Flowerlike hybrid horseradish peroxidase nanobiocatalyst for the polymerization of guaiacol

In this study, the catalytic activity and stability of flowerlike hybrid horseradish peroxidase (HRP) nanobiocatalyst (HRP-Cu 2+ ) obtained from Cu 2+ ions and HRP enzyme in the polymerization reaction of guaiacol were analyzed. We demonstrated that HRP-Cu 2+ and hydrogen peroxide (H 2 O 2 ) initiator showed significantly increased catalytic activity and stability on the polymerization of guaiacol compared to that of free HRP enzyme. Poly(guaiacol) was observed with quite high yields (88%) and molecular weights (38,000 g/mol) under pH 7.4 phosphate-buffered saline (PBS) conditions at 60 °C with 5 weight% of HRP-Cu 2+ loading. HRP-Cu 2+ also shows very high thermal stability and works even at 70 °C reaction temperature; free HRP enzyme denatures at that temperature. Furthermore, HRP-Cu 2+ provided considerable repeated use and showed some degree of catalytic activity, even after the fourth recycle, in the polymerization of guaiacol.

oxidation of guaiacol compared to using free HRP enzyme [26,27]. In addition, the aforementioned nanobiocatalyst was reported to show very high stability and reusability in contrast to free HRP enzyme. In these studies, only catalytic activity and stability of hybrid nanoflowers were examined, and no information related to the polymerization behavior of guaiacol or oxidation product was given. Guaiacol is a phenol derivative obtained from lignin. Guaiacol is used for the production of vanillin and as a building block for the synthesis of a variety of molecules [28]. Therefore, we believe that it is essential to show the polymerization tendency of guaiacol by using HRP-Cu 2+ hybrid nanoflowers.

Preparation of HRP-Cu 2+ hybrid nanoflowers
Firstly, 120 mM CuSO 4 solution was prepared in ultrapure water. CuSO 4 (60 µL) was blended with 9 mL of free HRP enzyme solution (0.2 mg/mL concentration) in pH 7.4 PBS buffer. The obtained mixture was stirred for 5 min to allow homogenization and was left to incubate for three days at +4 °C. After centrifugation, the blue precipitate was washed with water to remove inert waste. The obtained nanoflowers were then dried and used for polymerization experiments [27].

Representative polymerization procedure
Guaiacol and HRP-Cu 2+ were mixed in 5 mL of a buffer solution. After that, the temperature of the mixture was adjusted to the desired reaction temperature. The compound H 2 O 2 (70 µL, 34.5%-36.5%) was added to the resulting solution 15 times every 10 min to initiate the polymerization. At the end of the reaction, the precipitated polymer was centrifuged. The obtained product was washed with water and methanol and dried at 60 °C [29,30]. The colors of the obtained products were black.

Reusability experiments for the polymerization of guaiacol
The reusability of HRP-Cu 2+ was determined by consecutive polymerization reactions of guaiacol. After each reaction, 0.1 mL of HCl (0.1 M) was added to the mixture. The polymer product was centrifuged and separated from the reaction. Then, another run was started to monitor the next polymerization [31].

