Enzyme Reactor Based on Reversible pH-Controlled Catalytic

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Enzyme reactor based on reversible pH controlled catalytic polymer porous membrane Juan Qiao, Junfang Jiang, Lili Liu, Ji Shen, and Li Qi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01951 • Publication Date (Web): 28 Mar 2019 Downloaded from http://pubs.acs.org on March 28, 2019

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ACS Applied Materials & Interfaces

Enzyme Reactor Based on Reversible pH Controlled Catalytic Polymer Porous Membrane

Juan Qiaoa,b, Junfang Jianga,b, Lili Liua,c, Ji Shen a,b, Li Qi* a,b

a

Beijing National Laboratory for Molecular Sciences; Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (P.R. China)

b

School of Chemical Sciences, University of Chinese Academy of Sciences, Beijing 100049 (P.

R. China) c

College of Chemistry & Environmental Science, Hebei University, Baoding 071002 (P. R.

China)

ACS Paragon Plus Environment

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ACS Applied Materials & Interfaces 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

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ABSTRACT The challenge for polymeric enzyme reactors at present is to selectively control the enzymolysis rate in the complex conditions. Additionally, the fabrication methodology is hindered by complex processes, especially for achieving diverse stimuli-responsiveness and functions. Here, we reported a kind of pH sensitive polymer (poly styrene-co-maleic anhydride-acrylic acid, PSMAn-AA) based hybrid enzyme reactor. It comprised of magnetic nanoparticles and pH sensitive PS-MAn-AA porous polymer membrane making by breath figure method. The enzyme L-asparaginase (L-ASNase) could covalently bond on the surface of the pH sensitive porous polymer membrane (pH-PPM) and the resultant enzyme reactor were characterized by Fourier transform infrared spectroscopy and vibrating sample magnetometer. The apparent MichaelisMenten constants (Km and Vmax) of L-ASNase enzyme reactor at different pH were determined by a chiral ligand exchange capillary electrophoresis method with L-asparagine as the substrate. The Vmax value of L-ASNase enzyme reactor (0.67 mM / min) was almost 3 folds of that of free L-ASNase (0.23 mM / min) at pH 8.2. Its ability of precisely control the enzymolysis rate in the complex conditions is triggered primarily by pH of buffer solution, allowing controlled enzymatic reactions and displaying excellent stability and reusability of the proposed pH-PPM. This strategy for porous polymer membrane enzyme reactor fabrication has established a platform for enzyme efficiency adjusting. These valve-like distinguished features highlight the outstanding potential of stimuli-responsive enzyme reactor applied for enzyme immobilization and enzyme related disease therapeutic.

KEYWORDS pH stimuli-responsive polymer, porous polymer membrane, pH-controlled catalysis, confinement effect, covalent immobilization, L-ASNase enzyme reactor, chiral separation of D,L-amino acids


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1. Introduction Enzyme catalyzed reaction is a considerable research interest and has currently demonstrated their potential application in diverse areas owing to their crucial advantages. 1, 2 However, the free enzymes usually exhibit limited solution stability due to denaturation and deactivation occurs upon catalyze reaction.

3, 4

Moreover, recycling and reusability of the free enzymes in

solution is time-consuming separation and purification steps in the point view of practice. Thus enzyme reactors, in which the enzyme immobilized on different solid materials leading to enzyme mobility depression, have been proposed as an effective substitutable strategy to overcome these limitations. This strategy has been systematically investigated during these years and many kinds of materials have been applied for enzyme immobilization, including inorganic, 5

organic6,7 and hybrid carriers.8,9 The crucial advantages, including increased half-life, enhanced

stability and no purification processes, profited from much stabile enzyme structure and less denaturation provided by the immobilized enzyme. 10,11 Basically, several concerning aspects were required during materials selecting for enzyme immobilization: mild reaction conditions, materials with large surface area, universal process and platforms for enzyme immobilization. Meanwhile, besides the fundamental considering, some special aspects were desirable for enzyme immobilization and enzymolysis controlling to satisfy the increasing application demand. For example, enzyme immobilization via host-guest interactions of adamantane modified enzymes with a cyclodextrin modified surface to prevent enzyme release was described by Hernández group.

