Covalently Self‐Assembled Peptide‐Based Hydrolase Mimic for Realizing Exceptional Catalytic Longevity in Foreign Environments

As a de novo design of artificial enzymes, peptide assembly is receiving enormous attention. However, the development of durable peptide‐based biocatalysts that can resist undesirable deformation and loss of function in non‐native environments is challenging. Herein, a covalently self‐assembled, peptide‐based hydrolase mimic (referred to as a nanopepzyme) with exceptional stability regardless of the changes in the external environment is reported. The photocrosslinking of decapeptides, YYHHHHHHYY, leads to the formation of well‐defined nanospheres with multiple catalytic histidine residues protruding from their surfaces. The nanopepzyme not only exhibits extraordinary long‐term stability even after 6 months but also maintains its structures under adverse environmental conditions (pH, temperature, ion strength, and organic solvents). In addition, the nanopepzyme demonstrates hydrolase‐like activity and is effective as a significantly durable biocatalyst, as verified by the model reactions following incubation under various harsh conditions. This study expands the scope of peptide assembly for the preparation of peptide‐based biocatalysts that can be applied in considerably harsh foreign environments.

DOI: 10.1002/sstr.202200344 As a de novo design of artificial enzymes, peptide assembly is receiving enormous attention. However, the development of durable peptide-based biocatalysts that can resist undesirable deformation and loss of function in non-native environments is challenging. Herein, a covalently self-assembled, peptide-based hydrolase mimic (referred to as a nanopepzyme) with exceptional stability regardless of the changes in the external environment is reported. The photocrosslinking of decapeptides, YYHHHHHHYY, leads to the formation of welldefined nanospheres with multiple catalytic histidine residues protruding from their surfaces. The nanopepzyme not only exhibits extraordinary long-term stability even after 6 months but also maintains its structures under adverse environmental conditions (pH, temperature, ion strength, and organic solvents). In addition, the nanopepzyme demonstrates hydrolase-like activity and is effective as a significantly durable biocatalyst, as verified by the model reactions following incubation under various harsh conditions. This study expands the scope of peptide assembly for the preparation of peptide-based biocatalysts that can be applied in considerably harsh foreign environments.
Waals interactions. [6] Typically, the dynamic characteristics of noncovalent interactions cause spontaneous rearrangement of supramolecular building blocks through changes in the surrounding environment. [6b,7] Despite their intriguing dynamic nature in stimuli-responsive properties and switchable catalytic systems, [6b,7,8] the use of environments adverse to supramolecular assemblies can cause the undesirable deformation of peptide structures, similar to the denaturation of proteins, leading to the loss of function. Eventually, such low operational stability of catalytic peptide assemblies in non-native environments would not only lead to inactivation and inefficient recycling of catalysts but also impede the use of peptides in processes such as the biphasic reaction and solvent extraction, limiting their wide-scale application. Therefore, it still remains a significant challenge to develop peptide-based biocatalysts with high durability and resistance that can be used under a wide range of conditions and can be easily separated without deformation.
In this regard, the chemical fixation or covalent crosslinking of peptides would be a more effective approach to improve the stability of structures compared with noncovalent assembly. [9] However, it is still challenging to build a well-defined structure by directly assembling building blocks without templates or preorganization through robust covalent bonds owing to the lack of self-error-correcting mechanisms during the formation of irreversible covalent bonds. [10] Several studies have reported that the integration of tyrosine crosslinking in biomaterials strengthens structures and improves resistance to mechanical and chemical deformation. [11] In addition, recent studies have reported that crosslinking tyrosine-rich short peptides can lead to selfassembly into nanostructures. [9] Despite the success of initial studies on covalently crosslinked peptide nanostructures, biocatalysts based on monolithic peptide covalent assemblies that retain their structures and catalytic functions regardless of environmental changes have not yet been fully explored.
