Investigation of the Free-Radical Polymerization of Vinyl Monomers Using Horseradish Peroxidase (HRP) Nanoflowers

Investigation of the Free-Radical Polymerization of Vinyl Monomers Using Horseradish Peroxidase (HRP) Nanoflowers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Investigation of the Free-Radical Polymerization of Vinyl Monomers Using Horseradish Peroxidase (HRP) Nanoflowers Gulbahar Ozaydin, Muge Mirioglu, Seyma Dadi, Ismail Ocsoy, Ersen Gokturk This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4914498/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Jan, 2025 Read the published version in Polymer Bulletin → Version 1 posted 8 You are reading this latest preprint version Abstract In this study, we report the production of flower-shaped HRP-Cu 2+ hybrid nano biocatalyst from the complexation between horseradish peroxidase (HRP) enzyme and Cu 2+ ions, and investigate catalytic activity and stability of the obtained nanoflowers on the polymerization of some vinyl monomers (styrene, methylmethacrylate, acrylamide and N -isopropylacrylamide). Polymerizations of these monomers, except water soluble acrylamide, were accomplished under emulsion conditions using cationic, anionic and non-ionic surfactants in the presence of H 2 O 2 and 2,4-pentanedione mediator. Optimum polymerizations were achieved under the conditions of non-ionic surfactant (tween 40) used. HRP-Cu 2+ mediated polymerizations resulted in very high yields and molecular weights ( M n ) of the polymers. Optimum polymerization of styrene with 84% of yield ( M n = 319 kDa) was accomplished at room temperature. However, the highest polymerization yields for acrylamide (96%, M n = 171 kDa) and N -isopropylacrylamide (85%, M n = 185 kDa) was achieved at 70 ºC. Similarly, optimum polymerization of methylmethacrylate was accomplished with 84% of yield ( M n = 190 kDa) at 60 ºC. While free-HRP loses its catalytic activity at 60 ºC and above temperatures, HRP-Cu 2+ showed very high catalytic activity and stability even at 70 ºC. Increasing activity and stability of hybrid nanoflowers provide significant advantages for both scientific and industrial applications. Horseradish peroxidase polymerization styrene methylmethacrylate acrylamide N-isopropylacrylamide Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Enzymes are biomacromolecules that can be used in many synthetic processes and show excellent activity and selectivity [ 1 ]. Enzymes are environmentally benign biocatalysts demonstrating high catalytic activity under mild reaction conditions [ 2 ]. Depending on their structures, enzymes are capable of catalyzing many complex reactions, and also show high selectivity (enantio-, regio-, and chemoselectivity) [ 3 ]. Enzymatic polymerizations have been one of the extensively investigated methodologies for polymer synthesis as an alternative to the traditional methods due to their simplicity and environmentally friendly properties. Horseradish peroxidase (HRP) enzyme can be effectively used as a biocatalyst in free-radical polymerization reactions of some vinyl monomers and aromatic compounds (phenol, aniline, etc.) [ 4 ]. HRP enzyme is able to transfer electrons from hydrogen peroxide to the monomer to generate radicals that will initiate oxidation reactions and polymerizations [4b, 5]. However, denaturation of enzymes in organic and aqueous solutions and at high temperatures is an important drawback for enzymatic polymerization reactions [ 6 ]. To overcome these problems, immobilization techniques could be applied to improve the stability of enzymes, and some applications have been successful. Immobilization methods mainly improve stability and reusability of enzymes, and also make enzymes more economic. Recently, a variety of solid supports have been used for the immobilization of enzymes, such as silica, polymer supports, inorganic and organic nanomaterials, etc [ 7 ]. Immobilization methods are mainly applied in biomedical and biotechnological fields. Immobilization provides moderately increased stability. Reusability of enzymes can also be improved after immobilization. After immobilization, enzymes exhibit high stability even at higher temperatures, and lower or higher pH solutions [ 8 ]. However, it was also observed that there was a significant decrease in the activity of enzymes after immobilization. Due to the restrictions in the movements and conformations of enzymes after immobilization, their catalytic activities dramatically decrease [ 9 ]. In order to increase catalytic activities of immobilized enzymes, great efforts have been carried out to explore convenient immobilization methods that can increase both stability and activity of enzymes [ 8 ]. Hybrid nano biocatalysts were first presented in the literature by Zare and coworkers [ 10 ]. It was reported that flower-shaped hybrid organic-inorganic nano biocatalysts were obtained using bovine serum albumin and Cu 2+ ions. In addition to this study, many enzymes including laccase, lipase, and carbonic anhydrase were reported to generate different types of flower-shaped hybrid nano biocatalysts with metal ions, and these kinds of catalysts showed higher catalytic activity and stability compared to free-enzymes [ 8 , 11 ]. One of the interesting studies about flower-shaped hybrid nano biocatalysts was reported by Ocsoy and coworkers [ 12 ]. It was observed that complexation between HRP enzyme and Cu 2+ ions resulted in flower-shaped nanostructures, and obtained HRP-Cu 2+ nanoflowers showed higher catalytic activity and stability compared to free-HRP enzyme. It was also reported that the reusability of these catalysts was highly enhanced by this strategy. Here, we report the effects of HRP-Cu 2+ hybrid nanoflowers on the polymerization of some vinyl monomers (styrene, methylmethacrylate, acrylamide and N -isopropylacrylamide). Polymerizations, except water soluble acrylamide, were accomplished under emulsion conditions using cationic, anionic and nonionic surfactants. HRP-Cu 2+ /H 2 O 2 /2,4-pentanedione initiating system in emulsion medium was used for the polymerization of vinyl monomers. The influence of reaction variables including catalyst loading, temperature, types of surfactant, and peroxide concentration were investigated for the HRP-Cu 2+ catalyzed free radical polymerization of vinyl monomers. The obtained results showed that HRP-Cu 2+ mediated catalyst system can be efficiently used to polymerize a variety of vinyl monomers in emulsion media with very high yields and molecular weights. While free-HRP enzyme loses its catalytic activity at 60 ºC, HRP-Cu 2+ hybrid nanoflowers showed very high catalytic activity and stability even at 70 ºC. These improvements provide significant advantages for both scientific and industrial applications. Materials and Methods Chemicals Methanol (Isolab, catalog# 947046), styrene (Sigma Aldrich, catalog# 8076791000), acrylamide (Sigma Aldrich, catalog# 8008301000), methyl methacrylate (Sigma Aldrich, catalog# 8005901000), N -isopropylacrylamide (Sigma Aldrich, catalog# 415324-50G), 2,4-pentanedione (Sigma Aldrich, catalog# 8000231000), tween 40 (Sigma Aldrich, catalog# 8221851000), cetyltrimethylammonium bromide (Sigma Aldrich, catalog# 8141190500), sodiumdodecyl sulfate (Sigma Aldrich, catalog# 8220501000), pH 7.4 phosphate buffered saline (PBS, MP biomedicals, catalog# 2810305), hydrogen peroxide (Merck, catalog# 1.08597), coppersulfate pentahydrate (CuSO 4 .5H 2 O, Sigma-Aldrich, catalog#18304), iron (II) sulfate (FeSO 4 , Sigma-Aldrich, catalog#F8263), horseradish peroxidase (HRP, Sigma-Aldrich, 77332, lyophilized, powder, beige, ~ 150 U/mg) were purchased and used for the synthesis of the nanoflowers and polymers. Instrumentation Scanning electron microscope (SEM) images were monitored using Zeiss Gemini 500 instrument. Agilent Premium Compact (600 MHz) NMR spectrometer was used for 1 H and 13 C nuclear magnetic resonance (NMR) analyses of the polymers. Thermogravimetric analyses (TGA) of the obtained polymers were accomplished using Mettler-Toledo TGA/DSC 1 Star system thermal analyzer under nitrogen atmosphere (N 2 ). Approximately, 5–10 mg samples were taken for the analysis, and thermal decomposition temperatures were determined at a temperature range from room temperature (RT) to 600 ºC with 10 ºC/min. Differential scanning calorimetry (DSC) thermograms of the polymers were recorded using a Mettler-Toledo DSC 1 Star system instrument at a heating rate of 10 ºC/min from − 50 to 450 ºC under N 2 atmosphere. For gel permeation chromatography (GPC) analyses, Shimadzu LC-20AD instrument was used. GPC analyses were performed using Agilent PLgel mixed-B column with HPLC purity N,N' -dimethylformamide (DMF) mobile phase at a flow rate of 1 mL/min at 40 ºC. Polystyrene (PS) standards were used for calibration. GPC chromatograms of the synthesized polymer were given in Figure S13-S29 (supplementary information). Synthesis of HRP-Cu 2+ nanoflowers CuSO 4 solution was added to the HRP enzyme solution (0.02 mg/mL) in pH 7.4 PBS buffer. The solution was vortexed at RT for 3 min and incubated at + 4 ºC for 3 days. After incubation, light blue precipitate was separated by centrifugation and washed several times with water to remove impurities. The obtained HRP-Cu 2+ nanoflowers was dried in an oven at 40 ºC and used for the reactions [ 12 – 13 ]. Representative polymerization procedure of styrene 10 mL of pure water and styrene (6.0 mmol, 0.69 mL) was put in a test tube with screw cap. Dissolved oxygen in the solution was degassed by bubbling N 2 gas through the mixture. HRP-Cu 2+ , H 2 O 2 , surfactant and 2,4-pentanedione were added to the mixture, and N 2 gas was bubbled through the mixture again. The mixture was stirred at the desired temperature for 24 hours. Then, the mixture was poured into methanol, and obtained precipitate was filtered, washed with methanol and dried in an oven [ 14 ]. Polymerization of other vinyl monomers 10 mL of distilled water and 6.0 mmol of monomer (acrylamide, methylmethacrylate and N -isopropylacrylamide) were put in a test tube, and dissolved oxygen was removed by bubbling N 2 gas through the solution. 