12

Approaches based on confinement effect

of the enzyme modified on polymer micro-porous-membranes and its substrates for increasing collision chance during emzymolysis were also reported.13,14


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However, when applying these approaches, fixed conditions including the optimized pH of buffer solution, temperature for emzymolysis were carried out. In other words, the immobilized enzyme could not meet the demands of enzymolysis at complex conditions or regulating the hydrolysis rate by changing condition very conveniently. Thus, it leaves the opportunity for us to establish controlled catalytic enzyme reactors which could tune emzymolysis efficiency by changing the circumstance conditions. Stimuli-responsive polymer is a kind of organic material which could change its property with the variation of conditions, such as temperature, pH and light etc. Among these polymers, poly acrylic acid (PAA) is considered as a functional polymer material with pH-responsive phase transition between hydrophilic state and hydrophobic state. The phase transition of PAA may significantly affect enzyme activity during enzymolysis in response to buffer pH variation. On one hand, it is proposed that at a higher pH buffer solution, the PAA would be more hydrophilic and have negative charge on the polymer chain. It will easily stretch into the aqueous solution and provide larger cavity of the enzyme, which could enhance the impact between the substrate and enzyme. On the other hand, at a lower pH buffer solution, the negative charge on PAA chain would be neutralized and further lead to lower hydrolysis. Thus, an approach for controlled catalytic enzyme reactors fabrication should be designed based on pH responsive polymer (poly styrene-co-maleic anhydride-acrylic acid, PS-MAn-AA). The pH-responsive polymer porous membrane (pH-PPM) could be made by breath figure methods using PS-MAn-AA and magnetic nanoparticles. The MAn section in the polymer would provide anchor groups for the enzyme; the AA section would have the pH sensitive function; magnetic nanoparticles would make it easily separation of the enzyme and substrates. To evaluate the function of the established controlled catalytic enzyme reactors, L-asparaginase (L-ASNase), a kind of signiﬁcant enzyme drug


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obtained from Escherichia coli,15 was immobilized onto the pH-PPM enzyme reactor. L-ASNase has variety applications in malignant diseases as chemotherapeutic drug for children acute lymphoblastic leukemia. As an enzyme, L-ASNase can hydrolyze L-asparagine (L-Asn) 16 to Laspartic acid (L-Asp).17 Owing to be an enzyme of bacterial origin with large molecular weight,18 many problems have been evoked when L-ASNase was utilized as a drug, such as half-life decreases of L-ASNase and rigorous hypersensitivity reactions to human body, which restricted its application severely. Therefore, exploring L-ASNase immobilization with controlled catalysis in complex samples is still urgent needed. In this study, a kind of hybrid materials composed of PS-MAn-AA@magnetic nanoparticles has been developed. To evaluate the kinetics of the immobilized L-ASNase, a chiral ligand exchange capillary electrophoresis (CLE-CE) separation method has been established and optimized. Then, the characterization of the polymer and pH sensitivity has been investigated. Moreover, the enzyme immobilization process and the comparison of the free enzyme in solution with the immobilized one at different pH have been measured. The hybrid materials provide a platform for tunable enzyme reactor construction and pave a new way for enzyme hydrolysis application in disease treatment.

2. EXPERIMENTAL SECTION 2.1. Chemicals. The L-arginase (L-Arg) and other amino acids, dansyl chloride (Dns-Cl), acrylic acid (AA), benzyl benzodithioate (BBDT) and L-ASNase were bought from SigmaAldrich Chemical Corporation (St. Louis, USA). St, lithium carbonate, sodium chloride, boric acid, hydrochloric acid and other organic reagents were all analytical grade and obtained from Beijing Chemical Corporation (Beijing, China). Coomassie brilliant blue G-250, lithium


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perchlorate (LiClO4), azobisisobutryonitrile (AIBN) and ZnSO4 were obtained from Aladdin Chemistry Company (Shanghai, China). The ferric chloride hexahydrate (FeCl3·6H2O) and ferrous chloride tetrahydrate (FeCl2·4H2O) were purchased from Xilong Chemical Company (Guangdong, P.R. China). Tris (hydroxymethyl) aminomethane (Tris) was bought from J&K Scientific Ltd. (Beijing, China). MAn was brought from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). The ionic liquid tetramethylammomium bromide ([N4,4,4,4][Br]) was obtained from Lanzhou Institute of Chemical Physics (Lanzhou, China). The aqueous solutions were prepared with Milli Q water, then filtered through a 0.45 µm membrane before use and stored at 4 oC. 2.2. Instruments. The AAs separation and analysis were carried on the CE instrumental, which applied a 1229 HPCE high voltage power supply provided by Beijing Institute of New Technology and Application (Beijing, China) and a UV detector provided by Rilips Photoelectricity Factory (Beijing, China) and a HW-2000 chromatography workstation (Qianpu, Software, Nanjing, China). The fused-silica capillaries for separation used in this work were obtained from Yongnian Optical Fiber Factory (Hebei, China) of 75 μm (i.d.) and 70.0 cm (55.0 cm effective). The molecular weight of PS-MAn-AA was analysed by Gel permeation chromatography (GPC) experiments, which was carried out on a HPLC instrument. The HPLC instrument was composed of a Waters 1515 HPLC solvent pump, a Waters 2414 differential refractometer detector and a set of Waters Styragel columns. Tetrahydrofuran was used as the eluent and the flow rate was 1.0 mL/min with different polystyrene as calibration.


