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Nan Li1,2 Wei Zheng1,2 Ying Shen1,2 Li Qi1 Yaping Li1,2 Juan Qiao1 Fuyi Wang1 Yi Chen1 ∗ 1 Beijing
National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing, P. R. China 2 Graduate School, University of Chinese Academy of Sciences, Beijing, P. R. China Received July 23, 2014 Revised August 27, 2014 Accepted August 27, 2014
Research Article
Preparation of a novel polymer monolith with functional polymer brushes by two-step atom-transfer radical polymerization for trypsin immobilization Novel porous polymer monoliths grafted with poly{oligo[(ethylene glycol) methacrylate]co-glycidyl methacrylate} brushes were fabricated via two-step atom-transfer radical polymerization and used as a trypsin-based reactor in a continuous flow system. This is the first time that atom-transfer radical polymerization technique was utilized to design and construct polymer monolith bioreactor. The prepared monoliths possessed excellent permeability, providing fast mass transfer for enzymatic reaction. More importantly, surface properties, which were modulated via surface-initiated atom-transfer radical polymerization, were found to have a great effect on bioreactor activities based on Michaelis–Menten studies. Furthermore, three model proteins were digested by the monolith bioreactor to a larger degree within dramatically reduced time (50 s), about 900 times faster than that by free trypsin (12 h). The proposed method provided a platform to prepare porous monoliths with desired surface properties for immobilizing various enzymes. Keywords: Atom-transfer radical polymerization / Immobilized reactor / Porous polymer monolith / Surface grafting / Trypsin DOI 10.1002/jssc.201400794
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction The immobilization of enzymes on solid supports is an effective approach to improve the stability and reusability of enzymes, and facilitate the product isolation [1]. To date, various supports have been developed for immobilizing enzymes, such as membranes, nanoparticles, and monolithic materials [2]. Among them, porous polymer monoliths, which were initially developed as separation media, have rapidly become outstanding in many other fields due to their easy fabrication, excellent tolerance to extreme pH, and distinctly fast mass transfer [3]. In the 1990s, Petro and coworkers immobilized trypsin on poly(glycidyl methacrylate-co-ethylene dimethacrylate) [poly(GMA-co-EDMA)] monoliths [4]. As a result, the polymer monolithic reactor showed high proteolytic activities
Correspondence: Professor Li Qi, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2, zhongguancun Beiyijie, Beijing 100190, China E-mail: [email protected]
Abbreviations: ATRP, atom-transfer radical polymerization; BA, N␣ -benzoyl-L-arginine; BAEE, N␣ -benzoyl-L-arginine ethyl ester; EDMA, ethylene dimethacrylate; GMA, glycidyl methacrylate; OEGMA, oligo(ethylene glycol) methacrylate; Km , Michaelis constant; Vmax , maximum velocity C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
originated from its unique porous structure. After that, various polymer monolithic support based enzyme reactors were developed and summarized in reviews [5, 6]. Although the intrinsic structures of polymer monoliths [7] possess nonignorable superiority for enzyme immobilization, surface properties, such as density of functionalities and hydrophobicity, also have great effect on activity of the enzyme. Understanding the influence of surface properties of substrate on enzyme activity is highly useful for engineering the efficient immobilized enzymatic reactor. Surface grafting is an attractive strategy to incorporate the desired properties into polymer monoliths without changing composition of original matrix [8]. For example, Svec and colleagues utilized the photografting method with irradiation to introduce poly(2-vinyl-4,4-dimethylazlactone) chains on the surface of polymer monolith for subsequent enzyme immobilization. The resultant monolith bioreactor possessed high activity [9]. Although exciting progress has been made, introducing an alternative surface grafting approach for preparing polymer monolith bioreactor is still significant. Atom-transfer radical polymerization (ATRP), one of the most efficient and robust controlled/living radical polymerizations, is effective to prepare polymer substrates at ∗ Additional corresponding author: Professor Yi Chen, E-mail: [email protected]
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Figure 1. Procedures for the preparation of (A) monolithic support and (B) monolith bioreactor.
mild conditions for wide applications [10–12]. Moreover, surface-initiated (SI) ATRP is commonly used for producing well-defined polymer brushes with a controlled chain length [13, 14]. Additionally, it allows for designing the copolymer grafts with modulated properties by varying the feeding monomer composition [15]. However, as far as we know, there is no research so far on employing ATRP to prepare and modify polymer monoliths for immobilizing enzymes. Herein, poly{oligo[(ethylene glycol) methacrylate]-coglycidyl methacrylate} (P(OEGMA-co-GMA)) brushes grafted porous polymer monoliths were prepared by two-step ATRP method and used for immobilization of trypsin. In P(OEGMA-co-GMA), biocompatible POEGMA units were inserted to hydrophilize the functional polymer brushes, because the hydrophilic microenvironment had proven to be beneficial for trypsin-catalytic reaction [16]. The resultant polymer monoliths were used to hydrolysis of N␣ -benzoylL-arginine ethyl ester (BAEE). Copolymer compositions and chain length of copolymer grafts were modulated in SI-ATRP process and their influence on kinetic parameters of the bioreactors have been investigated in detail. Further, the prepared bioreactors with two different OEGMA levels were applied to fast and efficient protein digestion.