Results and discussion
The coordination between Cu 2+ ions and amide nitrogen atoms in the protein structure of HRP enzyme formed complexes is the principal step for the formation of hybrid nanoflowers (HRP-Cu 2+ ) [27]. The Figure 1 demonstrates the morphology of the obtained nanoflowers using a scanning electron microscope (SEM). According to the SEM image of the nanoflowers, the obtained particles are micrometer sized, but they have nanoscale characteristics [25,27]. Therefore, the obtained catalyst was named a nanoflower. The formation of hybrid nanoflowers (HRP-Cu 2+ ) from copper ions (Cu 2+ ) and horseradish peroxidase (HRP) enzyme increases the catalytic activity and stability due to 1) high surface areas of the obtained nanoflowers, 2) favorable HRP conformation in HRP-Cu 2+ , and 3) entrapped HRP. Entrapped horseradish peroxidase enzyme with Cu 2+ ions can have more available active sites of HRP in the nanoflowers, and therefore, HRP-Cu 2+ shows higher catalytic activity and stability in contrast to free HRP enzyme. Previously, it was reported that the activity of HRP-Cu 2+ in the oxidation of guaiacol was found to be about 300% higher compared to the activity of the free HRP enzyme [27]. Inspired by this work, we discovered that the potential utilization of HRP-Cu 2+ for the polymerization of guaiacol means high efficiency, stability, and reusability under mild reaction conditions. The variable parameters including reaction pH, temperature, and the amount of HRP-Cu 2+ were optimized for the polymerization of guaiacol ( Figure 2). All polymerizations were conducted in pH buffer solutions with the careful optimization of the amount of the catalyst and guaiacol to obtain the best polymerization conditions. To address the impact of the pH of the solution on the polymerization of guaiacol, we investigated three pH buffers: pH 7.0, 7.4, and 8.0 ( Table 1). The results have shown that the optimum conditions for the polymerization of guaiacol were achieved in pH 7.4 phosphate-buffered saline (PBS). Polymerization attempts using pH 7.0 and 8.0 buffers ( Table 1; entries 8 and 9) were also successful, however, obtained yields for those polymerizations were lower than the ones for the polymerization carried out in pH 7.4 PBS buffer.
The impact of reaction temperatures and the amount of HRP-Cu 2+ on the polymerization of guaiacol in pH 7.4 buffer were also investigated. When the polymerization was carried out with 5 weight% of catalyst loading at 60 °C in the presence of H 2 O 2 , the highest yield product (88%) was observed (Table 1; entry 4). Horseradish peroxidase is known to be thermally  deactivated at temperatures around 60 °C due to the denaturation of the enzyme [32][33][34]. However, the highest yielded polymerization of guaiacol with HRP-Cu 2+ was achieved at 60 °C. According to the findings, HRP-Cu 2+ hybrid nanoflowers still work even at 70 °C reaction temperature (Table 1; entry 5). This achievement provides using HRP-Cu 2+ for oxidative polymerization reactions carried out at higher reaction temperatures. The optimum polymerization of guaiacol with 88% yield and 38,000 g/mol molecular weight was accomplished in pH 7.4 buffer at 60 °C with 5 weight% of HRP-Cu 2+ loading (Table 1; entry 4). Increasing the amount of HRP-Cu 2+ slightly increased the yield of the product (Table 1;entries 6 and 7), but the molecular weights of the obtained polymers were almost the same as the ones obtained under the conditions given in entry 4 in Table 1. Due to the high cost of HRP enzyme, the optimum HRP-Cu 2+ concentration was determined to be 5% by weight of guaiacol. The effect of the addition of an organic solvent to the reaction medium on the polymerization yield and molecular weight of the obtained product was also investigated (Table 1; entries 10 and 11). The stability of HRP-Cu 2+ was aimed to be determined by adding some amount of water-miscible organic solvents to the reaction media, because HRP enzyme is known to denature in organic solvents [15]. Polymerization performed using a mixture of 4.5 mL of pH 7.4 PBS buffer and 0.5 mL of methanol (Table 1; entry 10) under 60 °C reaction temperature with 5 weight % of catalyst loading showed a dramatic decrease in the yield (31%) and molecular weight (9700 g/mol) of the obtained polymer. Further increase in the amount of methanol to 1.0 mL with 4.0 mL of pH 7.4 PBS buffer under the same reaction conditions (Table 1; entry 11) resulted in losing a considerable degree of the catalytic activity of HRP-Cu 2+ and gave a polymer with 13% yield and 6000 g/mol molecular weight (see Supplementary information for GPC data). The structure of the resultant polymer (Table 1; entry 4) was studied by FT-IR, 1 H, and 13 C NMR spectra. To understand the structure of the resulting product, 1 H NMR analysis was first investigated for polymer entry 4 in Table 1 (Figure 3). According to 1 H NMR spectrum of the obtained product, methyl proton signals of the methoxy group in the polymer structure appeared as a singlet peak at 3.74 ppm. The expected chemical shifts for the aromatic protons of the product were observed at 6.74, 6.90, and 8.90 ppm, which confirmed the structure of poly(guaiacol). The proton of the phenolic -OH was not detected in the 1 H NMR analysis of the product, suggesting that  -OH protons of the product probably interacted with DMSO-d6 to exchange hydrogen and deuterium atoms [35]. Therefore, -OH protons of the product disappeared in the 1 H NMR spectrum of the product. Figure 4 displays the 13 C NMR spectrum for the obtained poly(guaiacol) ( Table 1; entry 4). The presence of the methyl carbon of methoxy group can be recognized at δ= 56 ppm. The peaks observed between 112.7-148.1 ppm can be assigned as aromatic carbons of obtained poly(guaiacol). The 13 C NMR spectrum confirmed that the obtained product had a perfect agreement with the expected poly(guaiacol) structure.
