An ideal enzyme immobilization carrier: a hierarchically porous cellulose monolith fabricated by phase separation method

Materials

CA powder (M_n=5.0×10⁴; 39.2–40.2 wt% acetyl content) and picrylsulfonic acid solution (TNBS, 5% (w/v) in H₂O) were was purchased from Sigma-Aldrich Co., (St. Louis, MO, USA). Sodium hydroxide (NaOH), epichlorohydrin (EC), sodium thiosulfate, trimethylamine (Et₃N), HRP, hydrogen peroxide (H₂O₂), phenol, N,N-dimethylformide (DMF), 1-hexanol, ethanol, hydrochloric acid solution (HCl; 0.01 and 1.0 M), methanol and phosphate (PBS, 1/15 M, pH 7.0) buffer were obtained from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). Disuccinimidyl carbonate (DSC), 4-aminoantipyrine (4-AAP) and borate buffers (pH=8.0 and 9.0) were supplied from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Bradford protein assay kit was purchased from Thermo Fisher Scientific Inc. (Rockford, IL, USA). All the reagents were used as received without further purification.

Cellulose monolith was fabricated according to our previous paper. Briefly, the preparation method of cellulose monolith used in this study was described as follows. Firstly, CA monolith was prepared through TIPS method. The CA homogeneous solution with concentration of 200 mg/mL was obtained using the mixed solvent (DMF/1-hexanol (v/v)=1/1.5) by heating at 70°C, followed by being maintained at 20°C for 24 h to complete the phase separation. After solvent replacement with ethanol and drying in vacuo, CA monolith was fabricated successfully. Subsequently, CA monolith was immersed into a 2 M NaOH solution in methanol for 3 h at room temperature to perform the hydrolysis reaction. After neutralizing with 1 M HCl and washing with water and methanol thoroughly, cellulose monolith was obtained by drying in vacuo.

Instruments

Fourier-transform infrared (FT-IR) spectroscopy measurements using the attenuated total reflectance (ATR) method were performed by a Thermo Scientific Nicolet iS5 with iD5 ATR accessory (Waltham, MA, USA). Scanning electron microscopy (SEM) images were recorded by a Hitachi S-3000N instrument (Tokyo, Japan) at 15 kV. A thin gold film was sputtered on the samples before the images were collected. UV-Vis absorbance measurements were carried out using a Hitachi U-2810 UV-Vis spectrometer (Tokyo, Japan).

Synthesis of functionalized cellulose monolith for enzyme immobilization

In order to immobilize enzyme covalently onto the surface of cellulose monolith, certain functional groups are required through a series of chemical modifications. In this work, functionalized cellulose monolith with the efficient SC groups (SCCL monolith) for enzyme immobilization was obtained through the following steps. Firstly, epoxy-modified cellulose monolith (ECL monolith) was synthesized through the reaction with EC in NaOH aqueous solution for 2 h at 30°C, followed by reacting with ammonia aqueous solution for 24 h at 70°C to obtain primary amino functionalities onto the monolith (AMCL monolith) [29]. SCCL monolith was prepared via the reaction of amino groups with DSC anhydrous DMF solution with the addition of Et₃N for 24 h at room temperature.

The determination of the amount of corresponding functional groups on the surface of cellulose monolith were described as follows. For the calculation of epoxy group, 100 mg of ECL monolith was suspended into 10 mL of 1.0 M sodium thiosulfate aqueous solution. After stirring at room temperature for 5 h, 0.01 M HCl solution was used to titrate the released sodium hydroxide and phenolphthalein was employed as the indicator. The amount of epoxy group was calculated according to the volume of HCl solution consumed in the titration process. The amount of primary amine groups located on the surface of AMCL monolith was determined through a TNBS method. AMCL monolith (50 mg) was immersed into 10 mL of TNBS solution (10 mM) in borate buffer (100 mM, pH 9.0) and reacted for 2 h at room temperature. After rinsing, the alkaline hydrolysis was performed by addition of potassium hydroxide solution (3 mL, 2 M) and stirring for 3 h at 45°C, resulting in the release of the covalently combined 2,4,6-trinitrophenol from monolith. The absorbance of the alkaline solution was measured, which is proportional to the amount of primary amount content (spectrophotometry at λ=385 nm against a borate buffer blank; extinction coefficient of ε_{385 nm}=14 100 M⁻¹·cm⁻¹). To quantify the amount of SC groups, 20 mg of SCCL monolith was immersed into a 2 mL of ammonia water (0.1 M) for 5 min with stirring slightly at room temperature. After removal of the monolith, the amount of SC which is equal to the formed N-hydroxysuccinimide was determined by the measurement of UV absorbance at 260 nm (extinction coefficient of ε_{260 nm}=9700 M⁻¹·cm⁻¹).

