Engineering peptide-based biomimetic enzymes for enhanced catalysis

Abstract

Herein, we design and synthesize a novel hydrolase model by integrating the supramolecular self-assembly of an amphiphilic short peptide (Fmoc-FFH) and electrostatic complexation (with PEI) at an aqueous liquid–liquid interface to synthesize stable peptide–polymer Fmoc-FFH/PEI hybrid capsules (FPCs). After treatment with glutaraldehyde as a crosslinking agent, we can obtain novel Fmoc-FFH/PEI/GA hybrid capsules (FPGCs). The FPGCs with imidazolyl groups as the catalytic centers exhibit high catalytic activity for the hydrolysis of p-nitrophenyl acetate (PNPA). The resulting hydrolase model (FPCs or FPGCs) shows kinetics behavior typical of natural enzymes, and the catalytic activity is higher than that of a Fmoc-FFH hydrogel. The enhanced catalytic activity may be attributed to the high density of catalytic sites on the inner surface of the hybrid capsule. Additionally, the FPGCs retained 93% of their productivity after fifteen cycles, suggesting high stability and excellent recyclability. This novel hybrid capsule is expected to be applied as a substitute for natural hydrolases in industrial production applications.

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Introduction

Enzymes catalyze a considerable variety of reactions with extremely high catalytic efficiency and specificity under mild conditions.^1,2 However, problems such as low operational stability, high costs associated with preparation and purification, and particularly difficulties in recovery and recycling greatly limit the applications of enzymes.^3,4 To overcome the aforementioned limitations, mimetic enzymes have rapidly emerged as an active field of research.^5–11 Over the past several decades, the development of enzyme mimics has been the focus of considerable research efforts. However, the high complexities of enzymes have prevented researchers from replicating the enzymatic features. Therefore, it is completely unrealistic to develop synthetic chemical equivalents to natural enzymes in terms of structure, catalytic efficiency, specificity, selectivity, and so forth. To address this problem, a new strategy of supramolecular chemistry for enzymatic mimicking has recently been developed.^5,6,12–14

It is well known that various weak supramolecular interactions play important roles in the recognition of substrates, stabilization of transition states, and enhancement of catalytic activities.^15,16 Based on the unique advantages of supramolecular chemistry, researchers can construct highly ordered nanostructures to mimic the characteristics of natural enzymes.^9,17–22 In this respect, Liu and co-workers developed many artificial enzymes via self-assembly.^5,10,23,24 For instance, Zou et al.²³ reported a supramolecular vesicle with controllable glutathione peroxidase (GPx) activity via the self-assembly of supra-amphiphiles formed by host–guest recognition between cyclodextrin and adamantane derivatives. Additionally, Soberats et al.²⁵ prepared an artificial enzyme by combining the catalyst 2-aminobenzimidazole with resorcinarene cavitands to mimic the enzymatic hydrolysis of p-nitrophenylcholine carbonate. Zaramella et al.²⁶ synthesized an artificial enzyme through the self-assembly of histidine-containing peptides H1–H3 on the surface of AuMPC 1, which triggers their esterolytic activity. Recently, in our group, the catalytic triad (Ser/His/Asp) of natural hydrolase was introduced into the peptide segment of 9-fluorenylmethoxycarbonyl diphenylalanine (Fmoc-FF). Based on the self-assembly and co-assembly of Fmoc-peptides, a series of supramolecular nanofibers were obtained as artificial hydrolases.²⁷ These highly ordered nanostructures tend to have similar advantages: (i) dynamic supramolecular structures, (ii) amphiphilic supramolecular architectures, (iii) supramolecular interactions as driving forces for substrate recognition, and (iv) specific and/or hydrophobic microenvironments for catalysis, which provides new ideas and methods for the development of artificial enzymes.

Generally, the use of native enzymes in their free form always faces difficulties in recovery and reuse. Hence, the need to preserve the catalytic properties of enzymes becomes increasingly important. Immobilization may result in improved stability of the enzymes toward harsh reaction conditions and moreover enable their easier separation from the reaction media and their reuse.^28–35 Analogous to native enzymes, supramolecular enzyme mimics also face recycling and reuse problems in many cases due to the small size of the supramolecular assemblies. In this study, we attempt to address this problem via the immobilization of biomimetic enzymes.