Following these results, we evaluated the FT-IR analysis of guaiacol and poly(guaiacol) ( Table 1; entry 4) to confirm the occurrence of polymerization ( Figure 5). An absorption band that appeared around 1650 cm -1 in the FT-IR spectrum of the product was probably related to the formation of a benzoquinone type structure in the backbone. -OH stretching vibrations of guaiacol and the product were clearly matching around 3400 cm -1 . The absorption bands at 817 and 848 cm -1 in the FT-IR spectrum of the product were attributed to 1,2,4-trisubstituted benzene ring formation in the polymer structure [36]. These peaks verified that polymerization was propagated through ortho-ortho or ortho-para couplings of guaiacol and verifying the formation of phenylene/oxyphenylene repeat units.  Thermogravimetric analysis was also performed to analyze the thermal stabilities of the obtained products. According to the literature, 10% weight losses of poly(guaiacol) synthesized from manganese based catalysts were reported to be between 100-200 °C, and 41% of initial weights of those products were found to remain after pyrolysis under nitrogen atmosphere [36,37]. According to TGA thermograms, the obtained polymers synthesized from an HRP-Cu 2+ hybrid catalyst have shown considerably higher thermal stabilities under nitrogen atmosphere (see Supplementary information for TGA data). The polymer obtained from Table 1, entry 4 conditions started to decompose around 200 °C and lost 10% and 50% of its initial weight at 252 °C and 828 °C, respectively. Pyrolysis residue (carbonaceous char) of the poly(guaiacol) ( Table 1; entry 4) was found to be 48% at 900 °C. Since poly(guaiacol) has a long conjugated polyaromatic backbone, the obtained products demonstrated very high thermal stabilities.
Polymerization of guaiacol was also performed using free HRP enzyme in order to detect differences between activities of free HRP enzyme and HRP-Cu 2+ . The polymerization results of guaiacol by free HRP enzyme in the presence of H 2 O 2 are summarized in Table 2. The optimum polymerization of guaiacol with 51% yield and 9600 g/mol molecular weight was accomplished with 5 weight% of HRP enzyme in pH 7.4 PBS buffer at 30 °C (Table 2; entry 13). Increasing the reaction temperature to 40 °C ( Table 2; entry 14) resulted in a decrease of the polymerization yield (35%) since HRP is sensitive to heating and it denatures around 60 °C [32][33][34]. Enhancing HRP concentration to 10 weight% (Table 2; entry 15) slightly increased the yield (53%) and molecular weight (10,800 g/mol) of the product. However, the addition of 15 weight% of HRP (Table 2; entry 16) resulted in a small increase of the yield (56%) and a decrease of the molecular weight (8400 g/ mol) of the product compared to the conditions of entry 15 in Table 2. Since HRP is a very expensive enzyme, 5 weight% of HRP was decided to be in an optimum concentration in the polymerization of guaiacol (Table 2; entry 13). According to the obtained results, HRP-Cu 2+ was decided to have higher catalytic activity and stability in the polymerization of guaiacol compared to free HRP enzyme. Polymerization of guaiacol carried out with HRP-Cu 2+ gave poly(guaiacol) with higher yield and molecular weight compared to the free HRP enzyme. HRP-Cu 2+ catalyst exhibited higher stability at reaction temperatures of 60 °C and above, and it did not undergo denaturation in contrast to free HRP enzyme.
To examine the reusability of HRP-Cu 2+ , consecutive polymerizations of guaiacol were performed under pH 7.4 PBS buffer with 5 weight% HRP-Cu 2+ loading at 60 °C (Table 3). After each polymerization, the precipitated product was  centrifuged and separated from the reaction media. After that, another run was started up with the addition of guaiacol to the reaction media to see polymerization yield and molecular weight. The second run (Table 3; entry 17) showed that the activity of HRP-Cu 2+ was similar to the first run (Table 1; entry 4). Polymerization yield was 54% and the number average molecular weight (M n ) of the obtained product was 14,000 g/mol. HRP-Cu 2+ still showed some activity in the third run, and gave poly(guaiacol) with 21% yield and 6000 g/mol molecular weight (Table 3; entry 18). The HRP-Cu 2+ hybrid nanoflowers showed slight catalytic activity even in the fourth run and gave a polymer with 8% yield with 2900 g/mol molecular weight (Table 3; entry 19). However, no product was observed after the fourth run. HRP-Cu 2+ probably lost its catalytic activity due to the denaturation of HRP in nanoflowers and the deformation of nanoflower shapes of HRP-Cu 2+ after each run.

Conclusion
In conclusion, HRP-Cu 2+ obtained from the complexation between HRP enzyme and Cu 2+ ions exhibited enhanced catalytic activity and stability in the polymerization reaction of guaiacol compared to that of free HRP enzyme. Optimum polymerization of guaiacol was accomplished in pH 7.4 PBS buffer at 60 °C with 5 weight% of HRP-Cu 2+ loading in the presence of H 2 O 2 and resulted in poly(guaiacol) with 88% yield and 38,000 g/mol molecular weight. Polymerizations using HRP-Cu 2+ exhibited poly(guaiacol) with higher yield and molecular weight compared to using the free HRP enzyme. HRP-Cu 2+ hybrid nanoflowers also exhibited higher stability at 60 °C and higher reaction temperatures and did not undergo denaturation in contrast with free HRP enzyme. Furthermore, HRP-Cu 2+ showed some degree of catalytic activity even after the fourth recycle and can be efficiently used for oxidative polymerizations. 1