10 mg HRP-Cu 2+ , H 2 O 2 , 0.1 g of tween 40 and 2,4-pentanedione were added to the solution, and N 2 gas was passed through the mixture again. Tween 40 was not added to the polymerization of acrylamide. The mixture was stirred at the desired temperature for 24 hours. Then, the mixture was poured into methanol, and the obtained precipitate was filtered, washed with methanol and dried in an oven [ 2 , 15 ]. RESULTS and DISCUSSION The synthesis of HRP-Cu 2+ hybrid flower-shaped nano biocatalyst was achieved with the complexation of Cu 2+ ions and HRP enzyme. Flower-shaped nanostructures can be obtained with the appropriate nucleation that occurs as a result of the coordination of Cu 2+ ions with the amine groups in the structure of the HRP. The formation steps of HRP-Cu 2+ hybrid flower-shaped nano biocatalyst have been shown in detail in the literature [ 12 , 16 ]. The morphology of the synthesized HRP-Cu 2+ nano biocatalyst can be seen in the SEM (scanning electron microscopy) analysis shown in Fig. 1 . According to the SEM analysis result, the obtained HRP-Cu 2+ nano biocatalyst have flower-shaped and porous structure with a size of approximately 10 µm. It was reported that the mass percentage of copper ion (Cu 2+ ) in the HRP-Cu 2+ nanoflowers was approximately 10% and HRP-Cu 2+ was reported to show approximately 300% higher catalytic activity compared to free-HRP enzyme on the oxidation of guaiacol [ 12 ]. HRP-Cu 2+ also exhibited only 4% of stability loss in solution within 30 days. However, free-HRP enzyme showed approximately 65% of stability loss in the same period. Thus, HRP-Cu 2+ nanoflowers showed much higher stability compared to the free-HRP enzyme [ 12 ]. In order to detect catalytic activity of HRP-Cu 2+ nanoflowers on the polymerization of vinyl monomers, polymerization of styrene was first investigated (Fig. 2 ). After optimum polymerization condition of styrene using HRP-Cu 2+ was determined, polymerizations of other vinyl monomers (acrylamide, methylmethacrylate and N -isopropylacrylamide) were carried out. The results of the HRP-Cu 2+ mediated polymerization of styrene are summarized in Table 1 . Polymerizations were carried out under emulsion condition with cationic, anionic and non-ionic surfactants in the presence of H 2 O 2 and 2,4-pentanedione mediators. Polymerization of vinyl monomers did not occur in the absence of HRP-Cu 2+ or peroxide. The influence of HRP-Cu 2+ amount on the polymerization of styrene was first optimized in water with tween40 surfactant at RT (entries 1, 2 and 3 in Table 1 ). It was observed that utilization of 10 mg of HRP-Cu 2+ provided very good yield (84%) and the highest molecular weight ( M n = 319 kDa) of polystyrene (entry 2 in Table 1 ). Doubling the amount of HRP-Cu 2+ slightly improved the polymerization, and resulted in 87% of yield (entry 3 in Table 1 ). However, the number average molecular weight ( M n ) of the obtained polystyrene was 210 kDa which was lower compared to the condition of using 10 mg of HRP-Cu 2+ . Observation of low M n is probably due to the generation of more radical species during the polymerization with utilization of 20 mg of HRP-Cu 2+ [ 17 ]. Thereby, higher HRP-Cu 2+ concentration gave the lower molecular weight of polymer. In addition to that utilization of 10 mg HRP-Cu 2+ was more economic than that of 20 mg of it. Decreasing the amount of the catalyst from 10 mg to 5 mg significantly reduced the polymerization yield. Addition of 5.0 mg of catalyst in the polymerization resulted in 57% of polymer yield (entry 1 in Table 1 ). Table 1 Polymerization of styrene by HRP-Cu 2+ nano biocatalyst in the presence of H 2 O 2 and 2,4-pentanedione Entry a HRP-Cu 2+ (mg) H 2 O 2 (mmol) Surfactant T p (ºC) Yield (%) M n (kDa) Ð 1 5 1.0 0.1 g Tween40 RT 57 298 1.88 2 10 1.0 0.1 g Tween40 RT 84 319 2.03 3 20 1.0 0.1 g Tween40 RT 87 210 2.71 4 10 1.0 0.1 g Tween40 40 72 184 1.63 5 10 1.0 0.1 g Tween40 50 62 177 2.78 6 10 1.0 0.05 g Tween40 RT 65 140 2.39 7 10 1.0 0.2 g Tween40 RT 86 79 2.44 8 b 10 1.0 0.1 g Tween40 RT 84 309 1.57 9 10 0.5 0.1 g Tween40 RT 57 76 2.67 10 10 2.0 0.1 g Tween40 RT 88 56 4.62 11 10 1.0 0.1 g SDS RT 0 - - 12 10 1.0 0.1 g CTAB RT 37 16 5.79 a all polymerizations were carried out with 690 µL of styrene (6.0 mmol) in 10 mL water in the presence of 0.05 mmol of 2,4-pentanedione, tween 40 surfactant and H 2 O 2 in 24h, b The amount of 2,4-pentanedione was 0.1 mmol, other parameters were kept the same as condition a, SDS = sodiumdodecyl sulfate, CTAB = cetyltrimethylammonium bromide, T p = reaction temperature, M n : the number average molecular weight, Ð : polydispersity index. Reaction temperature is one of the important parameters for the polymerization of styrene due to its effects on the activity of enzymes. Therefore, polymerization of styrene at different reaction temperatures was performed to detect better catalytic activity of HRP-Cu 2+ nanoflowers. Polymerization of polystyrene carried out at room temperature (entry 2 in Table 1 ) provided higher yield and molecular weight compared to the reactions accomplished at 40 and 50 ºC (entries 4 and 5 in Table 1 ). Therefore, room temperature (RT) was decided to be optimum polymerization temperature for styrene. In order to facilitate the polymerization of styrene, different types and amounts of surfactants were also investigated to generate emulsion system. First, the influence of anionic (SDS), cationic (CTAB) and non-ionic (tween 40) surfactants on the HRP-Cu 2+ mediated polymerization of styrene were investigated. While addition of anionic SDS and cationic CTAB surfactants negatively affected the polymerization yields (entries 11 and 12 in Table 1 ), utilization of non-ionic tween 40 surfactant significantly enhanced the yield and the molecular weight of polystyrene. By adding 0.1 g of tween 40, the polymerization yield was 84% (entry 2 in Table 1 ). M n and polydispersity index ( Ð ) of the polystyrene were respectively 319 kDa and 2.03. Polymerization yield was dramatically decreased by lowering the amount of tween 40 from 0.1 g to 0.05 g (entry 6 in Table 1 ), and 65% of yield was observed. However, negligible increase in the polymerization yield (86%) occurred by adding 0.2 g tween 40 in the solution (entry 7 in Table 1 ). However, the number average molecular weight of the obtained polystyrene (79 kDa) significantly decreased compared to the polymerization achieved by adding 0.1 g of tween 40 (entry 2 in Table 1 ). Polymerization of styrene in the presence of cationic CTAB surfactant gave 37% of yield with 16 kDa molecular weight (entry 12 in Table 1 ). However, no polymer product was observed with utilization of anionic SDS in the polymerization (entry 11 in Table 1 ). Probably, cationic and anionic surfactants deactivate or limit the activity of HRP-Cu 2+ nanoflowers. These results suggest that non-ionic tween 40 surfactant provides better polymerization system for HRP-Cu 2+ mediated polymerization of styrene. In order to discover the role of H 2 O 2 concentration on the HRP-Cu 2+ mediated vinyl polymerizations, three different experiments with increasing H 2 O 2 concentration were conducted. Increasing H 2 O 2 concentration from 0.5 to 1.0 mmol resulted in increasing yield and M n . While polymerization performed using 1.0 mmol H 2 O 2 gave 84% of yield (entry 2 in Table 1 ), addition of 0.5 mmol H 2 O 2 resulted in 57% of yield (entry 9 in Table 1 ). M n of the polymer (76 kDa) obtained from adding 0.5 mmol H 2 O 2 was also lower compared to the 1.0 mmol H 2 O 2 concentration (319 kDa). Enhancing H 2 O 2 concentration from 1.0 to 2.0 mmol showed the maximum polymerization yield with 88% (entry 10 in Table 1 ). However, M n of the obtained polymer was 56 kDa which was significantly lower than that of the polymer obtained from entry 2. Higher peroxide concentration probably generated increasing number of active chains and/or termination reactions. Another reason could be inhibiting enzymatic activity with increasing H 2 O 2 concentration. Therefore, M n of the polymer decreased. Further increase in the concentration of peroxide was not considered since excess peroxide inhibits the activity of HRP enzyme [ 18 ]. 2,4-pentanedione mediator has also a great impact on the polymerization of vinyl monomers. For the initiation of the radical polymerization, β-diketone radicals can be generated from the oxidation of 2,4-pentanedione with H 2 O 2 in the presence of HRP-Cu 2+ nanoflowers [ 19 ]. Two different 2,4-pentanedione (acetylacetone) concentration was investigated during the polymerizations. Increasing acetylacetone concentration from 0.05 mmol to 0.1 mmol was found to be no significant effect on the polymerization yield and the molecular weight of the polymer (entries 2 and 8 in Table 1 ). The yield of the polymerization using 0.1 mmol of pentanedione (entry 8 in Table 1 ) was the same as the reaction carried out addition of 0.05 mmol of pentanedione. The number average molecular weights of the polymers were also found to be very close. Therefore, utilization of 0.05 mmol of pentanedione cocatalyst was determined to be sufficient for the polymerization of styrene. Previously, utilization of 20 mg of free-HRP enzyme for the polymerization of 12 mmol of styrene was reported to be resulted in up to 64.3% of yield with 3.97x10 5 Da at RT under mini-emulsion conditions with 12 mmol acetylacetone and 0.05 mmol of H 2 O 2 [ 20 ]. Utilization of cosolvents including THF (tetrahydrofuran), dioxane, DMF (dimethylformamide) and methanol in the polymerization of HRP enzyme initiated polymerization of styrene was also investigated without adding surfactant, and the yields of the polystyrene were very low with up to 21.2% under H 2 O 2 /acetylacetone catalyst system [ 14 ]. According to our findings, HRP-Cu 2+ nanoflowers demonstrated higher catalytic activity towards polymerization of styrene compared to the free-HRP enzyme. Structural characterization of the polystyrene was accomplished by FT-IR, 1 H NMR and 13 C NMR analyses. According to the FT-IR spectrum of polystyrene (entry 2 in Table 1 ), absorption bands observed around 2900 cm -1 and 3000 cm -1 indicate the presence of aliphatic and aromatic C-H stretching vibrations. C = C stretching vibrations belonging to the phenyl ring appeared around 1750 cm -1 (Figure S1 , supplementary information). 