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The morphology of ordered porous pH-PPM made by the breath figures method was detected using an S-4800 scanning electron microscope (SEM, Hitachi, Japan). The polymer and enzyme content on the pH-PPM was determined by a thermogravimetric analyzer (TGA 7, PerkinElmer, USA). The experiment was carried out at a heating rate of 20 °C/min in air with the temperature range from 50 °C to 750 °C. The structure of polymer and pH-PPM was confirmed by the Fourier transform infrared (FT-IR) measurement. The spectra were obtained in the wavenumber range from 4000 to 400 cm−1 on a Bruker Tensor-27 spectrophotometer. The magnetic property of the pH-PPM, which evaluated by vibrating sample magnetometer (VSM) was measured on a Lakeshore 7307 at room temperature. 2.3. CLE-CE analysis. Before using, the bare capillary shall be flashed with NaOH solution (0.1 M) and water for 30 min, respectively. After one injection of sample, the capillary was rinsed with 0.1 M HNO3, water, 0.1 M NaOH, water for 2 min in order. Then the capillary was filled with running buffer, which was ﬁltered by the 0.45 μm pores membrane filter. The samples of D,L-AAs (2.0 mg/mL) were prepared by 40.0 mM lithium carbonate buffer solution (pH 9.5). The derived Dns-D,L-AAs samples were introduced to capillary by siphoning for 8 s at 15.0 cm height and separated at -20 kV by the CLE-CE methods. Unless it was noted, the buffer solution at pH 8.2 was composed by 5.0 mM ammonium acetate, 100.0 mM boric acid, 3.0 mM Zn (II) and 9.0 mM [N4,4,4,4][L-Arg]. According to the previous literature, 8 the D,L-AAs were derived by Dns-Cl. Briefly, 40.0 μL lithium carbonate (40.0 mM), 40.0 μL D,L-AAs and 40.0 μL Dns-Cl (1.5 mg/mL in acetone) were added in a 200 μL vial and reacted at microwave irradiation (480 W, 6.0 min). Then,


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ethamine (5.0 μL, 2.0%) was used to terminate the reaction. The Dns-D,L-AAs stock solutions were kept at 4 oC. 2.4. Fabrication of the enzyme reactor. 2.4.1. Synthesis of the Fe3O4 magnetic nanoparticles. The Fe3O4 magnetic nanoparticles were prepared by reported methods: 8 the FeCl3·6H2O (5.4 g, 20.0 mmol) and FeCl2·4H2O (1.98 g, 10.0 mmol) were mixed in 100 mL deionized water, then 2.5 mL 25% ammonia was added to adjusted the pH of the mixture to 12.0. The mixture solution was vigorously stirred at 70 oC for 3 h. To remove the excess ammonia, the product was washed by water to neutral pH value. After that the product was washed several times with ethanol and dried at 45 oC for 24 h. 2.4.2. Synthesis of PS-MAn-AA. To obtain polymer PS-MAn-AA with well-organized structure, reversible addition-fragmentation chain transfer (RAFT) method was selected for polymer synthesis. Firstly, the PS-MAn was prepared and used as a macro-molecular chain transfer agent. The reactants including monomers MAn 10.0 mM and S 100.0 mM, chain transfer agent BBDT 0.1 mM, initiating agent AIBN 10.0 μM was added into a boiling flask-3-neck (50.0 mL) and dissolved by 5.0 mL 1,4-dioxane. The freeze-pumpthaw method was used for removing the oxygen in the solutions. Then, the reactant reacted at 60 oC for 24 h and products were collected. In detail, the products was precipitated by adding excess ether, then filtered and dried at 50 °C for 24 h with vacuum condition. Then, for synthesis the PS-MAn-AA, the PS-MAn 300.0 mg, 300.0 mg AA and AIBN 10.0 mg were dissolved in 5.0 mL 1,4-dioxane. The freeze-pump-thaw method was used to remove the oxygen in the reactant solution and then reacted at 60 °C for 12 h. The products were poured into excess of ether, filtered, and dried at 50 °C in a vacuum oven for 24 h.