2 Materials and methods 2.1 Reagents Milli-Q water, which was produced from a water purification system (Millipore), was filtered through a 0.45 m membrane before use. All solvents used in chromatographic system were HPLC grade (Beijing Chemical Plant). Other chemical reagents and instruments used in this study are presented in the Supporting Information. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.2 Preparing polymer monolith bioreactor As shown in Fig. 1A, the poly(ethylene dimethacrylate) (poly(EDMA)) monolith was prepared within a stainless-steel column (50 mm × 4.6 mm id) via the ATRP method according to our previous report [17]. Grafting of P(OEGMAco-GMA) brushes was performed with activators regenerated by electron transfer (ARGET) ATRP, which is special initiation process for ATRP without the need of rigorous deoxygenation [18]. The typical reaction conditions were as follows: CuBr2 (34 mg, 0.15 mmol), GMA (0.76 mL, 5.8 mmol), OEGMA (0.76 mL, 1.7 mmol), and 1,1,4,7,7pentamethyldiethylenetriamine (32 L, 0.15 mmol) were dissolved in 24 mL of THF. Subsequently, the mixture was sonicated for 10 s to form a homogenous solution. Then, 0.10 mL of tin(II)-2-ethylhexanoate was added and mixed by 10 s of sonication. The resultant grafting solution was pumped through the poly(EDMA) monolith at the flow rate of 0.10 mL/min. After 4 h of grafting polymerization at room temperature, THF and water was successively pumped through the monolith at the flow rate of 0.50 mL/min to wash out the resident compounds. Six porous polymer monoliths with different OEGMA and GMA contents were synthesized (A (0.76/1.50)−F (1.50/0.76)). The resulting monolithic support is abbreviated as X’(a/b), where X represents the sequence number of the monolith, a and b represent the feed volume of OEGMA and GMA in the surface grafting mixture. Backpressures (⌬P) of these monoliths in chromatographic system were examined and permeability (KF ) was calculated from the Darcy equation: KF =
Fm L ⌬Pr 2
(1)
Where Fm is the flow rate, is the viscosity of the mobile phase, L is the column length, and r is the monolith inner radius. www.jss-journal.com
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The immobilization of trypsin was achieved by in situ reaction between epoxide groups of GMA units and amino groups of trypsin (Fig. 1B). A fresh trypsin solution (1.0 mg/mL) containing 50 mM benzamidine, which prevented undesired autodigestion, was prepared in 50 mM TrisHCl buffer (pH 8.7). The above trypsin solution was pumped through the monolith at a flow rate of 0.05 mL/min. After immobilization reaction for 12 h at room temperature, the obtained monolith bioreactor was washed with 50 mM TrisHCl buffer containing 0.5 M NaCl for 2 h to remove the unreacted trypsin. Six bioreactors (A(0.76/1.50)−F(1.50/0.76)) have been prepared from six corresponding supports.
2.3 Determining the amount of immobilized trypsin The amount of bound trypsin was determined using Bradford method [2]. Briefly, the trypsin-immobilized monolith was pushed out from stainless column, followed by being chopped into a small column with the length of 0.2 cm. Then, the small monolithic section was immersed in 100 L of 100 mM NaOH for 2 h at room temperature. Meanwhile, trypsin standard solutions with various concentrations (800–4000 g/mL) were prepared in 100 mM NaOH. Individual wells of a 96-well microtiter plate were charged with 20 L of each trypsin standard solution and cleaved trypsin solution. Then, 180 L of the Bradford agent was added to each solution. After the resulting mixture was placed at room temperature for 5 min, absorbance at 595 nm was measured. Subsequently, amount of immobilized trypsin on the monolith bioreactor was calculated.
2.4 Determining the trypsin activity with BAEE as substrate The prepared monolith bioreactor was inserted into a continuous flow system (Supporting Information Fig. S1) to perform hydrolysis reaction. For evaluating the activity of immobilized trypsin, 5 L of substrate BAEE at certain concentration prepared in 50 mM Tris-HCl buffer (pH 7.4) was injected in system 1 with Tris-HCl buffer as mobile phase. Then, the effluent containing hydrolytic product N␣ -benzoyl-L-arginine (BA) was collected. Finally, 5 L of the obtained effluent was injected into system 2, followed by HPLC analysis, which was carried out with a RP C18 column. The peak area of BA was used to quantify the degree of hydrolytic reaction, since the adsorption of BA was much stronger than that of BAEE at 254 nm. Hydrolysis yield (H) was defined as: H(%) =
SBA × 100 SBA0
(2)
Where SBA represents peak area of BA originated from hydrolysis of BAEE, and SBA0 represents peak area of standard BA solution, which was prepared at the same concentration as product BA from complete hydrolysis of BAEE. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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For comparison, hydrolysis of BAEE using free trypsin was performed with in-solution incubation method (15 min of digestion time at 25⬚C) [19]. 2.5 Protein digestion A 250 mg/mL solution of cytochrome c, BSA, and lysozyme were prepared in 50 mM Tris-HCl buffer (pH 7.4). Five microliters of each protein solution was passed through the monolith bioreactor with Tris-HCl buffer as mobile phase at a flow rate of 0.20 mL/min, corresponding to a residence time of 50 s. The effluent containing the digests of protein was collected, followed by freeze-drying and then diluted with water to 200 L. The three proteins were also digested by conventional in-solution method. Free trypsin was added to the protein solution (trypsin/substrate ratio, 1:40 w/w), and the reaction proceeded at 37⬚C for 12 h. Protein digests (on monolith bioreactor and in-solution digestion) were further analyzed by MALDI-TOF-MS. The conditions for MALDI-TOF-MS analysis are described in the Supporting Information.