Enzyme immobilization on SCCL cellulose monolith

Firstly, 20 mg of the SCCL monolith was equilibrated in PBS buffer (1/15 M, pH 7.0) for 30 min, followed by immersing into a HRP solution (1.0 mL, 1.0 mg/mL). After incubating for 24 h at 15°C, monolith was rinsed for 3 times by PBS buffer to remove the physically adsorbed HRP and stored at 4°C in the same buffer solution until use. The amount of the immobilized HRP was determined through the concentration change of HRP solution before and after the immobilization experiment, which was calculated by Bradford protein assay method using the UV adsorption at 595 nm.

Activity assays of free and immobilized HRP

The activity of HRP in this work was measured through its ability to catalyze the chromogenic reaction of phenol, 4-AAP and hydrogen peroxide to form a benzoquinone derivative, which has UV adsorption at 510 nm. As for free enzyme, 5 μg/mL HRP in PBS buffer was used; for immobilized enzyme, 20 mg of HRP-immobilized SCCL monolith was employed. The specific conditions of the chromogenic reaction were as follows: 60 mM of phenol, 14 mM of 4-AAP and 2 mM of H₂O₂ were dissolved in PBS buffer solution (1/15 M, pH 7.0).

Thermal stability and reusability of immobilized HRP

Thermal stability of the immobilized and free HRP was measured after incubating at 60°C in PBS buffer (1/15 M, pH 7.0) for 160 min. After every 10 min time interval, HRP immobilized SCCL monolith was taken out and the residual enzyme activity was assayed as described above. The reusability of the immobilized HRP was evaluated through the residual activity measurement after each cycle, which was represented as a percentage of its initial activity. The monolith was rinsed 3 times by PBS buffer (1/15 M, pH 7.0) after use.

Introduction of reactive groups on cellulose monolith

The morphology such as pore size and its distribution of the solid support plays a key effect on the biological activity of the immobilized enzyme. Here, cellulose monolith with a hierarchically porous structure was utilized to immobilize HRP. Up to now, numerous efforts have been devoted to develop cellulose monolith with hierarchically porous structure and the solubilization step is a crucial factor for the fabrication process. Commonly used solvent systems include NaOH or LiOH/urea/water [37], [38], [39], [40], and Ca(SCN)₂/water [41], [42], [43]. However, the corresponding fabrications using these solvent systems always have problems like the use of toxic chemicals, high cost, and tedious procedures. Besides, freeze drying or supercritical drying are often required for isolation of the porous cellulose materials, which is also unsuitable for industrial applications due to the high cost and energy use.

Herein, we designed a cellulose monolith for immobilization of enzymes, which has been fabricated from a CA solution by the TIPS method [29]. The morphology of the cellulose monolith can be tuned easily through changing the fabrication parameters to adapt different practical applications. The resultant cellulose monolith used in this study possessed a three-dimensional open pore structure with average skeleton size of approximately 3.6 μm according to the results of SEM. It also exhibits mesoporous structure (average pore size of 11.2 nm) with large surface area (42.3 m² g⁻¹) calculated by BET equation from nitrogen adsorption/desorption isotherms. These results demonstrated a hierarchically porous structure was formed inside the resultant cellulose monolith.

Surface chemistry of the solid support is a significant factor for the enzyme immobilization. Epoxy and aldehyde groups are the well-investigated and most commonly functionalities used to covalently react with the primary amino groups to immobilize enzymes. However, for epoxy groups, the immobilization reaction is rather slow especially under mild conditions like neutral pH and low ionic strength. For aldehyde groups, although the immobilization time is much shorter, an extra reduction reaction for C=N bonds with NaBH₄/NaBH₃CN is necessary and therefore the whole coupling processes become tedious and time-consuming. In addition, quenching procedure with blocking agents is required for both functional groups to prevent nonspecific adsorption.

In this work, in order to improve the immobilization efficiency, SC functional group with high reactivity and selectivity was introduced on the surface of cellulose monolith through a series of chemical reactions. The formed carbonate bond has relatively high chemical stability. Moreover, the unreacted SC groups are easily deactivated into hydroxyl groups in aqueous solution with high pH with release of N-hydroxysuccinimide and CO₂. Therefore, no residual SC groups remain and the blocking procedure is unnecessary, which is much more convenient and efficient compared to epoxy and aldehyde groups.