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2.4.3. Fabrication of the magnetic pH-PPM. To obtain the pH-PPM applied for L-ASNase enzyme immobilization, 30.0 mg/mL PS-MAn-AA and 10.0 mg/mL the Fe3O4 magnetic nanoparticles were suspended in 1.0 mL chloroform. A clean flat glass plate was used for the preparation of pH-PPM. The mixed polymer and Fe3O4 nanoparticles solution was cast onto a clean flat glass under 95% humidity environment which prepared by a humidifier. Then the pHPPM formed on the plate soon afterwards the chloroform evaporated. 2.4.4. Immobilization of L-ASNase. The pH-PPM (10.0 mg), L-ASNase (0.05 mg/mL) and 2.0 mg LiClO4 were mixed in 1.0 mL 100 mM, pH 8.2 PBS. After that the solution was stirred for 3.0 h at 0 oC. Then the pH-PPM modified with L-ASNase was washed with a PBS buffer to remove the unreacted enzyme. Thus, the pH-PPM based L-ASNase enzyme reactor was obtained. The Bradford assay13 was used for the evaluation of immobilized L-ASNase amount onto the pH-PPM. The L-ASNase immobilized on the pH-PPM could be determined by detecting concentrations of L-ASNase solution before and after immobilization. 2.5. Kinetic study of L-ASNase@pH-PPM enzyme reactor. The free and immobilized activity of L-ASNase has been investigated by the kinetics study using L-Asn as a substrate. The Michaelis-Menten’s constant (Km) and maximum rate (Vmax) was calculated by the CLE-CE method (the detail CLE-CE separation method has been discussed in supporting information). Different D,L-Asn solutions with concentrations ranging from 17.35 μM to 6.66 mM were prepared and diluted with 50.0 mM PBS (pH 4.7 and pH 8.2). Then 20 μL L-ASNase solutions or the L-ASNase@pH-PPM were added into L-Asn solutions and reacted for 1.0 min at 37 °C. After hydrolysis, the enzyme reaction was stopped by boiling the solution for 10.0 min. For analysis, 40.0 μL up layer solution was mixed with derivatization solution as shown in Section 2.3.


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3. RESULTS AND DISCUSSION 3.1. Preparation of PS-MAn-AA. By means of RAFT polymerization and the breath figure method, PS-MAn-AA could be applied for the fabrication of pH-PPM owing to its multifunctional groups for enzyme immobilization. Firstly, an illustrative scheme of the PS-MAn-AA synthesis for obtaining controllable molecular weight efficiently is presented in Figure 1. The monomer AA was chosen to copolymerize with the polymer PS-MAn owing to its acidic sensitive property. The molecular weight and polymer distribution index (PDI) of the obtained PS-MAn and PS-MAn-AA were evaluated by GPC. The molecular weight of PS-MAn was 9.7 kD with the PDI 1.2, while PS-MAn-AA had a molecular weight about 13.8 kD with PDI 1.3. This clearly illustrated an incorporation of AA monomer into PS-MAn, as a result of living polymerization of RAFT. S O

O

O

+

N

N

C

S S N

N

n

m

C

O

AIBN, THF, 60 oC

S O

O

S HO

O

N

N

C

m

AIBN, THF, 60 oC

i

n

O

O

O HO

S

O

Figure 1. The synthesis procedure of PS-MAn-AA.


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3.2. Preparation and characterization of pH-PPM enzyme reactor. The simple fabricated pH-PPM consists of PS-MAn-AA and magnetic nanoparticles Fe3O4, which were mixed together in the chloroform solvent and stirred. Then, a closed case with 95 % humidity was setup. Finally, the mixture was cast onto a flat glass plate and the pH-PPM would be prepared after solvent evaporated (Figure 2A). The morphology of the pH-PPM was observed by the SEM and the result was displayed in Figure 2B. Ordered micro and nano porous could be found in the membrane which could be used for the enzyme immobilization. L-ASNase enzyme could be immobilized easily onto the pH-PPM by MAn block in the PS-MAn-AA for covalently immobilization (Figure 2A).