3 Results and discussion 3.1 Preparation and characterizations of porous polymer monoliths Firstly, the porous monolith was fabricated via ATRP using typical ATRP initiator (ethyl-2-bromopropionate). After polymerization, the bromine groups still remained on the surface of monolith and could be continuously served as initiator for the next step of ATRP. Compared with the conventional polymerization, ATRP method enabled rapid preparation of polymer monolith without heating apparatus. Additionally, the resultant monolith could be directly used for further modification without immobilization of initiator. Secondly, P(OEGMA-co-GMA) was grafted on the monolithic surface via (ARGET) ATRP. In grafting polymerization, tin(II)-2ethylhexanoate, an oil-soluble reductant, was selected to reduce Cu(II), producing Cu(I) as the active catalyst. Further, investigations of the morphologies and compositions were performed by SEM, mercury-intrusion porosimetry, and elemental analysis. The monolith B’(0.76/0.76) was used as a representative for the grafted monoliths. As displayed in Supporting Information Fig. S2, the SEM images showed that there were macropores existing in both ungrafted and P(OEGMA-co-GMA) grafted monolith. Beyond that, porous structure was slightly compressed in grafted monolith. As confirmed by mercury-intrusion porosimetry (Supporting Information Fig. S3), after grafting polymerization, the average pore size decreased from 30.3 to 9.5 m. A large number of macropores (>50 nm) in polymer monolithic structure was essential to achieve fast mass transfer between substrate molecules and product molecules in enzymatic reaction. The data in Supporting Information Table S1 show that the grafted monolith contained higher carbon and hydrogen percentages compared to the ungrafted one. It could be www.jss-journal.com
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Table 1. Backpressures and permeabilities of monolithic supports with different grafting compositions
Monolithic supporta)
A’(0.76/1.50) B’(0.76/0.76) C’(0.76/0.35) D’(0.00/0.76) E’(0.35/0.76) F’(1.50/0.76)
Monomer volume (mL) OEGMA
GMA
0.76 0.76 0.76 0.00 0.35 1.50
1.50 0.76 0.35 0.76 0.76 0.76
⌬P (MPa)b)
K (m2 )c)
1.4 1.2 1.1 0.7 1.0 2.1
0.59 × 10−10 0.75 × 10−10 0.75 × 10−10 1.10 × 10−10 0.80 × 10−10 0.39 × 10−10
a) Abbreviated as X’(a/b), where X’ represents the sequence number of monolithic support, a and b represent feed volume of OEGMA and GMA, respectively. b) Backpressure of the monolithic support, determined using methanol as mobile phase. The flow rate was 0.2 mL/min with detecting wavelength of 254 nm. c) Permeability of the monolithic support.
also found that relative content of bromine in grafted monolith dropped to less than 0.3% due to the increasing content of other elements. In addition, the amount of grafted polymer could be calculated based on elemental analysis results. In this work, the amount of grafted P(OEGMA-co-GMA) was 17.61 mg/m2 , which was calculated from the equation described in our previous work [20]. All of the characterizations demonstrated the successful grafting of P(OEGMA-co-GMA) on monolithic surface. Backpressures and permeabilities of porous polymer monoliths are shown in Table 1. As expected, the backpressure of the monolith increased with the addition of larger monomer volume. Moreover, all the prepared monoliths exhibited excellent permeability, which allowed them to realize low-pressure drop even in high-speed flow system. The hydrophilic property of polymer monoliths with different OEGMA contents was characterized by the chromatographic method. The test was carried out with medroxyprogesterone acetate, which is one of steroids and commonly used as the hydrophobic analytes in reversephase chromatography [21]. Supporting Information Fig. S4 shows the retention factors of medroxyprogesterone acetate on the monolithic supports (B (0.76/0.76) and D (0.00/0.76)−F’(1.50/0.76)). Shorter retention time of the model analyte has been found with the increasing OEGMA composition, demonstrating the better hydrophilicity of the resultant support. The data confirmed that the employed SI-ATRP method presented a simple approach to alter the OEGMA composition for modulating the hydrophilic property of the monolithic surface. 3.2 Reaction conditions for monolith bioreactor In a flow-through system, flow rate of mobile phase determines the reaction time, which is referred to the residence time of the substrate in monolithic column [22]. Therefore, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Effect of (A) flow rate and (B) temperature on the hydrolysis rate using bioreactor B(0.76/0.76). Conditions: substrate, 400 mM BAEE in Tris-HCl buffer (pH 7.4); temperature, 30⬚C; flow rate, 0.2 mL/min. The reaction conditions were kept constant except the investigated one.
hydrolysis rates of BAEE with flow rate ranging from 0.1– 0.5 mL/min (residence time: 20−110 s) were investigated. As displayed in Fig. 2A, the significant improvement in hydrolysis rate was observed when the flow rate was decreased from 0.5 to 0.2 mL/min, due to the prolonged reaction time. Further slowing down the flow rate to 0.2 mL/min led to complete conversion of BAEE into product BA. Thus the flow rate of 0.2 mL/min with short residence time (50 s) was selected for subsequent experiments. Moreover, the effect of temperature was also investigated by changing the column temperature from 10 to 50⬚C. We found that the maximum hydrolysis rate could be achieved at 30⬚C (Fig. 2B), which was slightly higher than that of its free counterpart (25⬚C). Supporting Information Fig. S5 represents the chromatograms of BAEE solution before and after hydrolysis under the optimum conditions, demonstrating that the prepared monolith bioreactor could effectively convert BAEE to BA with rapid hydrolysis rate. 3.3 Effect of comonomer composition on kinetic parameters of the monolith bioreactor Under the optimal reaction conditions, the activities of bioreactors were evaluated by Michaelis–Menten study, which could better reflect the changes of interactions between the substrate and the immobilized enzyme. From intercepts of Lineweaver–Burke plots (Supporting Information Fig. S6), kinetic parameters of the BAEE hydrolysis could be calculated [23] and the data are displayed in Table 2. www.jss-journal.com
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J. Sep. Sci. 2014, 37, 3411–3417 Table 2. Effects of GMA and OEGMA contents on kinetic parameters of BAEE hydrolysis
Bioreactora)
mtrypsin
Km (mM)
Vmax (M s−1 mg−1 )
A(0.76/1.50) B(0.76/0.76) C(0.76/0.35) D(0.00/0.76) E(0.35/0.76) F(1.50/0.76)
0.87 0.48 0.36 0.48 0.48 0.47
9.21 5.94 5.05 10.63 6.83 5.63
164.21 801.29 108.61 86.95 227.27 56.50
a) The bioreactor is named as X(a/b), where X represents sequence number of the bioreactor, a and b represent feed volume of OEGMA and GMA, respectively.