1 H and 13 C NMR spectra results of the synthesized polymer suggest that atactic polystyrene structure was obtained [ 14 ]. The phenyl ring protons are observed around 7.02 ppm and 6.50 ppm in the 1 H NMR spectrum of the polystyrene. The methine (CH) and methylene (CH 2 ) protons are respectively observed at 1.83 and 1.50 ppm (Figure S30, supplementary information). In the 13 C NMR spectrum, peaks observed at 145, 127 and 125 ppm indicates the phenyl ring carbons. 13 C peaks observed at 43 and 40 ppm attribute methylene (CH 2 ) and methine (CH) carbons, respectively (Figure S34, supplementary information). According to TGA and DSC analyses results, the polymer (entry 2 in Table 1 ) started to decompose around 350 ºC, and 50% of thermal decomposition occurred around 420 ºC (Figure S5, supplementary information). The glass transition temperature ( T g ) of the polymer was detected approximately at 110 ºC, and the thermal decomposition temperature was observed around 430 ºC (Figure S9, supplementary information). Table 2 Polymerization of other vinyl monomers by HRP-Cu 2+ in the presence of H 2 O 2 and 2,4-pentanedione Entry a Monomer T p (ºC) Yield (%) M n (kDa) Ð 13 Acrylamide RT 0 - - 14 Acrylamide 50 42 142 2.62 15 Acrylamide 70 96 171 2.66 16 b Methylmethacrylate RT 23 136 2.09 17 b Methylmethacrylate 50 73 160 3.16 18 b Methylmethacrylate 60 84 190 1.92 19 b N -isopropylacrylamide 70 85 185 2.82 a all polymerizations were carried out 6.0 mmol of monomer in 10 mL of water in the presence of 10 mg HRP-Cu 2+ , 0.05 mmol of 2,4-pentanedione and 1.0 mmol of H 2 O 2 in 24h, b 0.1 g of tween 40 surfactant was added for the polymerization mixtures. T p = polymerization temperature, M n : the number average molecular weight, Ð : heterogeneity index. Polymerizations of other vinyl monomers (acrylamide, methylmethacrylate and N -isopropylacrylamide) were also accomplished by HRP-Cu 2+ nanoflowers. All polymerizations were conducted using the optimized reaction condition obtained from the polymerization of styrene (entry 2 in Table 1 ). Unlike polymerization of other vinyl monomers, acrylamide was polymerized surfactant-free media due to solubility of polyacrylamide in water (Fig. 3 ). Only, reaction temperature parameter was altered to achieve these polymerizations. Polymerizations of these vinyl monomers at RT either did not take place or occurred with very low yields. Therefore, these polymerizations were carried out at higher reaction temperatures compared to styrene (Table 2 ). First, HRP-Cu 2+ mediated polymerization of acrylamide in water was conducted without adding tween 40 surfactant, and 70 ºC was found to be suitable temperature for the polymerization. Polyacrylamide was obtained with 96% of yield at 70 ºC (entry 15 in Table 2 ). Polymerization of acrylamide at 50 ºC resulted in lower yield and molecular weight (entry 14 in Table 2 ). Higher reaction temperatures above 70 ºC were not tried since HRP-Cu 2+ nanoflowers lost its catalytic activity. The M n and polydispersity index of the polymer obtained at 70 ºC were respectively 171 kDa and 2.66. FT-IR, 1 H NMR and 13 C NMR analyses were performed for the structural characterization of the obtained polyacrylamide (entry 15 in Table 2 ). According to the FT-IR analysis, a broad -OH stretching band around 3300 cm -1 was observed due to the moisture contents of the polymer (Figure S2, supplementary information). Carbonyl peak related to the amide (-CONH 2 ) functional group was observed around 1750 cm -1 . From the 1 H NMR analysis, the methylene (-CH 2 ) and methine (-CH) protons were respectively observed at 1.50 and 2.20 ppm (Figure S31, supplementary information). According to the 13 C NMR analysis, carbonyl (-C = O) carbon was seen at 179 ppm, methine (-CH) and methylene (-CH 2 ) carbons were also observed at 41 ppm and 35 ppm, respectively (Figure S35, supplementary information). According to the 13 C NMR spectrum results, polyacrylamide was decided to be atactic structure due to the observation of triplet peak (triad arrangements) for the methine carbon [15a]. TGA analysis result of the polyacrylamide (entry 15 in Table 2 ) was carried out between RT and 600 ºC under N 2 atmosphere, and showed three stages of thermal degradation [ 21 ]. About 15–20% of weight loss was observed up to 200 ºC due to removal of absorbed water. Between 200–340 ºC, about 20% mass loss occurred due to the removal of by-products including NH 3 , H 2 O and CO 2 . Finally, about 40% weight loss happened between 340–460 ºC due to removal of H 2 O and CO 2 from the remaining product (Figure S6, supplementary information). According to the DSC analysis of the polyacrylamide (entry 15 in Table 2 ), glass transition temperature ( T g ) and melting temperature ( T m ) of the polymer were observed at 165 ºC and 300 ºC, respectively (Figure S10, supplementary information). Methylmethacrylate (MMA) was also polymerized using HRP-Cu 2+ nanoflowers in the presence of H 2 O 2 , 2,4-pentanedione and tween 40 surfactant (Fig. 4 ). Optimum polymerization was accomplished with 84% of yield at 60 ºC (entry 18 in Table 2 ). The M n of the polymer was 190 kDa with 1.92 polydispersity index. Reaction temperature above 60 ºC was not tried because thermal initiation of MMA at temperatures above 60 ºC occurred without adding H 2 O 2 [ 22 ]. Polymerization of MMA carried out at RT and 50 ºC (entries 16 and 17 in Table 2 ) resulted in respectively 23% and 73% of yields which are lower than that of obtained from 60 ºC. Structural characterization of polyMMA (entry 18 in Table 2 ) was also shown by FT-IR, 1 H NMR and 13 C NMR analyses. Aliphatic C-H stretching vibrations around 2900 cm -1 , ester group carbonyl (-C = O) vibration band at 1700 cm -1 and C-O vibration band around 1100 cm -1 were observed from the FT-IR spectrum of the polymer (Figure S3, supplementary information). From the 1 H NMR analysis of the product, methyl protons (-OCH 3 ) belonging to the ester group was observed at 3.57 ppm. Methylene (-CH 2 ) and methyl (-CH 3 ) protons were respectively observed at 1.78 ppm and 0.82 ppm (Figure S32, supplementary information). According to the 13 C NMR spectrum, carbonyl (-C = O) carbon at 177 ppm, quaternary carbon (-C) attached to carbonyl at 54 ppm, methyl group carbon (-OCH 3 ) attached to ester group at 52 ppm, methylene (-CH 2 ) carbon at 44 ppm and methyl group (-CH 3 ) carbon at 16 ppm were observed (Figure S36, supplementary information). Obtained polyMMA demonstrated two stages of thermal degradation between RT and 600 ºC under N 2 atmosphere [ 23 ]. Approximately 5% of mass loss was observed between 200–250 ºC due to the degradation of the polymer side chains. The second mass loss about 80% occurred between 250–440 ºC due to the removal of by-products such as H 2 O and CO 2 from the polymer sample (Figure S7, supplementary information). The glass transition temperature ( T g ) of the polymer was detected at 125 ºC, and the degradation temperature ( T d ) of the polyMMA was observed around 370 ºC (Figure S11, supplementary information). Finally, polymerization of NIPAM was accomplished at 70 ºC similar to the acrylamide (Fig. 5 ). Polymerization yield was 85%, and M n of the polyNIPAM (PNIPAM) was 185 kDa with 2.82 PDI (entry 19 in Table 2 ). A broad -OH stretching band around 3300 cm -1 was observed in the FT-IR spectrum of the polymer due to the moisture content of the product (Figure S4, supplementary information). Aliphatic C-H stretching vibrations was observed at 2900 cm -1 , and amide carbonyl peak (-CONH 2 ) was detected around 1700 cm -1 in the spectrum. According to the 1 H NMR analysis of the product, methine (-CH) and methylene (-CH 2 ) protons were observed at 2.03 and 1.24 ppm, respectively. Proton peaks at 3.98 and 1.12 ppm were attributed to the methine (-CH) and two methyl group (-CH 3 ) protons of the isopropyl group, respectively (Figure S33, supplementary information). From the 13 C NMR spectrum of the polymer; carbonyl (-C = O) carbon, methine carbon (-CH) attached to carbonyl group and methylene carbon (-CH 2 ) were observed respectively at 174, 37 and 29 ppm. 13 C peaks at 41 and 22 ppm are related to the methine carbon (-CH) and two methyl carbons belonging to isopropyl group (Figure S37, supplementary information). According to the TGA thermogram of the PNIPAM, the polymer had three stages of thermal degradation similar to the polyacrylamide [ 24 ]. About 10% of mass loss up to 200 ºC was observed due to the removal of moisture. 