Herein, we used the imidazolyl-containing amphiphilic tripeptide Fmoc–Phe–Phe–His (Fmoc-FFH), which was obtained by incorporating imidazolyl groups into the supramolecular framework, to simulate the catalytic function of native hydrolase. Fmoc-FFH is an aromatic tripeptide that possesses an Fmoc-FF segment, which can self-assemble into nanofibers in an anti-parallel β-sheet arrangement.^36,37 We previously reported the hierarchical self-assembly of ordered materials at an aqueous liquid–liquid interface via the combination of an Fmoc-FF peptide solution with a cationic polymer solution.^38–40 Using this strategy, hybrid peptide/polymer capsules with high stability and recyclability were obtained. Similar to Fmoc-FF, Fmoc-FFH molecules also possess strong negative charges and can be dispersed in water in their monomeric state. Therefore, in this work, we used an aqueous solution of polyethyleneimine (PEI) to induce the self-assembly of Fmoc-FFH at a liquid–liquid interface, aiming to form hybrid Fmoc-FFH/PEI capsules with Fmoc-FFH nanofibers on the inner surface. Furthermore, the capsules were treated with glutaraldehyde to improve their stability. The catalytic performance (e.g., activity, stability, and recyclability) of the immobilized Fmoc-FFH nanofibers serving as biomimetic enzymes was evaluated.

Experimental

Chemicals and materials

Fmoc–diphenylalanine–histidine peptide (Fmoc-FFH) was purchased from the Shanghai Gil Biochemical Co., Ltd.; cationic polyethyleneimine (PEI), glutaraldehyde (GA), sodium hydroxide and hydrochloric acid were purchased from the Tianjin Guangfu Fine Chemical Research Institute. All other chemicals, such as p-nitrophenyl acetate (p-NPA), p-nitrophenol (p-NP), anhydrous ethanol and anhydrous acetonitrile, were of analytical grade and were obtained from commercial sources. All chemicals were used as received without any further purification. An Fmoc-FFH solution was prepared by dispersing the lyophilized peptide powder in ddH₂O by sonication, followed by adding an appropriate amount of a 0.5 M NaOH solution to the suspension with subsequent stirring for 30 min.

Synthesis of Fmoc-FFH/PEI capsules

A 1.0 wt% polyethyleneimine (PEI) solution was prepared by diluting a 50 wt% PEI solution in deionized water. The pH of the PEI solution was then adjusted to 8.0 by adding an appropriate amount of concentrated HCl. The lyophilized Fmoc-FFH peptide was dissolved in an alkaline aqueous solution (pH 9.0) at a final concentration of 4 mg mL⁻¹ by adding 0.5 M NaOH. Then, 1.5 mL of Fmoc-FFH solution was injected dropwise (5 μL) into 5.0 mL of a 1.0 wt% PEI solution (pH 8.0) through an Eppendorf pipette. After incubating for 30 min at room temperature, the excess PEI solution was then poured off, and the formed Fmoc-FFH/PEI capsules were washed twice with potassium phosphate buffer (10 mM, pH 8.0).

Synthesis of Fmoc-FFH/PEI/GA hybrid capsules

In a typical experiment, the formed Fmoc-FFH/PEI capsules were crosslinked with glutaraldehyde (10 mL, 1.0 wt%) for 2 h to obtain new capsules (Fmoc-FFH/PEI/GA capsules, FPGCs). The FPGCs were then washed twice with potassium phosphate buffer (10 mM, pH 8.0).

Characterization of hybrid capsules

Zeta potential measurements of the Fmoc-FFH solution and PEI solutions were performed using a Zetasizer Nano-ZS (Malvern Instruments, UK) at 25 °C. The pH value of the Fmoc-FFH (4 mg mL⁻¹) solution was maintained at 9.0. For the PEI solutions (1.0 wt%), different pH values from 3.0 to 12.0 were obtained by adding concentrated HCl or NaOH. A pH meter (Microbench pH 600, Singapore) was used to measure the pH values of all the solutions.