Figure 2. (A) The fabrication process of pH-PPM and schematic illustration pH sensitive property of L-ASNase@pH-PPM. (B) SEM image of the pH-PPM morphology (Scale bar, 5 µm). 3.3. Characterization of pH-PPM. For confirming the structures of polymers and magnetic membrane, FT-IR spectra of Fe3O4 nanoparticles, PS-MAn-AA and pH-PPM have been characterized (Figure 3A). The peaks at 574.7 cm-1 and 3417.7 cm-1 could be ascribed to the typically stretching vibration absorption of Fe-O from cores of Fe3O4 magnetic nanoparticles


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and the –OH absorption of the nanoparticles surface. The characterized stretching vibration absorption of CH2 in benzene ring at 2925.9 cm-1 and typical peak of C=O in maleic anhydride at 1847.7 cm-1, 1857.7 cm-1 which belong to the PS-MAn-AA were also be observed in the pHPPM sample. The data confirmed that the reactive pH-PPM has been successfully prepared. The TGA curves of Fe3O4 nanoparticles, pH-PPM and L-ASNase@pH-PPM were shown in Figure 3B. It could be observed that the weight loss of residual water was below 300 °C. The weight loss of pH-PPM and L-ASNase@pH-PPM between 400 °C and 750 °C was attributed to the degradation of polymer and L-ASNase. Owing to the different weight loss temperature of polymer and L-ASNase, the TGA curves were obviously different at the range of 670 °C and 750 °C.

Figure 3. (A) FT-IR spectra of Fe3O4 magnetic nanoparticles (a), PS-MAn-AA (b), and LASNase@pH-PPM (c). (B) TGA analysis of the Fe3O4 nanoparticles, pH-PPM, and LASNase@pH-PPM. The magnetic properties of these synthesized materials were investigated using VSM (Figure 4A). The saturation magnetization of the magnetic Fe3O4, pH-PPM, L-ASNase@pHPPM enzyme reactor were 1.71, 0.52 and 0.36 emu/g, respectively. Although the magnetism of the nanoparticles normally decreases with stepwise modification, these materials showed very


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low coercivity and could be magnetic collected quickly within 5 s (Figure 4B and 4C), which displayed the typical superparamagnetic property at room temperature. These results demonstrated that the pH-PPM possessed good magnetic responsiveness, which could be useful for separation and reuse of the immobilized L-ASNase.

Figure 4. (A)The magnetization curves detected by vibrating sample magnetometer of Fe3O4 (a), pH-PPM (b) and L-ASNase@pH-PPM enzyme reactor (c) at room temperature. Photographs of L-ASNase@pH-PPM enzyme reactor aqueous suspensions before (B) and after (C) complete magnetic capture within 5 s. 3.4. CLE-CE system. To evaluate the changing of D,L-amino acids, which are substrates of the kinetics study of enzyme, a unique CLE-CE system was constructed for the chiral separation of D,L-amino acids. Furthermore, the important parameters which could influence the chiral separation efficiency have been investigated in detail. The derivatized D,L-amino acids including Dns-D,L-Ala, Dns-D,L-Asn, Dns-D,L-Ser was selected as the test samples to evaluate the CLECE separation method. The optimization of CLE-CE separation conditions was studied in detail and displayed in Supporting Information, including the influence of buffer pH (Supporting Information, Figure S1 and Figure S2), ligand to Zn(II) ratio (Supporting Information, Figure S3 and Figure S4), concentration of complexes (Supporting Information, Figure S5 and Figure S6)


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on chiral separation efficiency. Finally, the optimized CLE-CE system, in which the running buffer at pH 8.2 was composed by 100.0 mM boric acid, 5.0 mM CH3COONH4, 3.0 mM Zn (II), 9.0 mM [N4,4,4,4][L-Arg]. The quantity analysis of Dns-D,L-Asn has been developed under the optimized CLE-CE conditions. The linear relationship (Supporting Information, Figure S7 and Figure S8) with favorable repeatability of Dns-D-Asn and Dns-L-Asn were obtained which would provide the analysis method for kinetics study of L-ASNase. The results displayed that good linearity of the linear regression analysis was obtained (r2 = 0.994 for Dns-D-Asn, r2 = 0.981 for Dns-L-Asn) in the range of 17.4-6.7×103 μM. The limit of detection (LOD) was both 8.7 μM for Dns-D-Asn and Dns-L-Asn. The relative standard deviations (RSD) of peak area repeatability and migration time of Dns-L-Asn were < 1.9 % and < 3.4 %, respectively. While, the RSD of peak area repeatability and migration time of Dns-D-Asn were < 2.1 % and < 3.7 %, respectively. 3.5. Optimization of L-ASNase immobilizing. With the polymer film formed, L-ASNase could be immobilized onto the surface of the pH-PPM. The MAn block in the polymer chains could react with amino groups in enzyme at a moderate condition to form covalent bonds easily. For optimizing the reaction conditions, the influence of polymer ratio of PS-MAn to AA, the ratio of PS-MAn-AA to Fe3O4 on pH-PPM fabrication, the immobilization reacting time and concentrations of L-ASNase on reacting efficiency have been investigated. According to the enzymolysis of L-ASNase as shown in Figure 5, the activity of the immobilized L-ASNase was calculated by the decrease of enzymatic substrate L-Asn after incubated with pH-PPM. To evaluate the enzymolysis activity of the pH-PPM enzyme reactor, the percentage of substrate L-Asn hydrolysis after incubated with L-ASNase@pH-PPM enzyme reactor, H (%), was applied and calculated.