3.3.1 Effect of GMA content For bioreactors with different GMA contents (A(0.76/1.50), B(0.76/0.76), and C(0.76/0.35)), the amount of trypsin immobilized on the monolithic supports was increased along with increasing GMA composition. As expected, increasing trypsin amount from 0.36 to 0.48 mg led to a dramatic increase of Vmax (maximum velocity) values, indicating an acceleration of the hydrolytic reaction. It was attributed to higher enzyme/substrate ratio. However, higher trypsin amount of 0.87 mg resulted in a decreased Vmax value. The phenomenon was ascribed to the fact that higher trypsin density on the monolithic surface would lead to less available active sites of immobilized trypsin for BAEE hydrolysis [24]. Additionally, longer P(OEGMA-co-GMA) brushes, which followed with addition of more GMA in polymerization mixture, would impede diffusion of the substrate into the polymer brushes, thus making the enzyme less accessible [25]. Such adverse effect of diffusion limitation also could cause the dramatic increasing of Km (Michaelis constant) values when raising the GMA content, which reflected the lower affinity between the substrate and the enzyme. The results further indicated that the SI-ATRP is a facile way to modulate the amount of immobilized enzyme on the monolithic surfaces, which had great impact on the activity of the bioreactor. 3.3.2 Effect of OEGMA content Km and Vmax values of the four bioreactors differing in OEGMA contents were investigated (Table 2). The OEGMA content affected the Km value from two aspects: on one hand, increasing the OEGMA content would cause diffusion limitation as described above, thus leading to an increase of Km value. However, on the other hand, larger OEGMA volume would provide better hydrophilic environment, which was in favor of stronger substrate–enzyme binding, resulting in decreased Km value [26]. Comparing among D(0.00/0.76), E(0.35/0.76), B(0.76/0.76), and F(1.50/0.76), we observed that a decrease of Km values followed with more OEGMA content, indicating that compared with the negative effect of elongated polymer brushes, beneficial effect of hydrophilicity on Km C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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value was dominated in the total variation of OEGMA contents (0.00−0.76 mL). For Vmax value, the extensively high OEGMA content resulted in overlong polymer brushes, leading to slower hydrolysis rate. It demonstrated that 0.76 mL of the initial GMA and OEGMA could provide the bioreactor B(0.76/0.76) with the best catalytic performance (Km = 5.94 mM, Vmax = 801.29 M s−1 mg−1 ). Compared with the free trypsin (Km = 4.03 mM, Vmax = 54.00 M s−1 mg−1 ), the proposed bioreactors exhibited much faster digestion velocity with only slightly decreased affinity.
3.4 Repeatability and stability of the polymer monolith bioreactor Repeatability test was carried out by repeated use. As shown in Supporting Information Fig. S7A, after ten runs of consecutive use, no obvious decrease in relative activity has been found, indicating a satisfactory repeatability of the digestion. Stability test was conducted by determining the activities of the bioreactor preserved at room temperature after various periods of time. As displayed in Supporting Information Fig. S7B, it has been observed that 80% of activity was still remained after the monolith bioreactor was used for six weeks. The results demonstrated that the proposed bioreactor was suitable for long-time storage.
3.5 Comparison of protein digestion between the monolith bioreactor and free trypsin The optimal bioreactor B(0.76/0.76) and a controlled bioreactor D(0.00/0.76), which did not contain the hydrophilic ingredient of OEGMA, were further applied to protein digestion at the flow rate of 0.2 mL/min (50 s of residence time). Cytochrome c, BSA, and lysozyme were selected as the model substrates. For comparison, in-solution digestions were also conducted. Figure 3 displays the mass spectra from the digestion of cytochrome c and BSA, which differ greatly in molecular weights. It could be observed that the maximum number of peptide peaks could be achieved by using bioreactor B(0.76/0.76) for both of the proteins. The results of the peptide identification for the three proteins were summarized in Supporting Information Table S2. It has been found that the bioreactor B(0.76/0.76) yielded the sequence coverages of 69, 38, and 64% for cytochrome c, BSA, and lysozyme, respectively, which were all higher than those obtained by free trypsin. More importantly, proteins were digested within only 50 s of residence time using monolith bioreactor, about 900 times faster than that performed in solution digestion (12 h). The rapid digestion rate demonstrated superiority of our proposed monolithic material for immobilizing enzymes. Additionally, compared with D(0.00/0.76) that did not contain the hydrophilic POEGMA unit, B(0.76/0.76) produced higher sequence coverages and more identified peptides, indicating that hydrophilization of polymer brushes by POEGMA www.jss-journal.com
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Figure 3. MALDI-TOF-MS spectra of (A) cytochrome c and (B) BSA digested by (a) free trypsin, (b) D(0.00/0.76), and (c) B(0.76/0.76), respectively. One missed cleavage was allowed in identification of peptides.
units led to better digestion performance of the monolith bioreactor.
4 Conclusion A new trypsin-based bioreactor was achieved through fabrication of P(OEGMA-co-GMA) brushes grafted porous polymer monolith via two-step ATRP method, followed by covalent coupling of trypsin. The adopted two-step ATRP method has been proved to be powerful to generate monolithic surfaces with modulated active sites and hydrophilicity for improving the digestion efficiency. As a result, the appropriate content of OEGMA and GMA could provide the resultant monolith bioreactor with high catalytic activity and high affinity from its kinetic characterizations. Furthermore, the bioreactor exhibited good repeatability and stability for practical application. In protein digestion, the bioreactor B(0.76/0.76) yielded a better result with evidently reduced digestion time, compared with the digestion conducted in an aqueous C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
solution. The proposed method presents a promising alternative for generating polymer monolithic support for enzyme immobilization. We gratefully acknowledge the financial support from National Natural Science Foundation of China (No. 21175138, No. 21375132, No. 21135006 and No. 21321003). The authors declared no conflict of interest.