In this article, we report a covalently self-assembled peptide-based biocatalyst, referred to as a nanopepzyme, which is a hydrolase mimic prepared using decapeptides (YYHHHHHHYY, denoted as YH6). The nanopepzyme was developed through tyrosine crosslinking with a resizable and fast-forming photocrosslinking system. With multiple catalytic hexahistidine residues protruding from its surface, the nanopepzyme demonstrated cleavage activity. Notably, the nanopepzyme exhibited extraordinary long-term stability even after 180 d and could withstand various harsh environment settings (pH, temperature, ion strength, and organic solvents) while retaining structural and catalytic properties. This study is expected to chart a new direction in the development of peptide-based biocatalysts that are operable in considerably harsh foreign environments.

Synthesis and Characterization of YH6 Nanopepzyme
For the design of a covalently self-assembled catalytic peptide building block, the peptide sequence was encoded with hexahistidine as a hydrolytic active motif at the center and tyrosine pairs as photocrosslinking motifs at both the termini. Consequently, the YYHHHHHHYY peptide sequence was designed. The covalently self-assembled YH6 nanopepzyme was synthesized through one-step photocrosslinking under visible light using a well-known ruthenium-catalyst (tris(2,2-bipyridyl)dichlororuthenium(II)hexahydrate, Ru(bpy) 3 Cl 2 ) ( Figure 1a). [12] Transmission electron microscopy (TEM) images indicated the formation of the spherical YH6 nanopepzyme (Figure 1b). Scanning electron microscopy (SEM) images revealed that the one-step photocrosslinking of peptide building blocks produced large quantities of the uniform YH6 nanopepzyme (Figure 1c and S1, Supporting Information). Dynamic light scattering (DLS) analysis revealed an average hydrodynamic diameter of 245.2 AE 65.9 nm in the wet state (Figure 1d), which is in close agreement with the microscopy data. In the formation system of the covalently self-assembled YH6 nanopepzyme, the yield calculated on the basis of the bicinchoninic acid (BCA) assay was 83% ( Figure S2, Supporting Information). Inductively coupled plasma-mass spectrometry (ICP-MS) analysis indicated that the quantity of ruthenium was 776.46 ppb, confirming that almost all the ruthenium was removed in the washed YH6 nanopepzyme. The variation of the concentration of peptides (0.5-2 mg mL À1 ) caused the average size of the YH6 nanopepzyme to range from 208.1 AE 45.9 to 378.5 AE 41.2 nm ( Figure S3, Supporting Information).
To confirm the occurrence of tyrosine crosslinking, various spectroscopic techniques were employed. In the UV-vis analysis ( Figure S4, Supporting Information), the strong absorbance peak at %280 nm, assigned to the π-π* transition of the tyrosine residue, shifted in a bathochromic manner owing to the visible light irradiation. This phenomenon is indicative of the electron delocalization of phenyl groups. [9b,11c,13a,b] Moreover, a shoulder peak appeared at 315 nm, and the YH6 nanopepzyme solution indicated a characteristic blue fluorescence emission band located at %400 nm (Figure 1e), further confirming the formation of dityrosine bonds. [9,11,13] The circular dichroism (CD) spectra of the monomer exhibited positive peaks at %209 and %228 nm and a weak valley at %214 nm ( Figure S5, Supporting Information), whereas the CD signal of the YH6 nanopepzyme almost vanished. This annihilation CD phenomenon was most likely caused by peptide aggregation via tyrosine crosslinking, similar to an aggregation-annihilation CD. [14] Fourier-transform infrared (FT-IR) spectra indicated that the YH6 peptide monomer exhibited an amide I peak at 1657 cm À1 , which was ascribed to the α-helix secondary structure conformation ( Figure S6 and Table S1, Supporting Information). [15] The FT-IR