15% of mass loss occurred between 200–330 ºC due to the possible removal of by-products such as NH 3 , H 2 O and CO 2 . About 40% of weight loss happened between 330–470 ºC removal of H 2 O and CO 2 from the remaining product (Figure S8, supplementary information). The PNIPAM only showed decomposition temperature at 400 ºC from DSC thermogram result, and no glass transition temperature was detected (Figure S12, supplementary information). CONCLUSION HRP-Cu 2+ mediated polymerization of four different vinyl monomers including styrene, acrylamide, methylmethacrylate and N -isopropylacrylamide was successfully investigated in the presence of H 2 O 2 and 2,4-pentanedione mediators under emulsion media. The obtained results showed that HRP-Cu 2+ mediated catalyst systems can be efficiently used to polymerize a variety of vinyl monomers in emulsion media with very high yields and molecular weights. Acrylamide monomer was polymerized surfactant-free media with very high yield and molecular weight due to solubility of polyacrylamide in water Non-ionic tween 40 surfactant provides better polymerization system for the HRP-Cu 2+ mediated polymerization of monomers compared to the anionic sodiumdodecyl sulfate and cationic cetyltrimethylammonium bromide surfactants. According to the structural characterization results, the synthesized polymers are mainly obtained as atactic structures. While free-HRP enzyme loses its catalytic activity at 60 ºC, HRP-Cu 2+ hybrid nanoflowers showed very high catalytic activity and stability even at 70 ºC. Increasing catalytic activity and stability of hybrid nanoflowers provide significant advantages for both scientific and industrial applications. Declarations Acknowledgement This work was supported by Hatay Mustafa Kemal University Coordinatorship of Scientific Research Projects (project # 21.GAP.058). Conflict of Interest Statement The authors declare no conflict of interest. Author contributions GO contributed to methodology, formal analysis and investigation. MM contributed to methodology, formal analysis and investigation. SD contributed to methodology, formal analysis, investigation, review and editing of manuscript. IO contributed to conceptualization, writing—review and editing. EG contributed to conceptualization, project administration, funding acquisition, writing—review and editing, and supervision. Supplementary materials The following analysis results are included as supporting information: FT-IR (Figures S1-S4), 1 H NMR (Figures S30-S33) and 13 C NMR (Figures S34-S37) spectra results; TGA (Figures S5-S8) and DSC (Figures S9-S12) thermograms; and GPC chromatograms (Figures S13-S29) of the synthesized polymers. References Miletić N, Nastasović A, Loos K (2012) Immobilization of biocatalysts for enzymatic polymerizations: Possibilities, advantages, applications. 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Gokturk E (2020) Flowerlike hybrid horseradish peroxidase nanobiocatalyst for the polymerization of guaiacol. Turkish Journal of Chemistry 44:1285-1292. a) Rana S, Yeh YC, Rotello VM (2010) Engineering the nanoparticle–protein interface: applications and possibilities. Current Opinion in Chemical Biology 14:828-834; b) Wang P (2009) Multi-scale Features in Recent Development of Enzymic Biocatalyst Systems. Applied Biochemistry and Biotechnology 152:343-352; c) Sheldon RA (2007) Enzyme Immobilization: The Quest for Optimum Performance. Advanced Synthesis & Catalysis 349:1289-1307. Altinkaynak C, Tavlasoglu S, Ocsoy I (2016) A new generation approach in enzyme immobilization: Organic-inorganic hybrid nanoflowers with enhanced catalytic activity and stability. Enzyme Microb Tech 93:105-112. a) Hanefeld U, Cao L, Magner E (2013) Enzyme immobilisation: fundamentals and application. Chemical Society reviews 42:6211-6212; b) Gupta MN, Mattiasson B (1992) Unique applications of immobilized proteins in bioanalytical systems. Methods of biochemical analysis 36:1-34. Ge J, Lei J, Zare RN (2012) Protein–inorganic hybrid nanoflowers. Nature Nanotechnology 7:428-432. Ocsoy I, Dogru E, Usta S (2015) A new generation of flowerlike horseradish peroxides as a nanobiocatalyst for superior enzymatic activity. Enzyme Microb Tech 75:25-29. Somturk B, Hancer M, Ocsoy I, Özdemir N (2015) Synthesis of copper ion incorporated horseradish peroxidase-based hybrid nanoflowers for enhanced catalytic activity and stability. Dalton Transactions 44:13845-13852. Gokturk E, Ocsoy I, Turac E, Sahmetlioglu E (2020) Horseradish peroxidase-based hybrid nanoflowers with enhanced catalytical activities for polymerization reactions of phenol derivatives. Polymers for Advanced Technologies 31:2371-2377. Singh A, Ma D, Kaplan DL (2000) Enzyme-Mediated Free Radical Polymerization of Styrene. Biomacromolecules 1:592-596. a) Kalra B, Gross RA (2002) HRP-mediated polymerizations of acrylamide and sodium acrylate. Green Chemistry 4:174-178; b) Teixeira D, Lalot T, Brigodiot M, Maréchal E (1999) β-Diketones as Key Compounds in Free-Radical Polymerization by Enzyme-Mediated Initiation. Macromolecules 32:70-72. Ocsoy I, Dogru E, Usta S (2015) A new generation of flowerlike horseradish peroxides as a nanobiocatalyst for superior enzymatic activity. Enzyme Microb Tech 75-76:25-29. Tsujimoto T, Uyama H, Kobayashi S (2001) Polymerization of Vinyl Monomers Using Oxidase Catalysts. Macromolecular Bioscience 1:228-232. Hollmann F, Arends IWCE (2012) Enzyme Initiated Radical Polymerizations. Polymers 4:759-793. Kohri M (2014) Development of HRP-mediated enzymatic polymerization under heterogeneous conditions for the preparation of functional particles. Polymer Journal 46:373-380. Shan J, Kitamura Y, Yoshizawa H (2005) Emulsion polymerization of styrene by horseradish peroxidase-mediated initiation. Colloid and Polymer Science 284:108-111. Leung WM, Axelson DE, Van Dyke JD (1987) Thermal degradation of polyacrylamide and poly(acrylamide-co-acrylate). Journal of Polymer Science Part A: Polymer Chemistry 25:1825-1846. Nising P, Meyer T, Carloff R, Wicker M (2005) Thermal Initiation of MMA in High Temperature Radical Polymerizations. Macromolecular Materials and Engineering 290:311-318. Nikolaidis AK, Achilias DS (2018) Thermal Degradation Kinetics and Viscoelastic Behavior of Poly(Methyl Methacrylate)/Organomodified Montmorillonite Nanocomposites Prepared via In Situ Bulk Radical Polymerization Polymers 10(5):491. Bauri K, Roy SG, Arora S, Dey RK, Goswami A, Madras G, De P (2013) Thermal degradation kinetics of thermoresponsive poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) copolymers prepared via RAFT polymerization. Journal of Thermal Analysis and Calorimetry 111:753-761. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterials.docx Cite Share Download PDF Status: Published Journal Publication published 24 Jan, 2025 Read the published version in Polymer Bulletin → Version 1 posted Editorial decision: Revision requested 02 Jan, 2025 Reviews received at journal 26 Dec, 2024 Reviewers agreed at journal 16 Dec, 2024 Reviewers agreed at journal 18 Sep, 2024 Reviewers invited by journal 16 Sep, 2024 Editor assigned by journal 19 Aug, 2024 Submission checks completed at journal 16 Aug, 2024 First submitted to journal 14 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4914498","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":345595898,"identity":"0f4ff99c-98a8-4a78-8cfe-26d88c7ace07","order_by":0,"name":"Gulbahar Ozaydin","email":"","orcid":"","institution":"Hatay Mustafa Kemal Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Gulbahar","middleName":"","lastName":"Ozaydin","suffix":""},{"id":345595899,"identity":"404f778a-78d5-402b-bbdf-ef865dacb2d7","order_by":1,"name":"Muge Mirioglu","email":"","orcid":"","institution":"Hatay Mustafa Kemal Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Muge","middleName":"","lastName":"Mirioglu","suffix":""},{"id":345595903,"identity":"982dbb65-73d5-4640-9bed-84f95ac46b13","order_by":2,"name":"Seyma Dadi","email":"","orcid":"","institution":"Abdullah Gul Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Seyma","middleName":"","lastName":"Dadi","suffix":""},{"id":345595904,"identity":"ca04c2c9-1e17-45ea-a918-a9342be72ab8","order_by":3,"name":"Ismail Ocsoy","email":"","orcid":"","institution":"Erciyes Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Ismail","middleName":"","lastName":"Ocsoy","suffix":""},{"id":345595906,"identity":"dbf23ce3-1020-4043-89ca-e3b5085fa239","order_by":4,"name":"Ersen Gokturk","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYBADAyBmZvgAJNnYidXCA9TCOAOkhZkULcw8DGDL8AN+6eMPHxf8sjG2l8h9bGzza5s8HzMD44ePObi1SPblGBvP7Esz45FIN07O7btt2MbMwCw5cxseB53hYZPm7TlswyORxnw4t+c2I1ALGzMvHi32Z9if/4Zrsey5bU9QC9DbZsw8Pw6bgbQkM/y4nUhQi8QZHmNp3oY0Y54zz5gNextuJ7cxMzbj9Qt/D/vDzzx/bAzb29OYJX78uW07v7354IePeLSAAWMbCoOxgYB6EPiDwRgFo2AUjIJRgAAAi0dG5/OXXzUAAAAASUVORK5CYII=","orcid":"","institution":"Hatay Mustafa Kemal Universitesi","correspondingAuthor":true,"prefix":"","firstName":"Ersen","middleName":"","lastName":"Gokturk","suffix":""}],"badges":[],"createdAt":"2024-08-14 14:51:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4914498/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4914498/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00289-025-05664-z","type":"published","date":"2025-01-24T15:58:25+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":64586439,"identity":"7d437499-dfd7-4a73-8bf5-6a079e40d477","added_by":"auto","created_at":"2024-09-16 08:10:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":241739,"visible":true,"origin":"","legend":"\u003cp\u003eSEM image of HRP-Cu\u003csup\u003e2+\u003c/sup\u003e hybrid nanoflowers.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4914498/v1/3efd77f3ed1087bf683c4589.png"},{"id":64585085,"identity":"97f8a89f-e003-47b8-910d-a9ad54815cfe","added_by":"auto","created_at":"2024-09-16 07:46:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":63922,"visible":true,"origin":"","legend":"\u003cp\u003eHRP-Cu\u003csup\u003e2+\u003c/sup\u003e mediated polymerization of styrene in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 2,4-pentanedione and tween 40 surfactant.\u003c/p\u003e","description":"","filename":"Figure2.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-4914498/v1/e8fd64091626a6631c09c662.png"},{"id":64585089,"identity":"11554c84-ff10-4c45-893e-47e434ec3159","added_by":"auto","created_at":"2024-09-16 07:46:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":57101,"visible":true,"origin":"","legend":"\u003cp\u003eHRP-Cu\u003csup\u003e2+\u003c/sup\u003e mediated polymerization of acrylamide in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 2,4-pentanedione.