For scanning electron microscopy (SEM) analysis, the hybrid capsules were first rinsed using ddH₂O for 5 min. The water was then removed by quickly freezing in liquid nitrogen and subsequent freeze drying under vacuum. The samples were then coated with gold using an Emitech K550 sputter coater (Hitachi High-Technologies Co., Japan) and then imaged using an S-4800 field-emission scanning electron microscope (SEM, Hitachi High-Technologies Co., Japan) at an accelerating voltage of 3 kV.

Enzyme activity assay

The enzyme activity was determined using p-nitrophenyl acetate (PNPA) as the substrates. Considering the UV spectrum of the hydrolytic product, which exhibits a strong absorption peak at 400 nm, the reaction rates were determined by monitoring the absorption increase at 400 nm with a Shimadzu 2450 UV-VIS-NIR spectrophotometer. Then, we can calculate the corresponding product concentration.

In a typical experiment, approximately fifty hybrid capsules or 250 μL of FHs (Fmoc-FFH hydrogels, both including 1 mg of lyophilized Fmoc-FFH powder) was added to 4750 μL of phosphate buffer solution (10 mM, pH 8.0). After incubating for 2 min at 37 °C, the reaction was initiated by adding 250 μL of a PNPA acetonitrile solution (100 mM); eventually, the substrate concentration became 5 mM. The absorption increase at λ = 400 nm due to the release of the product 4-nitrophenol (PNP) was recorded.

The self-hydrolysis of PNPA and its hydrolysis in the presence of Fmoc-FF/PEI capsules or Fmoc-FF/PEI/GA capsules were tested as the control experiment. The preparation of Fmoc-FF/PEI capsules and Fmoc-FF/PEI/GA capsules and the activity assay is the same as the procedure described above.

Effects of pH and temperature on the activity of the hybrid capsules and FHs

The optimal pH for the hybrid capsules and FHs was determined using PNPA as a substrate in the pH range of 6.0 to 10.0 at 37 °C. The pH buffer was a 10 mM phosphate-buffered solution (pH 8.0). The optimal temperature for the hybrid capsules and FHs was determined by measuring the initial reaction rate in the temperature range of 30 to 70 °C in phosphate buffer (10 mM, pH 8.0). All of these reactions were performed according to the method described in the Enzyme activity assay section.

Determination of kinetic parameters of Fmoc-FFH/PEI hybrid capsules

The kinetic parameters (K_cat and K_m) of the Fmoc-FFH/PEI hybrid capsules were determined by assaying the enzymatic activity in 10 mM phosphate buffer (pH 8.0) at 37 °C with seven different concentrations (10, 20, 25, 33, 50, and 100 mM) of PNPA, 4-nitrophenyl butyrate (PNPB) or 4-nitrophenyl phosphate (PNPP) as a substrate. The Michaelis–Menten equation was used to fit all the kinetic data to a Lineweaver–Burk plot, and SigmaPlot software (Systat Software, Chicago, IL, USA) was used to calculate the kinetic parameters. The initial reaction rate (V) at different substrate concentrations ([S]) was plotted according to the Lineweaver–Burk double-reciprocal model (eqn (1)).where V is the apparent initial catalytic rate, V_max is the maximum apparent initial reaction rate, [S] is the substrate concentration, K_m is the apparent Michaelis–Menten constant, K_cat measures the number of substrate molecules turned over per enzyme molecule per second, and [E] is the enzyme concentration. The kinetic parameters, including K_m, V_max, K_cat, and K_cat/K_m can be calculated from eqn (1) and (2).