Figure 1 shows the modification process of cellulose monolith. The hydroxyl groups of cellulose were transferred into epoxy groups and primary amine groups successively. Afterwards, the introduction of final functional SC groups on the skeleton surface of the cellulose monolith was performed. Specifically, the epoxy group was introduced through the reaction of the cellulose monolith with EC in NaOH solution [29]. The epoxy content of the ECL monolith was determined to be 270 μmol g⁻¹. Subsequently, the ECL monolith was converted into the AMCL monolith by the reaction with a large excess of ammonia solution, and the resultant amine content was determined as 126 μmol g⁻¹. The primary amine on the AMCL monolith was converted into an SC group by the reaction with a large excess of DSC, and the SC content was calculated as 95 μmol g⁻¹.

Fig. 1:

Introduction of reactive (a) epoxy, (b) primary amine, and (c) SC groups onto cellulose monolith and the SEM images of (d) cellulose, (e) ECL, (f) AMCL and (g) SCCL monoliths.

The introduction of these functional groups into the cellulose-based monolith were confirmed by FT-IR spectroscopy (Fig. 2). For the ECL monolith, the absorbance peak attributed to the stretching vibration of O–H became much smaller compared with that of the cellulose monolith. Additionally, new peaks located at 1000, 1020, and 1150 cm⁻¹ from the stretching vibration of C–OH and broad peaks at 806–871 and 907 cm⁻¹ due to the epoxy ring vibration were observed in the spectrum of the ECL monolith (Fig. 2b). In the spectrum of the AMCL monolith (Fig. 2c), the absorbance peaks attributed to the epoxy ring disappeared after the reaction with ammonia solution, supporting the introduction of the primary amine group. A characteristic peak at 1640 cm⁻¹ due to the stretching vibration of the SC group newly appeared in the FT-IR spectrum of the SCCL monolith (Fig. 2d). These results clearly demonstrated that the epoxy, primary amine, and SC groups were successfully introduced on the skeleton surface of the cellulose monolith. Moreover, through the modification process, a relatively long spacer (five atoms) was formed between the surface of cellulose monolith and the enzyme, which was favorable to maintain the conformation integrity and achieved an efficient enzyme immobilization result.

Fig. 2:

FT-IR spectra of (a) cellulose, (b) ECL, (c) AMCL and (d) SCCL monoliths.

Enzyme immobilization on SCCL monolith

The covalent immobilization of HRP on SCCL monolith was realized through the coupling reaction between the primary amino groups of HRP and SC functional groups located at the surface of cellulose monolith. According to the results of Bradford protein assay, the maximum HRP immobilization amount of SCCL monolith was 21.0 mg/g, which was relatively higher than other published solid supports due to the high surface area and hierarchically porous morphology of the monolith as well as the high hydrophilic property of the cellulose material [3], [7], [18], [44].

Thermal stability of free and immobilized HRP

It is well-known that enzyme is usually a kind of fragile biocatalyst and it is denaturated easily under harsh environment including high temperature. Immobilization is an efficient method to improve the thermal-resistance of enzyme. Figure 3 shows the thermal stability profile of the free and immobilized HRP incubated at 60°C. All the HRP activities were represented as the percentage of the maximum activity. It is observed that the immobilized HRP could maintain at relatively higher residual activity whereas the free HRP dropped sharply at same temperature. After 160 min, the activity of immobilized HRP still remained 75.4% but the free HRP almost lost all the activity (6.0%). These results demonstrated that immobilizing HRP on cellulose monolith greatly improved its thermal stability. The reason could be explained that through the covalent bonding onto surface of monolith, the conformational change of HRP was considerably restricted and its diffusion was largely limited, resulting in the improved thermal stability.

Fig. 3:

Thermal stability of (a) immobilized HRP and (b) free one at 60°C.

Reusability of immobilized HRP

One of the most important advantages of the immobilized enzyme is that it could be reused several time without significant activity loss. Here, the reusability of the immobilized HRP on SCCL monolith is shown in Fig. 4. The activity of HRP maintained 60.4% after 10 consecutive cycles, which was much higher than other published results. The outstanding performance was probably resulting from the hierarchically porous structure of the cellulose monolith. Macroporous structure could provide the HRP enough space to remain its active structure. On the other hand, the mesoporous structure with large surface area guaranteed the fast mass transfer with good product recovery and minimal loss of enzyme activity.

Fig. 4:

Reusability of immobilized HRP onto SCCL monolith.