to retain the activities of enzymes; 2) the PEG molecular net-cloth we used here has the non-swelling feature in aqueous phase which can effectively prevent the leakage of enzymes;19 3) different kinds of enzymes can be separately immobilized with different PEG layers without interactive interference, and we have demonstrated that three or more layer can be fabricated on one substrate, which is suitable for system containing more than two enzymes; 4) grafting polymer


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networks onto the flexible polymeric substrate can improve their mechanical strength, thus to avoid the leaching of enzymes and increase their operational stability;20 5) compared with other carriers such as protein or nucleic acid, the cost of the substrate (LDPE) in this work is very low; 6) this strategy is available for any surface containing C-H bonds, which may contribute to the application in the future.22 To demonstrate the superiority of this strategy, transglutaminase from guinea pig liver (TGase, EC 2.3.2.13) and trypsin from bovine pancreas (EC 3.4.21.4) were selected as model enzymes to fabricate the incompatible enzymes loaded film via visible light induced living photografting polymerization. TGase is a family of enzymes which can crosslink proteins by forming Nε-(γ-glutamyl) lysine bonds or incorporate primary amines into the protein substrate which contains γ-glutaminyl residues.23-25 Trypsin catalyzes preferentially the hydrolysis of the peptide bond at the carboxyl end of arginine or lysine residues in polypeptides.26 When TGase and trypsin coexist, TGase may modify the lysine or glutamine in trypsin, and trypsin may hydrolyze peptides in TGase.27 Then both of the enzymes’ activities may decrease. Hence, separately immobilizing them on one substrate can keep both of their activities and make them effectively catalyze a multi-steps reaction. The separate immobilization of TGase and trypsin on LDPE film is illustrated in Scheme 1b and 1c. Herein, this incompatible enzymes immobilization system was modified by using a photomask for ease of characterization. In Scheme 1b, PEGDA/trypsin solutions were cast onto the LDPE film with ITX semipinacol groups (LDPEITXSP) and then a strip photomask was put onto the film to spread out the PEGDA/trypsin solutions. During the grafting reaction under the visible light, only the region which visible light can pass through was initiated for crosslinking polymerization and trypsin can be gradually encapsulated into this strip PEG molecular net-cloth on LDPE surface to form the trypsin loaded


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film. In Scheme 1c, PEGDA/TGase solutions were cast onto the trypsin loaded film and then the same photomask was put onto the film to spread out the PEGDA/TGase solutions (the strip patterned regions obtained were perpendicular to the former shape by rotating the photomask 90 degrees). Because the small mesh of PEG molecular net-cloth size (3.0 × 3.0 × 3.0 nm3) and relative large size of TGase (76.6 kDa) and trypsin (24 kDa, 3.8 × 3.8 × 3.8 nm3),28 TGase is not able to diffuse into first PEG pattern layer and trypsin also cannot leak out from it. Thus at this time they could not interfere each other. After the second grafting reaction under the visible light, TGase was entrapped into the newly formed strip PEG network pattern and the TGasetrypsin loaded film (Type A) was finally prepared. To prove the feasibility of this work, the TGase-trypsin loaded film was then used to catalyze the synthesis of a new target anti-tumor drug LTV-azacytidine. As shown in Scheme 1d, the chemical structure of LTV-azacytidine can be divided into two parts: the prodrug and the targeted carrier (LTVSPWYGTGTQGTGR peptide). The prodrug is a derivative of 5azacytidine, which is usually employed for the treatment of breast cancer. However, this drug has some disadvantages in clinical application, such as leucopenia or liver damage.29 Therefore, many efforts have been made to find a way to deliver these chemotherapy agents directly to tumor cells while sparing the other cells in the body.30-32 One profitable way to reduce the side effect of 5-azacytidine is to conjugate the drug with the targeted moiety. Recently, the researches of A. Florczak et al.33 and E. Orban et al.34 demonstrated that the peptide containing LTVSPWY sequence was an effective targeting vehicle that can specifically bind to breast cancer cells but not interact with human primary cells from different tissue. Based on this result, we designed a new anti-tumor targeted drug LTV-azacytidine for the potential treatment of breast cancer.