\u003c/p\u003e","description":"","filename":"Figure3.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-4914498/v1/350dc480ca29d4b77c236d96.png"},{"id":64585083,"identity":"8ceb2b32-7537-4212-83b5-842b13dfcbd0","added_by":"auto","created_at":"2024-09-16 07:46:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":62155,"visible":true,"origin":"","legend":"\u003cp\u003eHRP-Cu\u003csup\u003e2+\u003c/sup\u003e mediated polymerization of methylmethacrylate in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 2,4-pentanedione and tween 40 surfactant.\u003c/p\u003e","description":"","filename":"Figure4.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-4914498/v1/51febf178f67de140e36267e.png"},{"id":64585506,"identity":"def22d09-533b-4779-b6b0-82c098773fde","added_by":"auto","created_at":"2024-09-16 07:54:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":70397,"visible":true,"origin":"","legend":"\u003cp\u003eHRP-Cu\u003csup\u003e2+\u003c/sup\u003e mediated polymerization of \u003cem\u003eN\u003c/em\u003e-isopropylacrylamide (NIPAM) in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 2,4-pentanedione and tween 40 surfactant.\u003c/p\u003e","description":"","filename":"Figure5.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-4914498/v1/e8b4e8baae6bcd8cd5abea02.png"},{"id":74858553,"identity":"2a22da6d-abd1-4a4c-aa72-3c500208e0de","added_by":"auto","created_at":"2025-01-27 16:11:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1244309,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4914498/v1/6c834cd8-8ab5-4da4-a143-4c4f85caddf4.pdf"},{"id":64585088,"identity":"69ec1472-39d0-4f77-8d7f-b4344265cf18","added_by":"auto","created_at":"2024-09-16 07:46:13","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4565153,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-4914498/v1/f1666a041c993f45b10b4c8f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Investigation of the Free-Radical Polymerization of Vinyl Monomers Using Horseradish Peroxidase (HRP) Nanoflowers","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eEnzymes are biomacromolecules that can be used in many synthetic processes and show excellent activity and selectivity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Enzymes are environmentally benign biocatalysts demonstrating high catalytic activity under mild reaction conditions [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Depending on their structures, enzymes are capable of catalyzing many complex reactions, and also show high selectivity (enantio-, regio-, and chemoselectivity) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEnzymatic polymerizations have been one of the extensively investigated methodologies for polymer synthesis as an alternative to the traditional methods due to their simplicity and environmentally friendly properties. Horseradish peroxidase (HRP) enzyme can be effectively used as a biocatalyst in free-radical polymerization reactions of some vinyl monomers and aromatic compounds (phenol, aniline, etc.) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. HRP enzyme is able to transfer electrons from hydrogen peroxide to the monomer to generate radicals that will initiate oxidation reactions and polymerizations [4b, 5]. However, denaturation of enzymes in organic and aqueous solutions and at high temperatures is an important drawback for enzymatic polymerization reactions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. To overcome these problems, immobilization techniques could be applied to improve the stability of enzymes, and some applications have been successful.\u003c/p\u003e \u003cp\u003eImmobilization methods mainly improve stability and reusability of enzymes, and also make enzymes more economic. Recently, a variety of solid supports have been used for the immobilization of enzymes, such as silica, polymer supports, inorganic and organic nanomaterials, etc [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Immobilization methods are mainly applied in biomedical and biotechnological fields. Immobilization provides moderately increased stability. Reusability of enzymes can also be improved after immobilization. After immobilization, enzymes exhibit high stability even at higher temperatures, and lower or higher pH solutions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, it was also observed that there was a significant decrease in the activity of enzymes after immobilization. Due to the restrictions in the movements and conformations of enzymes after immobilization, their catalytic activities dramatically decrease [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In order to increase catalytic activities of immobilized enzymes, great efforts have been carried out to explore convenient immobilization methods that can increase both stability and activity of enzymes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHybrid nano biocatalysts were first presented in the literature by Zare and coworkers [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It was reported that flower-shaped hybrid organic-inorganic nano biocatalysts were obtained using bovine serum albumin and Cu\u003csup\u003e2+\u003c/sup\u003e ions. In addition to this study, many enzymes including laccase, lipase, and carbonic anhydrase were reported to generate different types of flower-shaped hybrid nano biocatalysts with metal ions, and these kinds of catalysts showed higher catalytic activity and stability compared to free-enzymes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. One of the interesting studies about flower-shaped hybrid nano biocatalysts was reported by Ocsoy and coworkers [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It was observed that complexation between HRP enzyme and Cu\u003csup\u003e2+\u003c/sup\u003e ions resulted in flower-shaped nanostructures, and obtained HRP-Cu\u003csup\u003e2+\u003c/sup\u003e nanoflowers showed higher catalytic activity and stability compared to free-HRP enzyme. It was also reported that the reusability of these catalysts was highly enhanced by this strategy.\u003c/p\u003e \u003cp\u003eHere, we report the effects of HRP-Cu\u003csup\u003e2+\u003c/sup\u003e hybrid nanoflowers on the polymerization of some vinyl monomers (styrene, methylmethacrylate, acrylamide and \u003cem\u003eN\u003c/em\u003e-isopropylacrylamide). Polymerizations, except water soluble acrylamide, were accomplished under emulsion conditions using cationic, anionic and nonionic surfactants. HRP-Cu\u003csup\u003e2+\u003c/sup\u003e/H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/2,4-pentanedione initiating system in emulsion medium was used for the polymerization of vinyl monomers. The influence of reaction variables including catalyst loading, temperature, types of surfactant, and peroxide concentration were investigated for the HRP-Cu\u003csup\u003e2+\u003c/sup\u003e catalyzed free radical polymerization of vinyl monomers. The obtained results showed that HRP-Cu\u003csup\u003e2+\u003c/sup\u003e mediated catalyst system can be efficiently used to polymerize a variety of vinyl monomers in emulsion media with very high yields and molecular weights. While free-HRP enzyme loses its catalytic activity at 60 \u0026ordm;C, HRP-Cu\u003csup\u003e2+\u003c/sup\u003e hybrid nanoflowers showed very high catalytic activity and stability even at 70 \u0026ordm;C. These improvements provide significant advantages for both scientific and industrial applications.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003eMethanol (Isolab, catalog# 947046), styrene (Sigma Aldrich, catalog# 8076791000), acrylamide (Sigma Aldrich, catalog# 8008301000), methyl methacrylate (Sigma Aldrich, catalog# 8005901000), \u003cem\u003eN\u003c/em\u003e-isopropylacrylamide (Sigma Aldrich, catalog# 415324-50G), 2,4-pentanedione (Sigma Aldrich, catalog# 8000231000), tween 40 (Sigma Aldrich, catalog# 8221851000), cetyltrimethylammonium bromide (Sigma Aldrich, catalog# 8141190500), sodiumdodecyl sulfate (Sigma Aldrich, catalog# 8220501000), pH 7.4 phosphate buffered saline (PBS, MP biomedicals, catalog# 2810305), hydrogen peroxide (Merck, catalog# 1.08597), coppersulfate pentahydrate (CuSO\u003csub\u003e4\u003c/sub\u003e.5H\u003csub\u003e2\u003c/sub\u003eO, Sigma-Aldrich, catalog#18304), iron (II) sulfate (FeSO\u003csub\u003e4\u003c/sub\u003e, Sigma-Aldrich, catalog#F8263), horseradish peroxidase (HRP, Sigma-Aldrich, 77332, lyophilized, powder, beige, ~\u0026thinsp;150 U/mg) were purchased and used for the synthesis of the nanoflowers and polymers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eInstrumentation\u003c/h2\u003e \u003cp\u003eScanning electron microscope (SEM) images were monitored using Zeiss Gemini 500 instrument. Agilent Premium Compact (600 MHz) NMR spectrometer was used for \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC nuclear magnetic resonance (NMR) analyses of the polymers. Thermogravimetric analyses (TGA) of the obtained polymers were accomplished using Mettler-Toledo TGA/DSC 1 Star system thermal analyzer under nitrogen atmosphere (N\u003csub\u003e2\u003c/sub\u003e). Approximately, 5\u0026ndash;10 mg samples were taken for the analysis, and thermal decomposition temperatures were determined at a temperature range from room temperature (RT) to 600 \u0026ordm;C with 10 \u0026ordm;C/min. Differential scanning calorimetry (DSC) thermograms of the polymers were recorded using a Mettler-Toledo DSC 1 Star system instrument at a heating rate of 10 \u0026ordm;C/min from \u0026minus;\u0026thinsp;50 to 450 \u0026ordm;C under N\u003csub\u003e2\u003c/sub\u003e atmosphere. For gel permeation chromatography (GPC) analyses, Shimadzu LC-20AD instrument was used. GPC analyses were performed using Agilent PLgel mixed-B column with HPLC purity \u003cem\u003eN,N'\u003c/em\u003e-dimethylformamide (DMF) mobile phase at a flow rate of 1 mL/min at 40 \u0026ordm;C. Polystyrene (PS) standards were used for calibration. GPC chromatograms of the synthesized polymer were given in Figure S13-S29 (supplementary information).