broadband at the region of 600-630 cm À1 assigned to the imidazole C-N bending vibration was observed in both the YH6 monomer and the nanopepzyme. [16] The FT-IR peaks at 1617 cm À1 (aromatic ring C─C bond vibration) and 1515 cm À1 (aromatic ring C═C stretching vibration) decreased in intensity and shifted upon the formation of the YH6 nanopepzyme, [17] indicating electron delocalization of phenyl groups, which was likely caused by either the π-π* stacking between adjacent tyrosines or the decrease in the aromatic C─H number after the formation of dityrosine. [11e,18] Moreover, the peaks at 1184 cm À1 (phenolic C─OH stretching vibration), 1129 cm À1 (aromatic ring C-H bending), and the region of 700-860 cm À1 (C-H bending vibration, disubstituted benzenes) disappeared in the YH6 nanopepzyme. [11d,e,18,19] Simultaneously, the broad peak at %1045 cm À1 (C-O-C stretching vibration, isodityrosine) newly appeared, reflecting the crosslinking of tyrosines after the photocrosslinking. [19] To understand the growth mechanism of the YH6 nanopepzyme, its stepwise formation was analyzed by acquiring the UV-vis spectra, DLS profiles, and TEM images at various time intervals during the photoreaction. The intensity of the UV absorption spectral peak at 315 nm (Figure 1f ), which lies in the characteristic absorption wavelength range of tyrosine crosslinking, increased rapidly within 30 s. After 1 min of reaction time, the absorbance variation was negligible. The time evolution of the size of the YH6 nanopepzyme indicated that 173.8 nm of nanospheres were quickly formed within 20 s, grew to 244.5 nm within 30 s, and finally reached 245.2 nm in 10 min, which corroborates with the time-dependent TEM images (Figure 1g and S7, Supporting Information). Based on these results, the similarity in the time-dependent absorbance at 315 nm and the average size tendencies support the concept that tyrosine crosslinking is a crucial driving force for the formation of the YH6 nanopepzyme. Next, we analyzed the surface characteristics of the YH6 nanopepzyme to elucidate the presence of imidazole residues, which play a critical role in ester hydrolysis. To compare the surface characteristics, peptide nanoparticle (NP) based on YYGYY (YG) as the control was prepared using an identical synthesis method. Because imidazole is protonated at pH 5, the YH6 nanopepzyme demonstrated positive surface charges (5.11 mV) in relation to YG NPs (À20.97 mV) ( Figure 1h). Moreover, the pH-dependent zeta potential indicated that the isoelectric point (pI) value of the YH6 nanopepzyme is located pH of %7 owing to the presence of abundant histidine residues ( Figure S8, Supporting Information). This result suggests that the YH6 nanopepzyme has significant potential in catalysis because the half-protonated imidazole at a physiological pH can act as a general acid, general base, or nucleophile. [20] We further observed abundant histidine residues at the surface of the YH6 nanopepzyme by a binding assay using 5 nm-sized gold nanoparticles functionalized with nickel (II) nitraloacetic acid chelates (Ni-NTA Au NP) at a pH 8.5, whereat the zeta potential of the YH6 nanopepzyme was % À20 mV. The Ni-NTA complex is known to bind histidine tags with high affinity. [21] The control experiment conducted using the YG NP indicated that no Au NP binding events occurred because of a lack of binding moieties for Ni-NTA ( Figure 1i). However, scanning transmission electron  microscopy (STEM) and the corresponding elemental analysis of the YH6 nanopepzyme clearly revealed that Au NPs were predominately present and well distributed on their surface ( Figure 1j). This result verifies the presence of a His-rich surface and implies the applicability of the YH6 nanopepzyme in Ni-NTA purification/immobilization systems, similar to common recombinant proteins.