Recycling assay of Fmoc-FFH/PEI and Fmoc-FFH/PEI/GA hybrid capsules

Recycling studies of Fmoc-FFH/PEI and Fmoc-FFH/PEI/GA hybrid capsules were conducted on a 5 mL scale for the hydrolysis of PNPA using the same procedure described in the Enzyme activity assay section. The capsules were added to phosphate buffer (10 mM, pH 8.0), yielding a final enzyme concentration of 0.2 mg mL⁻¹. Then, 250 μL of a PNPA acetonitrile solution (100 mM) was added to a final concentration of 5 mM and shaken in a shaking bath (150 rpm) at 37 °C for 20 min. The capsules were collected by filtration after each reaction batch, washed three times with phosphate buffer (10 mM, pH 8.0) to remove residual product or substrate, and then dispersed in 5 mL of fresh phosphate buffer (10 mM, pH 8.0) for the next reaction cycle.

Storage stability and thermostability of hybrid capsules and FHs

Hybrid capsules and FHs were stored in phosphate buffer (10 mM, pH 8.0) at 4 °C for 30 days (or pH 7.0, pH 10 at room temperature for one day). The storage stability was compared in terms of relative activity, defined as the ratio of the activity of hybrid capsules or FHs after storage to their initial activity.

The thermostability of the enzyme was measured by analyzing the residual activity after incubating the hybrid capsules and FHs at 70 °C in 10 mM phosphate buffer (pH 8.0) for 1 h. All these reactions were performed according to the method described in the Enzyme activity assay section.

Results and discussion

Characterization of hybrid capsules

Fmoc-FFH can be dissolved in a basic solution (pH ≧ 9.0). As the pH decreases, the magnitude of the charge of Fmoc-FFH molecules decreases, allowing them to self-assemble into nanofibers through π–π interactions (Fig. S1†).¹⁰ A positively charged polymer, PEI, was used to induce and direct the self-assembly of Fmoc-FFH at an aqueous interface, leading to the rapid synthesis of stable Fmoc-FFH/PEI hybrid capsules (FPCs) and Fmoc-FFH/PEI/GA hybrid capsules (FPGCs, Fig. S3†).

To demonstrate the concept, an aqueous solution (pH 9.0) of Fmoc-FFH (4 mg mL⁻¹) was added dropwise (5 μL) to an aqueous solution (pH 8.0) of PEI (1.0 wt%) through a pipette. When the transparent liquid droplets came into contact with the PEI solution, white-colored capsules were formed within seconds. Fig. 1a and b present photographs of the synthetic FPCs and FPGCs, respectively, which are approximately 2 mm in diameter and exhibit good uniformity. To investigate the exterior and interior of the capsules, we fractured the capsules prior to SEM imaging. As shown in Fig. 1c, the exteriors of the FPCs have a smooth and compact surface structure, whereas their interiors exhibit a nanofibrous morphology (Fig. 1d). The compact membrane structure of the capsule exterior (Fig. 1c) is probably generated by the PEI-induced self-assembly of Fmoc-FFH and deposition of PEI. The porous and fibrous structure of the capsule interior is similar to that of FHs, suggesting that the internal layer may be formed by the self-assembly of Fmoc-FFH alone without PEI. As shown in Fig. 1e, the surface structure of the exterior of the FPGCs appears to be more compact, whereas the interior still exhibits a nanofibrous morphology that is slightly more compact (Fig. 1f). The reason for this result is that GA crosslinking primarily acts on polyethyleneimine, which mainly leads to changes of the external structure. In contrast, small amounts of PEI deposition occur in the capsule interior during the synthesis of stable FPGCs, which has a slight influence on the self-assembly of Fmoc-FFH.

Fig. 1 Morphology characterization of synthetic FPCs and FPGCs. (a) Photograph of hybrid capsules formed in a 1.0 wt% PEI solution (pH 8.0) by adding 4 mg mL⁻¹ Fmoc-FFH solution. (b) Photograph of hybrid capsules formed in 1.0 wt% PEI solution (pH 8.0) by adding 4 mg mL⁻¹ Fmoc-FFH solution and then crosslinking with glutaraldehyde. (c) SEM images of the exterior of FPCs. (d) SEM images of the interior of FPCs and partial enlarged details. (e) SEM images of the exterior of FPGCs and a cross-section of the capsule membrane with a thickness of 6.67 μm. (f) SEM images of the interior of the synthetic FPGCs and partial enlarged details.