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of HRP-Cu\u003csup\u003e2+\u003c/sup\u003e nanoflowers\u003c/h2\u003e \u003cp\u003eCuSO\u003csub\u003e4\u003c/sub\u003e solution was added to the HRP enzyme solution (0.02 mg/mL) in pH 7.4 PBS buffer. The solution was vortexed at RT for 3 min and incubated at +\u0026thinsp;4 \u0026ordm;C for 3 days. After incubation, light blue precipitate was separated by centrifugation and washed several times with water to remove impurities. The obtained HRP-Cu\u003csup\u003e2+\u003c/sup\u003e nanoflowers was dried in an oven at 40 \u0026ordm;C and used for the reactions [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRepresentative polymerization procedure of styrene\u003c/h2\u003e \u003cp\u003e10 mL of pure water and styrene (6.0 mmol, 0.69 mL) was put in a test tube with screw cap. Dissolved oxygen in the solution was degassed by bubbling N\u003csub\u003e2\u003c/sub\u003e gas through the mixture. HRP-Cu\u003csup\u003e2+\u003c/sup\u003e, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, surfactant and 2,4-pentanedione were added to the mixture, and N\u003csub\u003e2\u003c/sub\u003e gas was bubbled through the mixture again. The mixture was stirred at the desired temperature for 24 hours. Then, the mixture was poured into methanol, and obtained precipitate was filtered, washed with methanol and dried in an oven [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePolymerization of other vinyl monomers\u003c/h2\u003e \u003cp\u003e10 mL of distilled water and 6.0 mmol of monomer (acrylamide, methylmethacrylate and \u003cem\u003eN\u003c/em\u003e-isopropylacrylamide) were put in a test tube, and dissolved oxygen was removed by bubbling N\u003csub\u003e2\u003c/sub\u003e gas through the solution. 10 mg HRP-Cu\u003csup\u003e2+\u003c/sup\u003e, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 0.1 g of tween 40 and 2,4-pentanedione were added to the solution, and N\u003csub\u003e2\u003c/sub\u003e gas was passed through the mixture again. Tween 40 was not added to the polymerization of acrylamide. The mixture was stirred at the desired temperature for 24 hours. Then, the mixture was poured into methanol, and the obtained precipitate was filtered, washed with methanol and dried in an oven [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS and DISCUSSION","content":"\u003cp\u003eThe synthesis of HRP-Cu\u003csup\u003e2+\u003c/sup\u003e hybrid flower-shaped nano biocatalyst was achieved with the complexation of Cu\u003csup\u003e2+\u003c/sup\u003e ions and HRP enzyme. Flower-shaped nanostructures can be obtained with the appropriate nucleation that occurs as a result of the coordination of Cu\u003csup\u003e2+\u003c/sup\u003e ions with the amine groups in the structure of the HRP. The formation steps of HRP-Cu\u003csup\u003e2+\u003c/sup\u003e hybrid flower-shaped nano biocatalyst have been shown in detail in the literature [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The morphology of the synthesized HRP-Cu\u003csup\u003e2+\u003c/sup\u003e nano biocatalyst can be seen in the SEM (scanning electron microscopy) analysis shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. According to the SEM analysis result, the obtained HRP-Cu\u003csup\u003e2+\u003c/sup\u003e nano biocatalyst have flower-shaped and porous structure with a size of approximately 10 \u0026micro;m. It was reported that the mass percentage of copper ion (Cu\u003csup\u003e2+\u003c/sup\u003e) in the HRP-Cu\u003csup\u003e2+\u003c/sup\u003e nanoflowers was approximately 10% and HRP-Cu\u003csup\u003e2+\u003c/sup\u003e was reported to show approximately 300% higher catalytic activity compared to free-HRP enzyme on the oxidation of guaiacol [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. HRP-Cu\u003csup\u003e2+\u003c/sup\u003e also exhibited only 4% of stability loss in solution within 30 days. However, free-HRP enzyme showed approximately 65% of stability loss in the same period. Thus, HRP-Cu\u003csup\u003e2+\u003c/sup\u003e nanoflowers showed much higher stability compared to the free-HRP enzyme [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to detect catalytic activity of HRP-Cu\u003csup\u003e2+\u003c/sup\u003e nanoflowers on the polymerization of vinyl monomers, polymerization of styrene was first investigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). After optimum polymerization condition of styrene using HRP-Cu\u003csup\u003e2+\u003c/sup\u003e was determined, polymerizations of other vinyl monomers (acrylamide, methylmethacrylate and \u003cem\u003eN\u003c/em\u003e-isopropylacrylamide) were carried out.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results of the HRP-Cu\u003csup\u003e2+\u003c/sup\u003e mediated polymerization of styrene are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Polymerizations were carried out under emulsion condition with cationic, anionic and non-ionic surfactants in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 2,4-pentanedione mediators. Polymerization of vinyl monomers did not occur in the absence of HRP-Cu\u003csup\u003e2+\u003c/sup\u003e or peroxide. The influence of HRP-Cu\u003csup\u003e2+\u003c/sup\u003e amount on the polymerization of styrene was first optimized in water with tween40 surfactant at RT (entries 1, 2 and 3 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It was observed that utilization of 10 mg of HRP-Cu\u003csup\u003e2+\u003c/sup\u003e provided very good yield (84%) and the highest molecular weight (\u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e= 319 kDa) of polystyrene (entry 2 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Doubling the amount of HRP-Cu\u003csup\u003e2+\u003c/sup\u003e slightly improved the polymerization, and resulted in 87% of yield (entry 3 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, the number average molecular weight (\u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e) of the obtained polystyrene was 210 kDa which was lower compared to the condition of using 10 mg of HRP-Cu\u003csup\u003e2+\u003c/sup\u003e. Observation of low \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e is probably due to the generation of more radical species during the polymerization with utilization of 20 mg of HRP-Cu\u003csup\u003e2+\u003c/sup\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Thereby, higher HRP-Cu\u003csup\u003e2+\u003c/sup\u003e concentration gave the lower molecular weight of polymer. In addition to that utilization of 10 mg HRP-Cu\u003csup\u003e2+\u003c/sup\u003e was more economic than that of 20 mg of it. Decreasing the amount of the catalyst from 10 mg to 5 mg significantly reduced the polymerization yield. Addition of 5.0 mg of catalyst in the polymerization resulted in 57% of polymer yield (entry 1 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePolymerization of styrene by HRP-Cu\u003csup\u003e2+\u003c/sup\u003e nano biocatalyst in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 2,4-pentanedione\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEntry\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHRP-Cu\u003csup\u003e2+\u003c/sup\u003e (mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (mmol)\u003c/p\u003e \u003c/th\u003e \u003cth 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\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e309\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1 g Tween40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1 g Tween40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1 g SDS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1 g CTAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003eall polymerizations were carried out with 690 \u0026micro;L of styrene (6.0 mmol) in 10 mL water in the presence of 0.05 mmol of 2,4-pentanedione, tween 40 surfactant and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in 24h, \u003csup\u003eb\u003c/sup\u003eThe amount of 2,4-pentanedione was 0.1 mmol, other parameters were kept the same as condition a, SDS\u0026thinsp;=\u0026thinsp;sodiumdodecyl sulfate, CTAB\u0026thinsp;=\u0026thinsp;cetyltrimethylammonium bromide, \u003cem\u003eT\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e= reaction temperature, \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e: the number average molecular weight, \u003cem\u003e\u0026ETH;\u003c/em\u003e: polydispersity index.\u003c/p\u003e \u003cp\u003eReaction temperature is one of the important parameters for the polymerization of styrene due to its effects on the activity of enzymes. Therefore, polymerization of styrene at different reaction temperatures was performed to detect better catalytic activity of HRP-Cu\u003csup\u003e2+\u003c/sup\u003e nanoflowers. Polymerization of polystyrene carried out at room temperature (entry 2 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) provided higher yield and molecular weight compared to the reactions accomplished at 40 and 50 \u0026ordm;C (entries 4 and 5 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, room temperature (RT) was decided to be optimum polymerization temperature for styrene.