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H (%)=

Ao-AL-Asn Ao

* 100%

where A0 and AL-Asn are the values of L-Asn peak areas before and after enzyme reaction using L-ASNase@pH-PPM, respectively.

O

O L-ASNase

O

O

OH

OH NH2

NH2

OH

L-Asn

NH2 L-Asp

Figure 5. The enzyme reaction of L-Asn hydrolysis to L-Asp by L-ASNase enzyme. Firstly, different kinds of polymers have been synthesized with the ratio of PS-MAn to AA various from 2:1 to 1:2, and then applied for fabrication of pH-PPM. Prior to the immobilization step, the concentration ratio of PS-MAn to AA from 2:1 to 4:1 were detected. Figure 6A indicates that the best enzymolysis efficiency was obtained at the PS-MAn to AA in polymer ratio of 1:1. Figure 6B shows that the pH-PPM enzyme reactor fabricated using concentration ratio of PS-MAn-AA to Fe3O4 at 3:1 (30.0 mg/mL:10.0 mg/mL) could achieve the highest enzymolysis efficiency. The influence of immobilization concentration and reaction time of LASNase on the enzymolysis efficiency of the pH-PPM enzyme reactor also has been studied. Different L-ASNase solutions ranging from 0.03 mg/mL to 0.07 mg/mL and the enzyme immobilization time (1.0-5.0 h) were used for getting the highest enzymolysis efficiency. According to the results, the immobilization time for 3.0 h and L-ASNase concentration at 0.05 mg/mL was chosen finally (Figure 6C and Figure 6D). It is noted that the addition of LiClO4 in


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the enzyme immobilization process is worthwhile not only for efficient activation of anhydride group but also for promoting nucleophilic attack during the mild condition.

Figure 6. Optimization of polymer ratio of PS-MAn to AA (A), the ratio of PS-MAn-AA to Fe3O4 on pH-PPM fabrication (B) and the immobilization reacting time (C) concentrations of LASNase (D) on immobilization efficiency in the enzyme immobilization process. The pH-PPM was good candidate for L-ASNase immobilization because it could enhance the reusability of the enzyme and extend its application for online hydrolysis. The amount of immobilized L-ASNase was measured by the Coomassie blue assay. The calibration curve was developed by measuring the UV-vis absorbance (at 595 nm) of the L-ASNase solutions ranging from 1.25 μg/mL-0.01 mg/mL (y=7.72x +0.48, R2=0.988). The results depicted that the concentration of L-ASNase decreased about 2.1 μg/mL after enzyme immobilization according


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to the obtained calibration curve. It is calculated that about 2.1 μg (in 1.0 mL buffer solution) LASNase was immobilized onto 10.0 mg pH-PPM material, which is similar as the previously reported data.

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The results indicated that the pH-PPM was favorable for L-ASNase

immobilization and could provide perfect platform for enzyme immobilization and kinetics study. 3.6. The enzyme kinetics study. To prove the good property of L-ASNase@pH-PPM enzyme reactor, the kinetics study of free L-ASNase and immobilized L-ASNase at different pH were investigated by the decrease of substrate L-Asn. The hydrolysis efficiency of immobilized and free L-ASNase was estimated through the variation of the L-Asn peak area determined by the developed CLE-CEC technique. The constants of kinetic enzyme reaction Km and Vmax , which could evaluate the kinetics of the enzyme, were detected by Michaelis-Menten equation: [S] / V = Km / Vmax + [S] / Vmax In this equation, [S] is the concentration of the substrate (L-Asn), Km is Michaelis constant. V and Vmax are the initial and maximum velocities of enzyme reaction, respectively. The initial velocity means velocity determined at the very beginning of the enzymolysis reaction (1.0 min for this work). At that condition, the enzyme concentration approximately remained constant. It is well known that the activity and stability of an enzyme is strongly influenced by buffer solution pH. The proposed L-ASNase@pH-PPM enzyme reactor was a pH-sensitive membrane. Therefore, the buffer solution at different pH from 3.0 to 10.0 was optimized during hydrolysis. Figure 7 displayed that the enzymatic efficiency of L-ASNase@pH-PPM at pH 8.2 reached the highest value, which was consistent with the optimum pH of L-ASNase. 18