Stability of Covalently Self-Assembled YH6 Nanopepzyme
Peptide nanostructures made by noncovalent interactions are often susceptible to their surrounding environments (pH, temperature, and solvent), similar to proteins that undergo denaturation under non-native conditions. That is, the development of durable peptide nanostructures with enhanced stability, reusability, thermal stability, and accessibility of organic solvents may guarantee potential use even in considerably harsh environments. To confirm that crosslinking in peptide assembly enables accessing wide process conditions, we scrutinized the stability of the YH6 nanopepzyme. First, the long-term stability of the YH6 nanopepzyme dispersed in water was investigated through TEM and DLS analysis, following incubation for 180 d at room temperature (Figure 2a ,b). A comparison of the hydrodynamic average diameter and TEM images over a period of 180 d indicated the outstanding long-term storage stability of the YH6 nanopepzyme. Second, to study the thermal stability of the YH6 nanopepzyme, thermogravimetric analysis (TGA) was performed (Figure 2c and S9, Supporting Information). A major loss in the weight of the YH6 nanopepzyme occurred at >300°C, indicating higher thermal stability than that of monomers, which degrade at %220°C. Furthermore, the weight losses of the YH6 nanopepzyme and monomers were 39.9% and 63.9% at 400°C, respectively. Third, the structural stability of the YH6 nanopepzyme was investigated under various non-native settings. Surprisingly, even after 7 d of incubation in organic solvents (dimethyl sulfoxide [DMSO], toluene, acetonitrile [ACN], and methanol [MeOH]) and under various conditions (pH 2, pH 10, 1 M sodium chloride [NaCl], and 80°C), no apparent morphological transformation or disassembly was observed (Figure 2d and S10, Supporting Information). These results indicate the exceptional structural stability of the YH6 nanopepzyme, implying that potential use in a wide range of conditions is feasible.

Catalytic activity of YH6 Nanopepzyme
Following the investigation of the structural stability of the YH6 nanopepzyme, the catalytic activity of the YH6 nanopepzyme was investigated using a simple chromogenic substrate, 4-nitrophenyl acetate (4-NPA), by monitoring the appearance of the characteristic absorption peak of the yellow hydrolyzed product 4-nitrophenol (4-NP) at 400 nm ( Figure 3a). In the initial experiments using the 100 μM YH6 nanopepzyme, a significant increment in the intensity of the peak at 400 nm was observed ( Figure 3b). Furthermore, the hydrolytic activity exhibits concentration dependence of the YH6 nanopepzyme ( Figure S12, Supporting Information), indicating that the reaction was significantly accelerated by the catalysis of the YH6 nanopepzyme rather than the self-decomposition of 4-NPA. Control experiments performed using the YG NP, L-tyrosine, and Ru(bpy) 3 Cl 2 indicated a meager hydrolytic cleavage in relation to that using the YH6 nanopepzyme ( Figure 3c). Compared to L-histidine or YH6 monomer, covalently self-assembled YH6 nanopepzyme exhibited higher catalytic activity (Figure 3c), and hydrolytic activity increased as follows with respect to the number of histidine in the peptide sequences: YH6 > YH3 > YH2 > YH1 (Figure 3d, YYHYY, YH1; YYHHYY, YH2; YYHHHYY, YH3). These observations can be attributed to the cooperative effect of the imidazole groups closely present at the surface of the YH6 nanopepzyme. It is well known that the basicity of densely protruded imidazole groups is enhanced by deprotonation from adjacent histidine, facilitating the hydrolytic reaction. [20a] The catalytic efficiency was evaluated using the initial hydrolytic rates as a function of substrate concentration with a fixed amount of catalyst (50 μM). The catalysis yielded typical Michaelis-Menten kinetic plots, and the doublereciprocal plot was obtained based on the Michaelis-Menten equation (Figure 3e,f ). The Michaelis-Menten constant (K M ) and catalytic efficiency (k cat /K M , where k cat is the turnover number) values were 0.63 mM and 1.69 M À1 s À1 , respectively. The YH6 nanopepzyme demonstrated competent substrate affinity and catalytic efficiency comparable to previously reported metal cofactor-free supramolecular peptide assemblies (Table S2, Supporting Information).