The effect of the pH of the PEI solution on the synthesis of FPCs was investigated. The experiment indicated that stable capsules were formed at pH ≤ 11.0, while no capsule was formed at a pH of 12.0. The zeta potential of the PEI solution at pH values from 3 to 12 was measured to explain this phenomenon. As shown in Fig. 2, the PEI solution shows a nearly zero potential at pH 11.0, and the static charge gradually decreases over the pH range from 3 to 12. In this experiment, the Fmoc-FFH solution at a pH of 9.0 showed a negative zeta potential (−60.9 mV, Fig. 2); thus, it is difficult to self-assemble in this solution due to the electrostatic repulsion. When Fmoc-FFH molecules came into contact with positive PEI chains, the electrostatic repulsion was eliminated by the neutralization of charges. Self-assembly of Fmoc-FFH therefore occurred at the interface and formed capsules (Fig. S2†). A similar self-assembly mechanism was also found in other molecules, such as peptide amphiphiles.⁴¹

Fig. 2 Zeta potentials of PEI solution (1.0 wt%) at different pH values and of Fmoc-FFH (4 mg mL⁻¹) solutions at a pH of 9.0.

Additionally, the effect of glutaraldehyde on the activity recovery was investigated by adding glutaraldehyde at different final concentrations (10–125 mM). The results showed that the hybrid capsules retained 76.8 ± 2.5% of their activity after cross-linking with 25 mM glutaraldehyde at 25 °C for 2 h (Fig. 3). We hypothesized that the addition of glutaraldehyde led to a more compact structure, making enzyme activity weaker but the structure more stable.

Fig. 3 Effect of glutaraldehyde concentration on the recovery of activity of the Fmoc-FFH/PEI hybrid capsules. Data represent the means of three experiments, and error bars represent the standard deviation.

Enzyme activity assay

The specific activity was measured according to the increase in the absorption of the hydrolytic product p-nitrophenol at 400 nm. To investigate the effect of PEI, glutaraldehyde and microstructure of capsules on the self-hydrolysis of PNPA, we prepared the Fmoc-FF/PEI capsules and Fmoc-FF/PEI/GA capsules, which had a similar structure as FPCs and FPGCs, respectively. As shown in Fig. S6,† the results indicate these factors have no influence on the self-hydrolysis. Therefore, the self-hydrolysis of PNPA was used as the background in the catalytic reaction.

As shown in Fig. 4, the specific activity of FHs for the first 20 min reached 25.31 U g⁻¹, far below that of FPCs (54.79 U g⁻¹) and FPGCs (41.34 U g⁻¹), which is possibly due to the limited mass transfer of PNPA in the Fmoc-FFH hydrogel. In contrast, FPCs and FPGCs have porous structures, in which the substrate and products are able to pass through the capsule membrane. In comparison with FPCs, FPGCs have a more compact membrane, which limit the mass transfer of substrates.

Fig. 4 The specific activities of FHs, FPCs and FPGCs in a shaking bath (150 rpm, 37 °C). Data represent the means of three experiments, and error bars represent the standard deviation.

Effects of pH and temperature on the activity of the hybrid capsules

The Fmoc-FFH hybrid capsules and FHs both showed an optimal pH of 8.0 (Fig. 5). As expected, over the pH range from 6 to 10, the Fmoc-FFH hybrid capsules exhibited higher catalytic activity than the FHs. The optimal temperature of the hybrid capsules and FHs was 37 °C. Additionally, the result indicates that the hybrid capsules still have high enzyme activity at 60 °C, indicative of a high temperature resistance.

Fig. 5 Effects of pH (a) and temperature (b) on the specific activity of the FPCs, FPGCs and FHs.