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40.0

Enzymatic efficiency / %

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30.0

20.0

10.0

0.0

1 3.0

2 4.7

3 7.0

4 8.2

5 10.0

pH

Figure 7. Effect of buffer solution at different pH on L-ASNase@pH-PPM hydrolysis activity. For testify the precisely enzymolysis rate control in the complex conditions by pH of LASNase@pH-PPM enzyme reactor, the Km and Vmax of the free enzyme in solution and LASNase@pH-PPM at different pH (acidity and alkalinity) have been investigated. The results (Table 1) showed that comparing with the free enzyme, the L-ASNase immobilized on pH-PPM has a higher Vmax value at pH 4.7 and 8.2, respectively. Moreover, owing to the pH sensitivity of pH-PPM, the immobilized L-ASNase obtained three folds higher Vmax value at pH 8.2 than that at pH 4.7. Table 1. Lineweaver-Burk plot for (a) free L-ASNase solution and (b) L-ASNase immobilized on pH-PPM Free enzyme pH

L-ASNase@pH-PPM

Km

Vmax

Km

Vmax

(mM)

(mM / min)

(mM)

(mM / min)

4.7

0.20

0.11

0.58

0.21

8.2

0.36

0.23

0.19

0.67


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The mechanism of precisely enzymolysis rate control of the L-ASNase@pH-PPM could be explained by the membrane property (Figure 2A). The AA moiety of PS-MAn-AA provided obviously effect on the hydrolysis efficiency of L-ASNase under different pH value, as compared to the free enzyme in solution. At a higher pH buffer solution (pH 8.2), the AA moiety would be more hydrophilic and have negative charge on the polymer chains. At this situation, the polymer chains easily stretched into the aqueous solution and provided larger cavity of the enzyme, which could allow more substrates come into the porous and enhance the impact between the substrate and enzyme (Figure 2A). However, in a buffer solution at pH 4.7, the negative charge on AA moiety of polymer would be neutralized and change to be less hydrolysis. At this condition, the AA chains on the porous copolymer would curl together and embedded the L-ASNase in a much smaller cavity, which could prohibit the impact between the substrate and enzyme. Thus, the hydrolysis efficiency of immobilized L-ASNase could obviously decrease at lower pH buffer solution. Moreover, it should be noted that the enzymatic activity of free enzyme or immobilized enzyme might be partly influenced by the buffer pH, Table 1 and Figure 7 showed that comparing with free enzyme, the enzymatic activity (Vmax) of L-ASNase@pHPPM could obviously enhance not only at pH 4.7, but also at pH 8.2 (Table 1). That means the AA polymer chains of the pH-PPM in the stretching state or in the curling state indeed influenced the enzymatic activity of the immobilized L-ASNase. 3.7. Enzyme reactor stability and reusability. In the view of potential application of these membrane enzyme reactors for other enzymes and taking advantage of stimuli-responsive property of these functional enzyme reactors, we evaluated the stability and reusability of the pH-PPM enzyme reactors. Firstly, the relative activities of immobilized L-ASNase at the beginning and after four weeks have been investigated. The results displayed that after


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four weeks at the same storage condition, the immobilized L-ASNase still could maintain 73.2% relative activity, while the relative activity of free enzyme in solution only retained 20.3%. The results showed that the designed L-ASNase enzyme reactor possessed well stability for amino acids hydrolysis. Further, we investigated the reusability of the immobilized L-ASNase on pH-PPM. The L-Asn as substrate was hydrolysed repeatedly by the same pH-PPM enzyme reactor. The results displayed that the immobilized enzyme could maintain 48.0% relative activity after sixteen runs (Figure 8A). The batch repeatability of the fabricated L-ASNase@pH-PPM enzyme reactor was also investigated and the data was exhibited in Figure 8B. Moreover, a repeatable method for producing five enzyme reactors has been carried out. These enzyme reactors was exactly evaluated by the enzymatic efficiency (RSD=0.2%, n=5). The data means the batch repeatability of the fabricated L-ASNase enzyme reactor is good. Therefore, it could be confirmed that the pH-PPM enzyme reactors has been successfully achieved, recreated and could be taking into account the activity of regulating the hydrolysis rate by changing condition very conveniently.

Figure 8. Reusability (A) and batch fabrication repeatability (B) of L-ASNase@pH-PPM based enzyme reactor.