\u003c/p\u003e \u003cp\u003eIn order to facilitate the polymerization of styrene, different types and amounts of surfactants were also investigated to generate emulsion system. First, the influence of anionic (SDS), cationic (CTAB) and non-ionic (tween 40) surfactants on the HRP-Cu\u003csup\u003e2+\u003c/sup\u003e mediated polymerization of styrene were investigated. While addition of anionic SDS and cationic CTAB surfactants negatively affected the polymerization yields (entries 11 and 12 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), utilization of non-ionic tween 40 surfactant significantly enhanced the yield and the molecular weight of polystyrene. By adding 0.1 g of tween 40, the polymerization yield was 84% (entry 2 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e and polydispersity index (\u003cem\u003e\u0026ETH;\u003c/em\u003e) of the polystyrene were respectively 319 kDa and 2.03. Polymerization yield was dramatically decreased by lowering the amount of tween 40 from 0.1 g to 0.05 g (entry 6 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and 65% of yield was observed. However, negligible increase in the polymerization yield (86%) occurred by adding 0.2 g tween 40 in the solution (entry 7 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, the number average molecular weight of the obtained polystyrene (79 kDa) significantly decreased compared to the polymerization achieved by adding 0.1 g of tween 40 (entry 2 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Polymerization of styrene in the presence of cationic CTAB surfactant gave 37% of yield with 16 kDa molecular weight (entry 12 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, no polymer product was observed with utilization of anionic SDS in the polymerization (entry 11 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Probably, cationic and anionic surfactants deactivate or limit the activity of HRP-Cu\u003csup\u003e2+\u003c/sup\u003e nanoflowers. These results suggest that non-ionic tween 40 surfactant provides better polymerization system for HRP-Cu\u003csup\u003e2+\u003c/sup\u003e mediated polymerization of styrene.\u003c/p\u003e \u003cp\u003eIn order to discover the role of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration on the HRP-Cu\u003csup\u003e2+\u003c/sup\u003e mediated vinyl polymerizations, three different experiments with increasing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration were conducted. Increasing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration from 0.5 to 1.0 mmol resulted in increasing yield and \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e. While polymerization performed using 1.0 mmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e gave 84% of yield (entry 2 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), addition of 0.5 mmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e resulted in 57% of yield (entry 9 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e of the polymer (76 kDa) obtained from adding 0.5 mmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was also lower compared to the 1.0 mmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration (319 kDa). Enhancing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration from 1.0 to 2.0 mmol showed the maximum polymerization yield with 88% (entry 10 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e of the obtained polymer was 56 kDa which was significantly lower than that of the polymer obtained from entry 2. Higher peroxide concentration probably generated increasing number of active chains and/or termination reactions. Another reason could be inhibiting enzymatic activity with increasing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration. Therefore, \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e of the polymer decreased. Further increase in the concentration of peroxide was not considered since excess peroxide inhibits the activity of HRP enzyme [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e2,4-pentanedione mediator has also a great impact on the polymerization of vinyl monomers. For the initiation of the radical polymerization, β-diketone radicals can be generated from the oxidation of 2,4-pentanedione with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the presence of HRP-Cu\u003csup\u003e2+\u003c/sup\u003e nanoflowers [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Two different 2,4-pentanedione (acetylacetone) concentration was investigated during the polymerizations. Increasing acetylacetone concentration from 0.05 mmol to 0.1 mmol was found to be no significant effect on the polymerization yield and the molecular weight of the polymer (entries 2 and 8 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The yield of the polymerization using 0.1 mmol of pentanedione (entry 8 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) was the same as the reaction carried out addition of 0.05 mmol of pentanedione. The number average molecular weights of the polymers were also found to be very close. Therefore, utilization of 0.05 mmol of pentanedione cocatalyst was determined to be sufficient for the polymerization of styrene.\u003c/p\u003e \u003cp\u003ePreviously, utilization of 20 mg of free-HRP enzyme for the polymerization of 12 mmol of styrene was reported to be resulted in up to 64.3% of yield with 3.97x10\u003csup\u003e5\u003c/sup\u003e Da at RT under mini-emulsion conditions with 12 mmol acetylacetone and 0.05 mmol of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Utilization of cosolvents including THF (tetrahydrofuran), dioxane, DMF (dimethylformamide) and methanol in the polymerization of HRP enzyme initiated polymerization of styrene was also investigated without adding surfactant, and the yields of the polystyrene were very low with up to 21.2% under H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/acetylacetone catalyst system [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. According to our findings, HRP-Cu\u003csup\u003e2+\u003c/sup\u003e nanoflowers demonstrated higher catalytic activity towards polymerization of styrene compared to the free-HRP enzyme.\u003c/p\u003e \u003cp\u003eStructural characterization of the polystyrene was accomplished by FT-IR, \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR analyses. According to the FT-IR spectrum of polystyrene (entry 2 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), absorption bands observed around 2900 cm\u003csup\u003e-1\u003c/sup\u003e and 3000 cm\u003csup\u003e-1\u003c/sup\u003e indicate the presence of aliphatic and aromatic C-H stretching vibrations. C\u0026thinsp;=\u0026thinsp;C stretching vibrations belonging to the phenyl ring appeared around 1750 cm\u003csup\u003e-1\u003c/sup\u003e (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, supplementary information).\u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR spectra results of the synthesized polymer suggest that atactic polystyrene structure was obtained [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The phenyl ring protons are observed around 7.02 ppm and 6.50 ppm in the \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of the polystyrene. The methine (CH) and methylene (CH\u003csub\u003e2\u003c/sub\u003e) protons are respectively observed at 1.83 and 1.50 ppm (Figure S30, supplementary information). In the \u003csup\u003e13\u003c/sup\u003eC NMR spectrum, peaks observed at 145, 127 and 125 ppm indicates the phenyl ring carbons. \u003csup\u003e13\u003c/sup\u003eC peaks observed at 43 and 40 ppm attribute methylene (CH\u003csub\u003e2\u003c/sub\u003e) and methine (CH) carbons, respectively (Figure S34, supplementary information).\u003c/p\u003e \u003cp\u003eAccording to TGA and DSC analyses results, the polymer (entry 2 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) started to decompose around 350 \u0026ordm;C, and 50% of thermal decomposition occurred around 420 \u0026ordm;C (Figure S5, supplementary information). The glass transition temperature (\u003cem\u003eT\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e) of the polymer was detected approximately at 110 \u0026ordm;C, and the thermal decomposition temperature was observed around 430 \u0026ordm;C (Figure S9, supplementary information).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePolymerization of other vinyl monomers by HRP-Cu\u003csup\u003e2+\u003c/sup\u003e in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 2,4-pentanedione\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEntry\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonomer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eT\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e (\u0026ordm;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYield (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e (kDa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003e\u0026ETH;\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcrylamide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcrylamide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcrylamide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e171\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethylmethacrylate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethylmethacrylate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethylmethacrylate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e190\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eN\u003c/em\u003e-isopropylacrylamide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003eall polymerizations were carried out 6.0 mmol of monomer in 10 mL of water in the presence of 10 mg HRP-Cu\u003csup\u003e2+\u003c/sup\u003e, 0.05 mmol of 2,4-pentanedione and 1.0 mmol of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in 24h, \u003csup\u003eb\u003c/sup\u003e0.1 g of tween 40 surfactant was added for the polymerization mixtures. \u003cem\u003eT\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e= polymerization temperature, \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e: the number average molecular weight, \u003cem\u003e\u0026ETH;\u003c/em\u003e: heterogeneity index.