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C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Nan Li1,2 Wei Zheng1,2 Ying Shen1,2 Li Qi1 Yaping Li1,2 Juan Qiao1 Fuyi Wang1 Yi Chen1 ∗ 1 Beijing
National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing, P. R. China 2 Graduate School, University of Chinese Academy of Sciences, Beijing, P. R. China Received July 23, 2014 Revised August 27, 2014 Accepted August 27, 2014
Research Article
Preparation of a novel polymer monolith with functional polymer brushes by two-step atom-transfer radical polymerization for trypsin immobilization Novel porous polymer monoliths grafted with poly{oligo[(ethylene glycol) methacrylate]co-glycidyl methacrylate} brushes were fabricated via two-step atom-transfer radical polymerization and used as a trypsin-based reactor in a continuous flow system. This is the first time that atom-transfer radical polymerization technique was utilized to design and construct polymer monolith bioreactor. The prepared monoliths possessed excellent permeability, providing fast mass transfer for enzymatic reaction. More importantly, surface properties, which were modulated via surface-initiated atom-transfer radical polymerization, were found to have a great effect on bioreactor activities based on Michaelis–Menten studies. Furthermore, three model proteins were digested by the monolith bioreactor to a larger degree within dramatically reduced time (50 s), about 900 times faster than that by free trypsin (12 h). The proposed method provided a platform to prepare porous monoliths with desired surface properties for immobilizing various enzymes. Keywords: Atom-transfer radical polymerization / Immobilized reactor / Porous polymer monolith / Surface grafting / Trypsin DOI 10.1002/jssc.201400794
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction The immobilization of enzymes on solid supports is an effective approach to improve the stability and reusability of enzymes, and facilitate the product isolation [1]. To date, various supports have been developed for immobilizing enzymes, such as membranes, nanoparticles, and monolithic materials [2]. Among them, porous polymer monoliths, which were initially developed as separation media, have rapidly become outstanding in many other fields due to their easy fabrication, excellent tolerance to extreme pH, and distinctly fast mass transfer [3]. In the 1990s, Petro and coworkers immobilized trypsin on poly(glycidyl methacrylate-co-ethylene dimethacrylate) [poly(GMA-co-EDMA)] monoliths [4]. As a result, the polymer monolithic reactor showed high proteolytic activities
Correspondence: Professor Li Qi, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2, zhongguancun Beiyijie, Beijing 100190, China E-mail: [email protected]
Abbreviations: ATRP, atom-transfer radical polymerization; BA, N␣ -benzoyl-L-arginine; BAEE, N␣ -benzoyl-L-arginine ethyl ester; EDMA, ethylene dimethacrylate; GMA, glycidyl methacrylate; OEGMA, oligo(ethylene glycol) methacrylate; Km , Michaelis constant; Vmax , maximum velocity C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
originated from its unique porous structure. After that, various polymer monolithic support based enzyme reactors were developed and summarized in reviews [5, 6]. Although the intrinsic structures of polymer monoliths [7] possess nonignorable superiority for enzyme immobilization, surface properties, such as density of functionalities and hydrophobicity, also have great effect on activity of the enzyme. Understanding the influence of surface properties of substrate on enzyme activity is highly useful for engineering the efficient immobilized enzymatic reactor. Surface grafting is an attractive strategy to incorporate the desired properties into polymer monoliths without changing composition of original matrix [8]. For example, Svec and colleagues utilized the photografting method with irradiation to introduce poly(2-vinyl-4,4-dimethylazlactone) chains on the surface of polymer monolith for subsequent enzyme immobilization. The resultant monolith bioreactor possessed high activity [9]. Although exciting progress has been made, introducing an alternative surface grafting approach for preparing polymer monolith bioreactor is still significant. Atom-transfer radical polymerization (ATRP), one of the most efficient and robust controlled/living radical polymerizations, is effective to prepare polymer substrates at ∗ Additional corresponding author: Professor Yi Chen, E-mail: [email protected]
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Figure 1. Procedures for the preparation of (A) monolithic support and (B) monolith bioreactor.
mild conditions for wide applications [10–12]. Moreover, surface-initiated (SI) ATRP is commonly used for producing well-defined polymer brushes with a controlled chain length [13, 14]. Additionally, it allows for designing the copolymer grafts with modulated properties by varying the feeding monomer composition [15]. However, as far as we know, there is no research so far on employing ATRP to prepare and modify polymer monoliths for immobilizing enzymes. Herein, poly{oligo[(ethylene glycol) methacrylate]-coglycidyl methacrylate} (P(OEGMA-co-GMA)) brushes grafted porous polymer monoliths were prepared by two-step ATRP method and used for immobilization of trypsin. In P(OEGMA-co-GMA), biocompatible POEGMA units were inserted to hydrophilize the functional polymer brushes, because the hydrophilic microenvironment had proven to be beneficial for trypsin-catalytic reaction [16]. The resultant polymer monoliths were used to hydrolysis of N␣ -benzoylL-arginine ethyl ester (BAEE). Copolymer compositions and chain length of copolymer grafts were modulated in SI-ATRP process and their influence on kinetic parameters of the bioreactors have been investigated in detail. Further, the prepared bioreactors with two different OEGMA levels were applied to fast and efficient protein digestion.