Catalytic Stability of YH6 Nanopepzyme
Next, we scrutinized the catalytic stability of the YH6 nanopepzyme by performing tests in recycling, long-term storage, and environmental change. In the reusability test, the YH6 nanopepzyme maintained 89% of its activity even after ten cycles without any morphological transformation or disassembly (Figure 4a and S14, Supporting Information); the decrease in activity was possibly caused by the loss of catalyst during the recycling process owing to centrifugation. As depicted in Figure 4b, the YH6 nanopepzyme exhibited outstanding long-term catalytic stability while maintaining a cleavage activity of almost 99% even after 180 d. To further validate the catalytic stability of the YH6 nanopepzyme, various non-native settings were implemented. First, the effect of temperature on the catalytic activity was evaluated following incubation for 1 h at various temperatures ( Figure 4c). The elevation in temperature induced a denaturation  in natural esterase, resulting in the loss of its activity completely at >60°C. However, the YH6 nanopepzyme preserved cleavage activity even upon exposure to a wide range of temperatures owing to the covalent-bond-derived resistance. The effects of salt concentration on the catalytic activity were analyzed based on a series of assay conditions with various salt concentrations (Figure 4d). The results indicate that the activity of natural esterase diminished to 32.4% at high salt concentrations owing to the destabilization of the ionic bonds that hold conformations. In contrast, the YH6 nanopepzyme displayed a relative activity of 84.1% in a 900 mM NaCl solution.
To explore the effect of foreign environmental conditions on catalytic stability, we incubated esterase and the YH6 nanopepzyme at pH 2, pH 10, 80°C, and toluene for 7 d; the relative catalytic activity at pH 7.4 was analyzed once a day. In the case of esterase, an apparent loss of activity was observed at both pH 2 and pH 10 within a day (Figure 4e-f ). Surprisingly, the YH6 nanopepzyme retained at least 95% of its activity, indicating its high stability over a broad range of pH. In the thermal stability tests, the relative activity of esterase decreased sharply within a day (Figure 4g). In contrast, the YH6 nanopepzyme exhibited outstanding thermal stability with a relative activity of 99% even after incubation at 80°C for 7 d. Furthermore, a gradual decrease in the hydrolytic activity of esterase stored in toluene was observed, while the loss of activity in the YH6 nanopepzyme was negligible even after immersion in an organic solvent for 7 d (Figure 4h). Taken together, these results suggested that the covalent assembly of functional peptides could be an alternative approach to engineering and developing peptide-based biocatalysts with exceptional stability in various environments.

Conclusion
We developed a highly durable peptide-based biocatalyst through covalent self-assembly induced by tyrosine crosslinking and verified their stability under adverse environmental conditions. The YH6 nanopepzyme can be readily formed and regulated through straightforward photocrosslinking systems. Further, they show catalytic activity in ester hydrolysis due to the proximity effect of the surface-exposed histidine residues. Owing to the covalent self-assembly, the YH6 nanopepzyme demonstrated extraordinary catalytic stability even upon recycling, long-term storage, or incubation under harsh conditions. We believe that this study will greatly expand the scope of peptide assembly for the preparation of robust peptide nanostructures that can be applied in considerably harsh foreign environments.