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To

incorporate

the

target

sequence

(LTVSPWY)

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with

the

prodrug,

a

LTVSPWYGTGTQGTGRGTGDDR peptide (LTV-peptide) was designed as the precursor. LTV-peptide is composed of a sequence of targeted region LTVSPWY, one trypsin cleavable site R, one TGase connecting site Q, three spacer regions GTG and one hydrophilic region DDR. Here we chose TGase to site selectively conjugate the primary amine in prodrug with the γglutaminyl residue in LTV-peptide. The hydrophilic DDR sequence was used to promote the solubility of the peptide and decrease the cost of the peptide production. After the LTV-peptide dissolving into aqueous medium, it is necessary to cut off this sequence to increase the proportion of effective drugs in unit mass. So in this work, trypsin was used to cut off the DDR peptide at the cleavable site R, resulting in the release of the hydrophilic residues. As shown in Scheme 1d, the conjugation of the prodrug with LTV-peptide and the removal of the DDR region can be achieved simultaneously by the separately immobilized TGase and trypsin. To the best of our knowledge, this is the first time to provide a method to separately immobilize different kinds of enzymes using the living graft polymerization strategy, and the first time to synthesize LTV-azacytidine which is a potential target drug in cancer chemotherapy. Results and Discussion


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Figure 1. XPS C 1s and S 2p core-level spectra of the (a, b) LDPE-ITXSP and (c, d) LDPE-gP(PEGDA). “Living” Property of the Immobilization. One important consideration to ensure one layer can be grafted on the other layer is the “living” properties of visible light induced living photografting polymerization. To confirm this “living” property, XPS was used to determine the chemical composition of the modified LDPE films. Figure 1 shows the C 1s and S 2p core-level spectra of LDPE-ITXSP and LDPE-g-P(PEGDA). In Figure 1a, the C 1s core-level spectrum of LDPE-ITXSP film can be curve-fitted into three peaks (285.0 eV for the C−H/C−C species, 286.6 eV for the C−O/C−S species, and 288.8 eV for the C=O species),35, 36 In Figure 1b, S 2p signal at a binding energy of about 168.5 eV, characteristic of covalently bonded sulfur, indicated that ITXSP dormant groups were successfully introduced onto the LDPE surface. In Figure 1c, the remarkable growth of oxygen-to-carbon signal ratio (O/C) demonstrated that PEG


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network had been successfully grafted onto LDPE films. What’s more, In Figure 1d, S 2p signal can be detected on the surface of PEG layers, indicating that there still had ITXSP residues at the end of the PEG chains which can reflect the “living” property of the polymerization.

Figure 2. Typical AFM images for TGase-trypsin loaded film (a) after first grafting reaction, (b) after second grafting reaction. To obtain the thickness of the first and second layer of the TGase-trypsin loaded film, AFM measurements were conducted and the results are shown in Figure 2a and 2b. The first grafting layer (trypsin layer) with the height of 2.1 µm and the second grafting layer (TGase layer) with the height of 1.8 µm were obtained with the same irradiation time (30 min). Compared with the traditional layer-by-layer method, the grafted PEG layers in this work are much thicker, which can immobilize more enzymes onto the substrate. Moreover, Figure 2b clearly shows that one layer can be grafted onto the other layer which also demonstrates the “living” character of visible light induced living photografting polymerization.


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Figure 3. Optical microscope images of (a) patterned LDPE-g-P(PEGDA) and (b) TGase-trypsin loaded film after Coomassie brilliant blue stained. The Distribution of Enzymes in PEG Network. The immobilization ratio and the distribution of enzymes on LDPE film were researched, respectively. After the grafting reaction, this TGasetrypsin loaded film was immersed into the Tris-HCl buffer (pH=7.0) for 24 h. By using the Bradford method, we found that only 10% of the initial added enzymes were detected in the buffer, which means 90% of enzymes can be immobilized into the network successfully. To further investigate the distribution of the two enzymes in PEG network, film grafted P(PEGDA) without adding enzymes (Figure 3a) and TGase-trypsin loaded film (Figure 3b) were both stained by Coomassie brilliant blue. After washing with acetone and ethanol, these stained microarrays were characterized by optical microscope. Due to the fact that Coomassie brilliant blue can’t strongly interact with PEG networks shown in Figure 3a, the blue microarrays in Figure 3b indicated that both of the two enzymes were embedded into the PEG net-cloths uniformly.


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Figure 4. Activities of (a) free trypsin and trypsin immobilized in Type A (the structure of Type A can be seen in Scheme 1c) and Type B (TGase and trypsin were mixed together and immobilized in the same PEG net-cloth layer on LDPE-ITXSP film) method at various temperatures and (b) free TGase and TGase immobilized in Type A and Type B method at various temperatures.