2 Materials and methods 2.1 Reagents Milli-Q water, which was produced from a water purification system (Millipore), was filtered through a 0.45 m membrane before use. All solvents used in chromatographic system were HPLC grade (Beijing Chemical Plant). Other chemical reagents and instruments used in this study are presented in the Supporting Information. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.2 Preparing polymer monolith bioreactor As shown in Fig. 1A, the poly(ethylene dimethacrylate) (poly(EDMA)) monolith was prepared within a stainless-steel column (50 mm × 4.6 mm id) via the ATRP method according to our previous report [17]. Grafting of P(OEGMAco-GMA) brushes was performed with activators regenerated by electron transfer (ARGET) ATRP, which is special initiation process for ATRP without the need of rigorous deoxygenation [18]. The typical reaction conditions were as follows: CuBr2 (34 mg, 0.15 mmol), GMA (0.76 mL, 5.8 mmol), OEGMA (0.76 mL, 1.7 mmol), and 1,1,4,7,7pentamethyldiethylenetriamine (32 L, 0.15 mmol) were dissolved in 24 mL of THF. Subsequently, the mixture was sonicated for 10 s to form a homogenous solution. Then, 0.10 mL of tin(II)-2-ethylhexanoate was added and mixed by 10 s of sonication. The resultant grafting solution was pumped through the poly(EDMA) monolith at the flow rate of 0.10 mL/min. After 4 h of grafting polymerization at room temperature, THF and water was successively pumped through the monolith at the flow rate of 0.50 mL/min to wash out the resident compounds. Six porous polymer monoliths with different OEGMA and GMA contents were synthesized (A (0.76/1.50)−F (1.50/0.76)). The resulting monolithic support is abbreviated as X’(a/b), where X represents the sequence number of the monolith, a and b represent the feed volume of OEGMA and GMA in the surface grafting mixture. Backpressures (⌬P) of these monoliths in chromatographic system were examined and permeability (KF ) was calculated from the Darcy equation: KF =
Fm L ⌬Pr 2
(1)
Where Fm is the flow rate, is the viscosity of the mobile phase, L is the column length, and r is the monolith inner radius. www.jss-journal.com
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The immobilization of trypsin was achieved by in situ reaction between epoxide groups of GMA units and amino groups of trypsin (Fig. 1B). A fresh trypsin solution (1.0 mg/mL) containing 50 mM benzamidine, which prevented undesired autodigestion, was prepared in 50 mM TrisHCl buffer (pH 8.7). The above trypsin solution was pumped through the monolith at a flow rate of 0.05 mL/min. After immobilization reaction for 12 h at room temperature, the obtained monolith bioreactor was washed with 50 mM TrisHCl buffer containing 0.5 M NaCl for 2 h to remove the unreacted trypsin. Six bioreactors (A(0.76/1.50)−F(1.50/0.76)) have been prepared from six corresponding supports.
2.3 Determining the amount of immobilized trypsin The amount of bound trypsin was determined using Bradford method [2]. Briefly, the trypsin-immobilized monolith was pushed out from stainless column, followed by being chopped into a small column with the length of 0.2 cm. Then, the small monolithic section was immersed in 100 L of 100 mM NaOH for 2 h at room temperature. Meanwhile, trypsin standard solutions with various concentrations (800–4000 g/mL) were prepared in 100 mM NaOH. Individual wells of a 96-well microtiter plate were charged with 20 L of each trypsin standard solution and cleaved trypsin solution. Then, 180 L of the Bradford agent was added to each solution. After the resulting mixture was placed at room temperature for 5 min, absorbance at 595 nm was measured. Subsequently, amount of immobilized trypsin on the monolith bioreactor was calculated.
2.4 Determining the trypsin activity with BAEE as substrate The prepared monolith bioreactor was inserted into a continuous flow system (Supporting Information Fig. S1) to perform hydrolysis reaction. For evaluating the activity of immobilized trypsin, 5 L of substrate BAEE at certain concentration prepared in 50 mM Tris-HCl buffer (pH 7.4) was injected in system 1 with Tris-HCl buffer as mobile phase. Then, the effluent containing hydrolytic product N␣ -benzoyl-L-arginine (BA) was collected. Finally, 5 L of the obtained effluent was injected into system 2, followed by HPLC analysis, which was carried out with a RP C18 column. The peak area of BA was used to quantify the degree of hydrolytic reaction, since the adsorption of BA was much stronger than that of BAEE at 254 nm. Hydrolysis yield (H) was defined as: H(%) =
SBA × 100 SBA0
(2)
Where SBA represents peak area of BA originated from hydrolysis of BAEE, and SBA0 represents peak area of standard BA solution, which was prepared at the same concentration as product BA from complete hydrolysis of BAEE. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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For comparison, hydrolysis of BAEE using free trypsin was performed with in-solution incubation method (15 min of digestion time at 25⬚C) [19]. 2.5 Protein digestion A 250 mg/mL solution of cytochrome c, BSA, and lysozyme were prepared in 50 mM Tris-HCl buffer (pH 7.4). Five microliters of each protein solution was passed through the monolith bioreactor with Tris-HCl buffer as mobile phase at a flow rate of 0.20 mL/min, corresponding to a residence time of 50 s. The effluent containing the digests of protein was collected, followed by freeze-drying and then diluted with water to 200 L. The three proteins were also digested by conventional in-solution method. Free trypsin was added to the protein solution (trypsin/substrate ratio, 1:40 w/w), and the reaction proceeded at 37⬚C for 12 h. Protein digests (on monolith bioreactor and in-solution digestion) were further analyzed by MALDI-TOF-MS. The conditions for MALDI-TOF-MS analysis are described in the Supporting Information.