Synthesis of YH6 Nanopepzymes: To eliminate any preformed assembly, peptide solutions used in all experiments were freshly prepared and used immediately. In a typical synthesis of peptide NPs, the reaction solution was prepared by mixing aqueous peptide (0.2 mL, 5 mg mL À1 ), APS www.advancedsciencenews.com www.small-structures.com Small Struct. 2023, 4, 2200344 (0.6 mL, 10 mM), and Ru(bpy) 3 Cl 2 solutions (0.2 mL, 0.87 mM) in a quartz cuvette (1 cm). The resulting solution was immediately exposed to a white light lamp system (OSRAM, DULUX L LED, 18 W, 2 EA) with cooling fans at room temperature for 10 min. After photocrosslinking of peptides, the products were collected by centrifugation (Centrifuge 5425, Eppendorf ) at 13 500 rpm for 2 min and washed 3 times with water. Thereafter, the synthesized peptide NPs were dispersed in water at a desired concentration for further use. For control experiments, other peptide NPs (YG, YH1, YH2, YH3) were synthesized by the identical protocol. The synthetic yield of the YH6 nanopepzyme was evaluated using the BCA protein assay kit and the standard curve of the YH6 monomer treated with the BCA working reagent. The total content of unreacted peptide monomers in the supernatant after centrifugation at 13 500 rpm for 2 min was determined by the BCA protein assay kit. Next, 50 μL of the supernatant was mixed with 1 mL of the BCA working reagent and incubated for 30 min at 25°C. Next, absorbance at 562 nm was recorded and the concentrations of remaining peptide monomers were determined using the standard curve of the YH6 peptide monomers. The concentrations of the remaining peptide monomers in the supernatant were calculated to be 0.17 mg mL À1 ; the percentage of peptides that participated in the reaction was 83%. To determine the amount of remaining ruthenium in the YH6 nanopepzyme, the washed YH6 nanopepzyme was analyzed through ICP-MS (Perkin Elmer NexION 300X and NexION 2000). The amount of ruthenium (776.46 ppb) in the YH6 nanopepzyme was negligible. Electron Microscopy Imaging: The morphologies of the peptide products were investigated by TEM (Bio-TEM, Hitachi, HT 7700), field emission-TEM (FE-TEM; Titan G2 ChemiSTEM Cs Probe, FEI), and FE-SEM (Hitachi, SU8220 and SU8230). For TEM, 10 μL spots of the sample were deposited on carbon-coated copper grids Electron Microscopy Sciences). After 1 min of incubation, excess samples were removed with filter paper. The grids were then washed with water and dried in a drying oven before TEM. For SEM, centrifuged YH6 nanopepzyme pellets were deposited on a Si wafer. The samples were then dried in a drying oven overnight.
Size and Zeta-Potential Analyses: The average size and zeta potential of the peptide NPs were measured using ELSZ-2000ZS (Otsuka Electronics). For DLS analysis, 0.8 mL of the YH6 nanopepzyme solution at an appropriate concentration was loaded in microcuvettes. For zeta-potential analysis, 0.1 mg mL À1 samples of the YH6 nanopepzyme in various pH buffer solutions were prepared and incubated for 30 min. The samples were loaded in quartz capillary zeta cells, and the zeta potentials were measured at room temperature. At least three measurements were performed on each experiment.
Spectroscopic Analyses: For UV-vis analysis, 1 mL samples of the reaction mixture at various irradiation times were immediately analyzed by a NanoDrop 2000c UV-Vis spectrometer (Thermo Fisher Scientific). For fluorescence analysis, washed YH6 nanopepzymes were dispersed in a pH 10 buffer (1 mg mL À1 ), and fluorescence emission spectrum was measured using an Infinite M200 Pro spectrofluorometer (TECAN) under 315 nm excitation. FT-IR spectra of YH6 nanopepzyme and monomer were obtained by Frontier (PerkinElmer). For CD analysis, 1 mg mL À1 of aqueous peptide sample was loaded into a quartz cuvette cell with 1 mm length. The CD spectrum was collected from triplicate scans, at 25°C from 280 to 180 nm at a scanning rate of 30 nm min À1 , using a J-1500 CD spectrometer (JASCO) equipped with a Peltier thermostatic cell holder and a nitrogen purging facility. Each spectrum was baseline subtracted with the CD signals from water.