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3.8. Application of L-ASNase@pH-PPM. As an effective antitumor agent, L-ASNase has been applied in the treatment of acute lymphoblastic leukemia.15, 18 To further promote the application, it is meaningful to explore the L-ASNase@pH-PPM enzyme reactor for hydrolysis of L-Asn in human serum samples. The D,L-Asn spiked human serum samples were incubated with the LASNase@pH-PPM based enzyme reactor and the results were shown in Figure 9. By using CLECE technique, the baseline chiral separation of D,L-Asn in human serum samples could be achieve and the peak of substrate L-Asn decreased significantly because of the enzymolysis (Figure 9c). The results demonstrated that the proposed L-ASNase@pH-PPM enzyme reactor indeed has the great potential in real clinical application.

Dns-D-Asn

4.0

Abs / mAu

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


3.0

Dns-L-Asn

c

2.0

b 1.0

a

0.0 20.0

21.0

22.0

23.0

24.0

Migration time / min

Figure 9. Electropherograms of L-Asn enzymolysis in human serum samples by LASNase@pH-PPM enzyme reactor: (a) human serum sample and (b) human serum sample spiked with 62.5 μM D,L-Asn (c) human serum sample spiked with 62.5 μM D,L-Asn enzymolysis by L-ASNase@pH-PPM enzyme reactor at 37 °C for 5 min. Buffer condition: 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM Zn (II) and 9.0 mM AAIL [N4,4,4,4][L-Arg], pH 8.2; Capillary: 75 μm id × 60 cm length (45 cm effective); injection: siphoned for 8 s at 15 cm; voltage: - 20 kV; UV detection: 254 nm; 25 oC.


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4. CONCLUSION In this work, we have presented a new methodology to fabricate enzyme reactors to precisely control the enzymolysis rate in the complex conditions. For this purpose, the polymer based pH sensitive enzyme reactor has been developed and applied in L-ASNase immobilization. Moreover, MAn groups and pH sensitive part (AA section) were available to establish interactions with amine groups of L-ASNase and pH sensitive polymeric pores. Importantly, upon self-assemble of AA section, the enzyme immobilized at the porous surface maintained a reasonable activity, which could be tuned by the buffer pH. This methodology allowed us to fabricate pH-PPM for catalytic control enzymolysis during enzyme reaction in a straightforward manner. Moreover, this kind of pH sensitive enzyme reactor could pave a new area for stimuliresponsive materials applying in enzyme reactor fabrication and show bright future for improving enzyme property and efficiency.

ASSOCIATED CONTENT Supporting Information. The optimization of CLE-CE separation conditions was displayed in the Supporting Information.

AUTHOR INFORMATION Corresponding

Author:

*Tel:

+86-10-82627290.

Fax:

+86-10-62559373.

E-mail:

[email protected].

ACKNOWLEDGMENT We are grateful for the financial support from National Natural Science Foundation of China (Grants 21575144, 21874138, 21635008, 21621062, 21727809) and Chinese Academy of


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Sciences (QYZDJ-SSW-SLH034). We also thank Dr. Liping Zhao for her kind help in SEM characterization.

ABBREVIATIONS PS-MAn-AA, poly styrene-co-maleic anhydride-acrylic acid; L-ASNase, L-asparaginase; pHPPM, pH-responsive polymer porous membrane; L-Asn, L-asparagine; L-Asp, L-aspartic acid; L-Arg, L-arginase; Dns-Cl, dansyl chloride; AIBN, azobisisobutryonitrile; [N4,4,4,4][Br], tetramethylammomium bromide; GPC, Gel permeation chromatography; VSM, vibrating sample magnetometer; RAFT, reversible addition-fragmentation chain transfer; BBDT, benzyl benzodithioate; CLE-CE, chiral ligand exchange capillary electrophoresis.

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16. Cachumba, J. J. M.; Antunes, F. A. F.; Peres, G. F. D.; Brumano, L. P.; Santos, J. C. D.; Silva, S. S. D., Current applications and different approaches for microbiall-asparaginase production. Braz. J. Microbiol. 2016, 47 (Suppl 1), 77-85. 17. Jean-François, J.; Fortier, G., Immobilization of L-asparaginase into a biocompatible poly(ethylene glycol)-albumin hydrogel: I: Preparation and in vitro characterization. Biotechnol. Appl. Biochem. 1996, 23 (3), 221-226. 18. Kumar, S.; Dasu, V. V.; Pakshirajan, K. Studies on pH and Thermal Stability of Novel Purified L-Asparaginase from Pectobacterium Carotovorum MTCC 14281 Microbiology, 2011, 80(3), 355-362.

The table of contents Graphic


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Enzyme Reactor Based on Reversible pH-Controlled Catalytic

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