The Activities of the Immobilized Enzymes. Figure 4a shows the activities of free trypsin and the activities of trypsin immobilized in Type A and Type B (TGase and trypsin mixed immobilization) at various temperatures. It can be observed that the activities of free trypsin and the immobilized trypsin in Type A group were 6102±353 and 5608±101 U/mg respectively at 20 °C. As the temperature increased to 40 °C, the activities of free trypsin and the immobilized trypsin in Type A group increased to 7514±857 and 6807±574 U/mg, respectively. However, when the temperature was further increased to 50 °C, the activity of the immobilized trypsin in Type A group was 4901±202 U/mg which was similar to the free trypsin group (4639±201 U/mg). Temperature significantly influenced the activities by affecting the stability of the trypsin


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and the kinetic of the reaction. When temperature was below 50 °C, it almost had no adverse effect on the stability of trypsin. Because the reaction was mainly dominated by the diffusion of substrates, it was reasonable that the activities of free trypsin were higher at temperature below 40 °C. At 50 °C, the stability of free trypsin was significantly affected by high temperature while it had little influence on the immobilized trypsin because their conformation could be stabilized by the PEG network.37 Although the diffusion rate of immobilized trypsin in Type A group was still lower than the free trypsin group, the structure of the trypsin in Type A group was more stable than the free trypsin group at 50 °C. Therefore, the immobilized trypsin showed similar activity to free trypsin at 50 °C. Trypsin activities in Type B group were little lower than the activities in Type A group. It could be due to that some of the lysine or glutamine in trypsin was modified by TGase which may partly deactivated the trypsin. Figure 4b shows the activities of free TGase and the activities of TGase immobilized in Type A and Type B method at various temperatures. Similar rules can be observed that the immobilized TGase showed similar activities to free TGase at 50 °C. However, TGase activities in Type B group were also lower than the activities in Type A group at any temperatures which could be due to the hydrolysis of TGase’s structure by trypsin. Overall, the optimum temperature of TGase-trypsin loaded film was between 30-40°C, so 35°C was selected as the operating temperature.


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Figure 5. Operational stability of (a) trypsin and (b) TGase for dual-enzymes loaded film (Type A) at 35 °C. Operational Stability of the TGase-Trypsin Loaded Film. The ultimate aim of immobilizing enzymes is to increase their operational stability to make them reuse for long time, and thus to make the bioconversion process economically feasible.38-44 To further characterize its operational stability, the reusability of dual-enzymes loaded film was studied at 35 °C. In Figure 5a, trypsin immobilized in net-cloth showed good catalytic ability in the first 5 batches, and there was still 49% of the initial activity remained after 10 batches. In Figure 5b, TGase immobilized in netcloth showed little decrease of activity in the first 4 batches. Overall, this dual-enzymes loaded film can be used for four cycles without obvious activity decrease.


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Figure 6. MALDI-TOF/MS of (a) LTV-peptide, (b) LTV-azacytidine and (c) product catalyzed by trypsin loaded film. Synthesis of LTV-Azacytidine. By using this TGase-trypsin loaded film, the targeted antitumor drug LTV-azacytidine was finally synthesized with the yield of 32%. In Figure 6a, the identity of the LTV-peptide was characterized by MALDI-TOF/MS spectrometry (calcd for C96H148N30O35, M=2282.1 Da; found, m/z 2282.1). In Figure 6b, the molecular weight of LTVazacytidine was detected, which met the calculated molecular weight (calcd for C88H133N25O30, M=2019.9 Da; found, m/z 2020.7 [M+H]+). To further demonstrate the specific functions of the enzymes, Fig. 6c shows the mass spectrometry of the product which was obtained by cleavage of GTGDDR moiety from LTV-peptide after digesting by trypsin loaded film. This analysis supported our expectation that trypsin can cut off the hydrophilic residue of the LTV-peptide (calcd for C74H113N21O24, M=1679.8 Da; found, m/z 1680.7 [M+H]+). All the results prove that LTV-azacytidine can be successfully synthesized by TGase-trypsin loaded film.


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Conclusion A new protocol was developed to separately encapsulate TGase and trypsin in PEG netcloths grafted on LDPE film by the visible light induced living photografting polymerization method. To demonstrate the potential application of this strategy, this dual-enzymes loaded film was used to catalyze the synthesis of a new target anti-tumor drug LTV-azacytidine. The results proved that more than 90 % of the two enzymes can be successfully encapsulated into PEG netcloth, and this catalytic film showed good operational stability at the first four batches. Compared with the two enzymes in the mixing co-immobilization strategy, both of them in the separately immobilization method showed higher activities. We believed that this novel incompatible enzymes immobilization strategy can establish a powerful platform for potential biomedical and industrial applications. And the final catalysate, LTV-azacytidine, might be developed as a potential agent in cancer chemotherapy. Experimental Section Introducing ITXSP Groups on LDPE Films (Scheme 1a). ITX can be planted on LDPE films through the sandwich structure following the reported method.19 Firstly, ITX solution (acetone as the solvent, 40 µL, 3 mmol mL-1) was dispersed onto the LDPE film and then the film was placed between two quartz plates. Secondly, this sandwich system was placed under the UV lamp (wavelength 254 nm, 9 mW cm-2) for 3 min at room temperature to obtain LDPE-ITXSP. After the reaction, the film was extracted with acetone for 24 h to remove the residual ITX. Preparation of TGase-Trypsin Loaded Film. Trypsin was added into 100 mM Tris-HCl (pH=7.0) by shaking to form a concentration of 11.5 mg mL-1 solution. Then 37.5 µL of trypsin solution was mixed with 22.5 µL PEGDA to form a precusor solution containing 7.19 mg mL-1