Ni-NTA Au NP Binding Assay: A 10 μL sample of the as-synthesized YH6 nanopepzyme dispersed in a 20 mM Tris-HCl buffer (pH 8.5) with 150 mM NaCl was added to 90 μL of a Ni-NTA Au NP solution and incubated for 3 h. Next, peptide NPs were separated by centrifugation at 13 500 rpm for 2 min and washed with water. The samples were analyzed through TEM. As a control experiment, a YG peptide NP was measured in an identical method.
Stability Test: The long-term stability of the YH6 nanopepzyme dispersed in water was investigated by TEM and DLS analysis before and after incubation for 180 d at room temperature. TGA was performed using a Q500 auto-thermogravimetric analyzer (TA Instruments) to investigate the thermal stability of the YH6 nanopepzyme. Samples of 5 mg of dried YH6 peptide monomer and nanopepzyme were heated in a nitrogen atmosphere from 40 to 1000°C at a heating rate of 10°C min À1 . To investigate the chemical stability in various environments, the YH6 nanopepzyme was exposed to organic solvents (DMSO, toluene, acetonitrile, methanol), various pH values (pH 2, 10), aqueous 1 M NaCl solution, and 80°C. Each sample was incubated for 1 week, and morphological transformation was explored through TEM analysis.
Catalytic activity Measurement: The catalytic activity of the YH6 nanopepzyme was determined using the hydrolysis of 4-NPA as a model reaction. Calibration curves for the products were collected by injecting known quantities of 4-NP. The extinction coefficient of 4-NP at pH 7.4 was 13 100 M À1 cm À1 . In the initial step, 0.55 mM YH6 nanopepzyme was prepared in 1 mL of PBS buffer (10 mM, pH 7.4), and 4-NPA (25 mM) was freshly prepared in acetonitrile. For the reaction, 1 mL of the reaction mixture was prepared by mixing 4-NPA, the PBS buffer solution, and the YH6 nanopepzyme at the desired concentration in a 1 cm-long quartz cuvette. The final concentration of 4-NPA was 0.5 mM, and the content of ACN was 2%. The final concentration of the YH6 nanopepzyme was varied from 50 to 100 μM. For comparison, hydrolytic effects of YH6 monomer, L-histidine, YG NP, L-tyrosine, and Ru(bpy) 3 Cl 2 were also analyzed. Hydrolytic activity was measured using a UV-vis spectrometer at 400 nm. To determine the catalytic efficiency of the YH6 nanopepzyme, initial rate was recorded at varying concentrations of 4-NPA (0-0.9 mM), whereas the concentration of the catalyst was fixed to 50 μM. The plot of the substrate-dependent initial rate of hydrolysis activity was fitted using the Michaelis-Menten equation (V 0 = V max [S]/(K M þ [S]), and kinetic traces of the rate constants were calculated based on reciprocal values using the Lineweaver Burk equation.
Catalytic Stability: For the reusability test, 1 mL of reaction mixture containing 100 μM YH6 nanopepzyme, PBS buffer, and 0.5 mM 4-NPA was prepared. After 10 min of reaction, the catalyst was collected by centrifugation at 13 500 rpm for 1 min, and the absorbance of the supernatant solution was recorded. After each reaction cycle, the YH6 nanopepzyme was washed with DI water for the next round of catalysis, and the same hydrolytic reaction was performed. To evaluate the long-term storage stability, the YH6 nanopepzyme was stored at room temperature, and the catalytic activity was monitored for 180 d. Temperature dependence was investigated after incubation under various thermal conditions (20-80°C) for 1 h. The ion strength effect was evaluated upon the addition of various concentrations of NaCl (0-900 mM) to the reaction system, and relative activity was measured. To estimate the effect of various conditions on the catalytic stability of the YH6 nanopepzyme, samples were incubated in pH 2, pH 10, 80°C, and toluene, respectively. The relative activity of each sample at pH 7.4 was recorded at daily intervals for 7 d. As a control experiment, esterase was subjected to an identical treatment and relative activity (based on 10 μg mL À1 ) was recorded.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.