3 Results and discussion 3.1 Preparation and characterizations of porous polymer monoliths Firstly, the porous monolith was fabricated via ATRP using typical ATRP initiator (ethyl-2-bromopropionate). After polymerization, the bromine groups still remained on the surface of monolith and could be continuously served as initiator for the next step of ATRP. Compared with the conventional polymerization, ATRP method enabled rapid preparation of polymer monolith without heating apparatus. Additionally, the resultant monolith could be directly used for further modification without immobilization of initiator. Secondly, P(OEGMA-co-GMA) was grafted on the monolithic surface via (ARGET) ATRP. In grafting polymerization, tin(II)-2ethylhexanoate, an oil-soluble reductant, was selected to reduce Cu(II), producing Cu(I) as the active catalyst. Further, investigations of the morphologies and compositions were performed by SEM, mercury-intrusion porosimetry, and elemental analysis. The monolith B’(0.76/0.76) was used as a representative for the grafted monoliths. As displayed in Supporting Information Fig. S2, the SEM images showed that there were macropores existing in both ungrafted and P(OEGMA-co-GMA) grafted monolith. Beyond that, porous structure was slightly compressed in grafted monolith. As confirmed by mercury-intrusion porosimetry (Supporting Information Fig. S3), after grafting polymerization, the average pore size decreased from 30.3 to 9.5 m. A large number of macropores (>50 nm) in polymer monolithic structure was essential to achieve fast mass transfer between substrate molecules and product molecules in enzymatic reaction. The data in Supporting Information Table S1 show that the grafted monolith contained higher carbon and hydrogen percentages compared to the ungrafted one. It could be www.jss-journal.com
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Table 1. Backpressures and permeabilities of monolithic supports with different grafting compositions
Monolithic supporta)
A’(0.76/1.50) B’(0.76/0.76) C’(0.76/0.35) D’(0.00/0.76) E’(0.35/0.76) F’(1.50/0.76)
Monomer volume (mL) OEGMA
GMA
0.76 0.76 0.76 0.00 0.35 1.50
1.50 0.76 0.35 0.76 0.76 0.76
⌬P (MPa)b)
K (m2 )c)
1.4 1.2 1.1 0.7 1.0 2.1
0.59 × 10−10 0.75 × 10−10 0.75 × 10−10 1.10 × 10−10 0.80 × 10−10 0.39 × 10−10
a) Abbreviated as X’(a/b), where X’ represents the sequence number of monolithic support, a and b represent feed volume of OEGMA and GMA, respectively. b) Backpressure of the monolithic support, determined using methanol as mobile phase. The flow rate was 0.2 mL/min with detecting wavelength of 254 nm. c) Permeability of the monolithic support.
also found that relative content of bromine in grafted monolith dropped to less than 0.3% due to the increasing content of other elements. In addition, the amount of grafted polymer could be calculated based on elemental analysis results. In this work, the amount of grafted P(OEGMA-co-GMA) was 17.61 mg/m2 , which was calculated from the equation described in our previous work [20]. All of the characterizations demonstrated the successful grafting of P(OEGMA-co-GMA) on monolithic surface. Backpressures and permeabilities of porous polymer monoliths are shown in Table 1. As expected, the backpressure of the monolith increased with the addition of larger monomer volume. Moreover, all the prepared monoliths exhibited excellent permeability, which allowed them to realize low-pressure drop even in high-speed flow system. The hydrophilic property of polymer monoliths with different OEGMA contents was characterized by the chromatographic method. The test was carried out with medroxyprogesterone acetate, which is one of steroids and commonly used as the hydrophobic analytes in reversephase chromatography [21]. Supporting Information Fig. S4 shows the retention factors of medroxyprogesterone acetate on the monolithic supports (B (0.76/0.76) and D (0.00/0.76)−F’(1.50/0.76)). Shorter retention time of the model analyte has been found with the increasing OEGMA composition, demonstrating the better hydrophilicity of the resultant support. The data confirmed that the employed SI-ATRP method presented a simple approach to alter the OEGMA composition for modulating the hydrophilic property of the monolithic surface. 3.2 Reaction conditions for monolith bioreactor In a flow-through system, flow rate of mobile phase determines the reaction time, which is referred to the residence time of the substrate in monolithic column [22]. Therefore, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Effect of (A) flow rate and (B) temperature on the hydrolysis rate using bioreactor B(0.76/0.76). Conditions: substrate, 400 mM BAEE in Tris-HCl buffer (pH 7.4); temperature, 30⬚C; flow rate, 0.2 mL/min. The reaction conditions were kept constant except the investigated one.
hydrolysis rates of BAEE with flow rate ranging from 0.1– 0.5 mL/min (residence time: 20−110 s) were investigated. As displayed in Fig. 2A, the significant improvement in hydrolysis rate was observed when the flow rate was decreased from 0.5 to 0.2 mL/min, due to the prolonged reaction time. Further slowing down the flow rate to 0.2 mL/min led to complete conversion of BAEE into product BA. Thus the flow rate of 0.2 mL/min with short residence time (50 s) was selected for subsequent experiments. Moreover, the effect of temperature was also investigated by changing the column temperature from 10 to 50⬚C. We found that the maximum hydrolysis rate could be achieved at 30⬚C (Fig. 2B), which was slightly higher than that of its free counterpart (25⬚C). Supporting Information Fig. S5 represents the chromatograms of BAEE solution before and after hydrolysis under the optimum conditions, demonstrating that the prepared monolith bioreactor could effectively convert BAEE to BA with rapid hydrolysis rate. 3.3 Effect of comonomer composition on kinetic parameters of the monolith bioreactor Under the optimal reaction conditions, the activities of bioreactors were evaluated by Michaelis–Menten study, which could better reflect the changes of interactions between the substrate and the immobilized enzyme. From intercepts of Lineweaver–Burke plots (Supporting Information Fig. S6), kinetic parameters of the BAEE hydrolysis could be calculated [23] and the data are displayed in Table 2. www.jss-journal.com
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J. Sep. Sci. 2014, 37, 3411–3417 Table 2. Effects of GMA and OEGMA contents on kinetic parameters of BAEE hydrolysis
Bioreactora)
mtrypsin
Km (mM)
Vmax (M s−1 mg−1 )
A(0.76/1.50) B(0.76/0.76) C(0.76/0.35) D(0.00/0.76) E(0.35/0.76) F(1.50/0.76)
0.87 0.48 0.36 0.48 0.48 0.47
9.21 5.94 5.05 10.63 6.83 5.63
164.21 801.29 108.61 86.95 227.27 56.50
a) The bioreactor is named as X(a/b), where X represents sequence number of the bioreactor, a and b represent feed volume of OEGMA and GMA, respectively.