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trypsin with pH = 7.0. Then 60 µL of PEGDA/trypsin solution was cast onto a LDPE-ITXSP film and a strip-type photomask was put onto the film to spread out the PEGDA/trypsin solution and form a “sandwich” structure. This sandwich structure was irradiated with visible light (xenon lamp plus filter with a through light band of 380–700 nm, and a light intensity of 3 mW/cm2 at λ = 420 nm) for 30 min at room temperature. After the grafting reaction, the reaction film was immersed into 100 mM Tris-HCl buffer (pH=7.0) for 24 h and washed with Tris-HCl buffer alternately three times to remove the unimmobilized trypsin (Scheme 1b). And this trypsin immobilized film was denoted as LDPE-trypsin. To prepare the TGase-trypsin seperatedly loaded film (Type A), TGase was added into 100 mM Tris-HCl (pH=7.0) by shaking to form a concentration of 3.5 mg mL-1 solution. Then 37.5 µL of TGase solution was mixed with 22.5 µL PEGDA to form a precusor solution containing 2.19 mg mL-1 TGase with pH = 7.0. Then 60 µL of PEGDA/TGase solution was cast onto a LDPE-trypsin film and the same photomask (rotated the photomask to make the strip-type patterned regions perpendicular to the former shape) was put onto the film to spread out the PEGDA/TGase solution and form a “sandwich” structure. This sandwich structure was also irradiated with visible light (xenon lamp plus filter with a through light band of 380–700 nm, and a light intensity of 3 mW/cm2 at λ = 420 nm) for 30 min at room temperature. After the grafting reaction, the reaction film was immersed into 100 mM Tris-HCl buffer (pH=7.0) for 24 h and washed with Tris-HCl buffer alternately three times to remove the unimmobilized TGase (Scheme 1c). To prepare the TGase-trypsin loaded film (Type B), two-enzyme solution was prepared by mixing trypsin solution and TGase solution together (1:1 (v/v)). After mixing for 10 min, 60 µL of PEGDA/two-enzyme solution (45:75 (v/v)) was cast onto both sides of the LDPE-ITXSP film


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and then the strip-type photomask was put onto the film to form a “sandwich” structure. This sandwich structure was also irradiated with visible light (xenon lamp plus filter with a through light band of 380–700 nm, and a light intensity of 3 mW/cm2 at λ = 420 nm) for 30 min at room temperature. After the grafting reaction, the reaction film was immersed into 100 mM Tris-HCl buffer (pH=7.0) for 24 h and washed with Tris-HCl buffer alternately three times to remove the unimmobilized enzymes. Synthesis of LTV-Azacytidine (Scheme 1d). LTV-azacitydine can be obtained according to following steps. Prodrug (Figure S1, 10 µmol) was dissolved into 1 mL sodium bicarbonate solution to adjust the solution pH = 7.4 (Solution A). LTV-peptide (2 µmol) was dissolved into Tris-HCl buffer (3 mL, 0.1 M, pH=7.0) and then mixed with Solution A (1 mL) to form the substrate solution. The solution was prewarmed at 35 °C for 5 min, and then TGase-trypsin loaded film (Type A) was added. The catalytic reaction was performed in water bath with shaking at 35 °C. After 10 hours of incubation, the reaction was stopped by taking out the TGase-trypsin loaded film. The solvent was removed by freeze drying and the crude product was purified by Welchrom C18 to remove the impurities. The yield (Mp / M0) of LTV-azacytidine was determined after the purification. Here, Mp is the moles of LTV-azacytidine obtained after purification, M0 is the moles of LTV-peptide added before reaction.

Received: ((will be filled in by the editorial staff)) Revised: ((will be filled in by the editorial staff)) Published online: ((will be filled in by the editorial staff)) ASSOCIATED CONTENT


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Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.XXXXXX. Materials and methods. Synthesis of prodrug. The hydrolysis of LTV-azacytidine. Cytotoxicity of LTV-azacytidine. Potentials for more enzymes (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (C. Z.) *E-mail: [email protected] (W. Y.) Present Addresses §

Nanyang Environment & Water Research Institute (NEWRI), Nanyang Technological

University (NTU), 637141, Singapore. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (Grant No. 51521062, 51221002, 51103009, 51473015), the Innovation and Promotion Project of Beijing University of Chemical Technology, and the Beijing Natural Science Foundation (Grant No. 2162035).

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Separated Immobilization of Incompatible Enzymes on Polymer

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