Determination of kinetic parameters of hybrid capsules

Michaelis–Menten kinetics is an important characteristic that distinguishes biological enzyme catalysts from general chemistry catalysts. We selected FPCs and FPGCs with high activity as the research focus and compared the hydrolysis rates under different concentrations of PNPA or PNPB solution. As shown in Fig. 6, the results show that FPCs and FPGCs have catalytic characteristics typical of natural enzymes, namely, they simulate the substrate recognition and the catalytic process of natural enzymes to some extent.

Fig. 6 Lineweaver–Burk plots for the hydrolysis of PNPA (a) and PNPB (b) catalyzed by FPCs and FPGCs.

Based on the Lineweaver–Burk double-reciprocal model fitting, we can calculate the Michaelis constants for artificial hydrolytic enzymes. As shown in Table 1, the values of K_cat/K_m indicate that the FPCs have a higher catalytic efficiency compared to FPGCs, which is in accordance with the result as described before. In comparison with PNPA, PNPB has a higher affinity (smaller K_m values) to Fmoc-FFH nanofibers (Table 2). It also has a slightly larger K_cat/K_m value, indicative of a higher catalytic efficiency. However, we find that the capsule can not catalyze the hydrolysis reaction of PNPP under the same conditions.

Table 1 Kinetic parameters of capsules using PNPA as substrate

Catalyst	Km (mM)	Kcat (mM min^−1 mg^−1)	Kcat/Km (10^−4 min^−1 mg^−1)
FPCs	11.09	0.031	27.95
FPGCs	7.39	0.017	23.01

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Table 2 Kinetic parameters of capsules using PNPB as substrate

Catalyst	Km (mM)	Kcat (mM min^−1 mg^−1)	Kcat/Km (10^−4 min^−1 mg^−1)
FPCs	3.09	0.011	35.59
FPGCs	1.66	0.005	27.38

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Reusability assay of hybrid capsules

The reusability of the hybrid capsules was evaluated through PNPA hydrolysis for successive batches. As shown in Fig. 7, the 93% productivity of FPGCs retained after fifteen cycles (total time: 300 min) is considerably higher than the 44% productivity of FPCs retained after five cycles (total time: 100 min), suggesting the high stability and excellent recyclability of FPGCs. However, as expected, it is difficult to recover the Fmoc-FF hydrogels via the simple centrifugation due to the small size of Fmoc-FFH nanofibers.

Fig. 7 Recycling and reuse of FPCs (a) and FPGCs (b) for the hydrolysis of PNPA (20 min per cycle).

Fig. 8 The storage stability (a) and thermal stability (b) of FPCs and FPGCs. The specific activities of the initial enzymes were normalized to 100%.

Storage stability and thermal stability of hybrid capsules

The storage stability of the hybrid capsules was also determined. After storage at 4 °C for 30 days, the residual activity of the FPGCs was 85.45%, demonstrating a better storage stability than that (71.83% residual activity) of the FPCs. As shown in Fig. 8, the hybrid capsules also have good thermal stability. After incubating the hybrid capsules for 1 h at 70 °C in 10 mM phosphate buffer (pH 8.0), the residual activities of the FPCs and FPGCs were 87.49% and 94.00%, respectively. Additionally, the capsules were stored in an phosphate buffer (pH 7.0 or pH 10) for 24 h. As shown in Fig. S7,† there is no obvious change in the catalytic activity of FPCs and FPGCs, indicating the high basic tolerability.

Conclusions

In conclusion, we designed and synthesized a novel hydrolase model: hybrid capsules (FPGCs). Because of the hydrophobic microenvironment provided by the Fmoc-FFH aromatic ring segments and the high density of catalytic histidines, this model exhibits higher catalytic activity for the hydrolysis of PNPA compared to Fmoc-FFH hydrogels. In addition, the resulting hydrolase model exhibits kinetics behaviour that is typical of natural enzymes as well as high stability and excellent recyclability, and it is expected to be applied as a substitute for natural hydrolases in industrial production applications.

Acknowledgements

This work was supported by the Natural Science Foundation of China (no. 21476165 and 21306134).

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Footnote

† Electronic supplementary information (ESI) available: Supplementary figures. See DOI: 10.1039/c6ra05778k

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