3.3.1 Effect of GMA content For bioreactors with different GMA contents (A(0.76/1.50), B(0.76/0.76), and C(0.76/0.35)), the amount of trypsin immobilized on the monolithic supports was increased along with increasing GMA composition. As expected, increasing trypsin amount from 0.36 to 0.48 mg led to a dramatic increase of Vmax (maximum velocity) values, indicating an acceleration of the hydrolytic reaction. It was attributed to higher enzyme/substrate ratio. However, higher trypsin amount of 0.87 mg resulted in a decreased Vmax value. The phenomenon was ascribed to the fact that higher trypsin density on the monolithic surface would lead to less available active sites of immobilized trypsin for BAEE hydrolysis [24]. Additionally, longer P(OEGMA-co-GMA) brushes, which followed with addition of more GMA in polymerization mixture, would impede diffusion of the substrate into the polymer brushes, thus making the enzyme less accessible [25]. Such adverse effect of diffusion limitation also could cause the dramatic increasing of Km (Michaelis constant) values when raising the GMA content, which reflected the lower affinity between the substrate and the enzyme. The results further indicated that the SI-ATRP is a facile way to modulate the amount of immobilized enzyme on the monolithic surfaces, which had great impact on the activity of the bioreactor. 3.3.2 Effect of OEGMA content Km and Vmax values of the four bioreactors differing in OEGMA contents were investigated (Table 2). The OEGMA content affected the Km value from two aspects: on one hand, increasing the OEGMA content would cause diffusion limitation as described above, thus leading to an increase of Km value. However, on the other hand, larger OEGMA volume would provide better hydrophilic environment, which was in favor of stronger substrate–enzyme binding, resulting in decreased Km value [26]. Comparing among D(0.00/0.76), E(0.35/0.76), B(0.76/0.76), and F(1.50/0.76), we observed that a decrease of Km values followed with more OEGMA content, indicating that compared with the negative effect of elongated polymer brushes, beneficial effect of hydrophilicity on Km C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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value was dominated in the total variation of OEGMA contents (0.00−0.76 mL). For Vmax value, the extensively high OEGMA content resulted in overlong polymer brushes, leading to slower hydrolysis rate. It demonstrated that 0.76 mL of the initial GMA and OEGMA could provide the bioreactor B(0.76/0.76) with the best catalytic performance (Km = 5.94 mM, Vmax = 801.29 M s−1 mg−1 ). Compared with the free trypsin (Km = 4.03 mM, Vmax = 54.00 M s−1 mg−1 ), the proposed bioreactors exhibited much faster digestion velocity with only slightly decreased affinity.
3.4 Repeatability and stability of the polymer monolith bioreactor Repeatability test was carried out by repeated use. As shown in Supporting Information Fig. S7A, after ten runs of consecutive use, no obvious decrease in relative activity has been found, indicating a satisfactory repeatability of the digestion. Stability test was conducted by determining the activities of the bioreactor preserved at room temperature after various periods of time. As displayed in Supporting Information Fig. S7B, it has been observed that 80% of activity was still remained after the monolith bioreactor was used for six weeks. The results demonstrated that the proposed bioreactor was suitable for long-time storage.
3.5 Comparison of protein digestion between the monolith bioreactor and free trypsin The optimal bioreactor B(0.76/0.76) and a controlled bioreactor D(0.00/0.76), which did not contain the hydrophilic ingredient of OEGMA, were further applied to protein digestion at the flow rate of 0.2 mL/min (50 s of residence time). Cytochrome c, BSA, and lysozyme were selected as the model substrates. For comparison, in-solution digestions were also conducted. Figure 3 displays the mass spectra from the digestion of cytochrome c and BSA, which differ greatly in molecular weights. It could be observed that the maximum number of peptide peaks could be achieved by using bioreactor B(0.76/0.76) for both of the proteins. The results of the peptide identification for the three proteins were summarized in Supporting Information Table S2. It has been found that the bioreactor B(0.76/0.76) yielded the sequence coverages of 69, 38, and 64% for cytochrome c, BSA, and lysozyme, respectively, which were all higher than those obtained by free trypsin. More importantly, proteins were digested within only 50 s of residence time using monolith bioreactor, about 900 times faster than that performed in solution digestion (12 h). The rapid digestion rate demonstrated superiority of our proposed monolithic material for immobilizing enzymes. Additionally, compared with D(0.00/0.76) that did not contain the hydrophilic POEGMA unit, B(0.76/0.76) produced higher sequence coverages and more identified peptides, indicating that hydrophilization of polymer brushes by POEGMA www.jss-journal.com
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Figure 3. MALDI-TOF-MS spectra of (A) cytochrome c and (B) BSA digested by (a) free trypsin, (b) D(0.00/0.76), and (c) B(0.76/0.76), respectively. One missed cleavage was allowed in identification of peptides.
units led to better digestion performance of the monolith bioreactor.
4 Conclusion A new trypsin-based bioreactor was achieved through fabrication of P(OEGMA-co-GMA) brushes grafted porous polymer monolith via two-step ATRP method, followed by covalent coupling of trypsin. The adopted two-step ATRP method has been proved to be powerful to generate monolithic surfaces with modulated active sites and hydrophilicity for improving the digestion efficiency. As a result, the appropriate content of OEGMA and GMA could provide the resultant monolith bioreactor with high catalytic activity and high affinity from its kinetic characterizations. Furthermore, the bioreactor exhibited good repeatability and stability for practical application. In protein digestion, the bioreactor B(0.76/0.76) yielded a better result with evidently reduced digestion time, compared with the digestion conducted in an aqueous C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
solution. The proposed method presents a promising alternative for generating polymer monolithic support for enzyme immobilization. We gratefully acknowledge the financial support from National Natural Science Foundation of China (No. 21175138, No. 21375132, No. 21135006 and No. 21321003). The authors declared no conflict of interest.
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