\u003c/p\u003e \u003cp\u003ePolymerizations of other vinyl monomers (acrylamide, methylmethacrylate and \u003cem\u003eN\u003c/em\u003e-isopropylacrylamide) were also accomplished by HRP-Cu\u003csup\u003e2+\u003c/sup\u003e nanoflowers. All polymerizations were conducted using the optimized reaction condition obtained from the polymerization of styrene (entry 2 in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Unlike polymerization of other vinyl monomers, acrylamide was polymerized surfactant-free media due to solubility of polyacrylamide in water (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Only, reaction temperature parameter was altered to achieve these polymerizations. Polymerizations of these vinyl monomers at RT either did not take place or occurred with very low yields. Therefore, these polymerizations were carried out at higher reaction temperatures compared to styrene (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). First, HRP-Cu\u003csup\u003e2+\u003c/sup\u003e mediated polymerization of acrylamide in water was conducted without adding tween 40 surfactant, and 70 \u0026ordm;C was found to be suitable temperature for the polymerization. Polyacrylamide was obtained with 96% of yield at 70 \u0026ordm;C (entry 15 in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Polymerization of acrylamide at 50 \u0026ordm;C resulted in lower yield and molecular weight (entry 14 in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Higher reaction temperatures above 70 \u0026ordm;C were not tried since HRP-Cu\u003csup\u003e2+\u003c/sup\u003e nanoflowers lost its catalytic activity. The \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e and polydispersity index of the polymer obtained at 70 \u0026ordm;C were respectively 171 kDa and 2.66.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFT-IR, \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR analyses were performed for the structural characterization of the obtained polyacrylamide (entry 15 in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). According to the FT-IR analysis, a broad -OH stretching band around 3300 cm\u003csup\u003e-1\u003c/sup\u003e was observed due to the moisture contents of the polymer (Figure S2, supplementary information). Carbonyl peak related to the amide (-CONH\u003csub\u003e2\u003c/sub\u003e) functional group was observed around 1750 cm\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFrom the \u003csup\u003e1\u003c/sup\u003eH NMR analysis, the methylene (-CH\u003csub\u003e2\u003c/sub\u003e) and methine (-CH) protons were respectively observed at 1.50 and 2.20 ppm (Figure S31, supplementary information). According to the \u003csup\u003e13\u003c/sup\u003eC NMR analysis, carbonyl (-C\u0026thinsp;=\u0026thinsp;O) carbon was seen at 179 ppm, methine (-CH) and methylene (-CH\u003csub\u003e2\u003c/sub\u003e) carbons were also observed at 41 ppm and 35 ppm, respectively (Figure S35, supplementary information). According to the \u003csup\u003e13\u003c/sup\u003eC NMR spectrum results, polyacrylamide was decided to be atactic structure due to the observation of triplet peak (triad arrangements) for the methine carbon [15a].\u003c/p\u003e \u003cp\u003eTGA analysis result of the polyacrylamide (entry 15 in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) was carried out between RT and 600 \u0026ordm;C under N\u003csub\u003e2\u003c/sub\u003e atmosphere, and showed three stages of thermal degradation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. About 15\u0026ndash;20% of weight loss was observed up to 200 \u0026ordm;C due to removal of absorbed water. Between 200\u0026ndash;340 \u0026ordm;C, about 20% mass loss occurred due to the removal of by-products including NH\u003csub\u003e3\u003c/sub\u003e, H\u003csub\u003e2\u003c/sub\u003eO and CO\u003csub\u003e2\u003c/sub\u003e. Finally, about 40% weight loss happened between 340\u0026ndash;460 \u0026ordm;C due to removal of H\u003csub\u003e2\u003c/sub\u003eO and CO\u003csub\u003e2\u003c/sub\u003e from the remaining product (Figure S6, supplementary information). According to the DSC analysis of the polyacrylamide (entry 15 in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), glass transition temperature (\u003cem\u003eT\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e) and melting temperature (\u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e) of the polymer were observed at 165 \u0026ordm;C and 300 \u0026ordm;C, respectively (Figure S10, supplementary information).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMethylmethacrylate (MMA) was also polymerized using HRP-Cu\u003csup\u003e2+\u003c/sup\u003e nanoflowers in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 2,4-pentanedione and tween 40 surfactant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Optimum polymerization was accomplished with 84% of yield at 60 \u0026ordm;C (entry 18 in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e of the polymer was 190 kDa with 1.92 polydispersity index. Reaction temperature above 60 \u0026ordm;C was not tried because thermal initiation of MMA at temperatures above 60 \u0026ordm;C occurred without adding H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Polymerization of MMA carried out at RT and 50 \u0026ordm;C (entries 16 and 17 in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) resulted in respectively 23% and 73% of yields which are lower than that of obtained from 60 \u0026ordm;C. Structural characterization of polyMMA (entry 18 in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) was also shown by FT-IR, \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR analyses. Aliphatic C-H stretching vibrations around 2900 cm\u003csup\u003e-1\u003c/sup\u003e, ester group carbonyl (-C\u0026thinsp;=\u0026thinsp;O) vibration band at 1700 cm\u003csup\u003e-1\u003c/sup\u003e and C-O vibration band around 1100 cm\u003csup\u003e-1\u003c/sup\u003e were observed from the FT-IR spectrum of the polymer (Figure S3, supplementary information). From the \u003csup\u003e1\u003c/sup\u003eH NMR analysis of the product, methyl protons (-OCH\u003csub\u003e3\u003c/sub\u003e) belonging to the ester group was observed at 3.57 ppm. Methylene (-CH\u003csub\u003e2\u003c/sub\u003e) and methyl (-CH\u003csub\u003e3\u003c/sub\u003e) protons were respectively observed at 1.78 ppm and 0.82 ppm (Figure S32, supplementary information). According to the \u003csup\u003e13\u003c/sup\u003eC NMR spectrum, carbonyl (-C\u0026thinsp;=\u0026thinsp;O) carbon at 177 ppm, quaternary carbon (-C) attached to carbonyl at 54 ppm, methyl group carbon (-OCH\u003csub\u003e3\u003c/sub\u003e) attached to ester group at 52 ppm, methylene (-CH\u003csub\u003e2\u003c/sub\u003e) carbon at 44 ppm and methyl group (-CH\u003csub\u003e3\u003c/sub\u003e) carbon at 16 ppm were observed (Figure S36, supplementary information).\u003c/p\u003e \u003cp\u003eObtained polyMMA demonstrated two stages of thermal degradation between RT and 600 \u0026ordm;C under N\u003csub\u003e2\u003c/sub\u003e atmosphere [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Approximately 5% of mass loss was observed between 200\u0026ndash;250 \u0026ordm;C due to the degradation of the polymer side chains. The second mass loss about 80% occurred between 250\u0026ndash;440 \u0026ordm;C due to the removal of by-products such as H\u003csub\u003e2\u003c/sub\u003eO and CO\u003csub\u003e2\u003c/sub\u003e from the polymer sample (Figure S7, supplementary information). The glass transition temperature (\u003cem\u003eT\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e) of the polymer was detected at 125 \u0026ordm;C, and the degradation temperature (\u003cem\u003eT\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e) of the polyMMA was observed around 370 \u0026ordm;C (Figure S11, supplementary information).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, polymerization of NIPAM was accomplished at 70 \u0026ordm;C similar to the acrylamide (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Polymerization yield was 85%, and \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e of the polyNIPAM (PNIPAM) was 185 kDa with 2.82 PDI (entry 19 in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A broad -OH stretching band around 3300 cm\u003csup\u003e-1\u003c/sup\u003e was observed in the FT-IR spectrum of the polymer due to the moisture content of the product (Figure S4, supplementary information). Aliphatic C-H stretching vibrations was observed at 2900 cm\u003csup\u003e-1\u003c/sup\u003e, and amide carbonyl peak (-CONH\u003csub\u003e2\u003c/sub\u003e) was detected around 1700 cm\u003csup\u003e-1\u003c/sup\u003e in the spectrum. According to the \u003csup\u003e1\u003c/sup\u003eH NMR analysis of the product, methine (-CH) and methylene (-CH\u003csub\u003e2\u003c/sub\u003e) protons were observed at 2.03 and 1.24 ppm, respectively. Proton peaks at 3.98 and 1.12 ppm were attributed to the methine (-CH) and two methyl group (-CH\u003csub\u003e3\u003c/sub\u003e) protons of the isopropyl group, respectively (Figure S33, supplementary information). From the \u003csup\u003e13\u003c/sup\u003eC NMR spectrum of the polymer; carbonyl (-C\u0026thinsp;=\u0026thinsp;O) carbon, methine carbon (-CH) attached to carbonyl group and methylene carbon (-CH\u003csub\u003e2\u003c/sub\u003e) were observed respectively at 174, 37 and 29 ppm. \u003csup\u003e13\u003c/sup\u003eC peaks at 41 and 22 ppm are related to the methine carbon (-CH) and two methyl carbons belonging to isopropyl group (Figure S37, supplementary information).\u003c/p\u003e \u003cp\u003eAccording to the TGA thermogram of the PNIPAM, the polymer had three stages of thermal degradation similar to the polyacrylamide [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. About 10% of mass loss up to 200 \u0026ordm;C was observed due to the removal of moisture. 15% of mass loss occurred between 200\u0026ndash;330 \u0026ordm;C due to the possible removal of by-products such as NH\u003csub\u003e3\u003c/sub\u003e, H\u003csub\u003e2\u003c/sub\u003eO and CO\u003csub\u003e2\u003c/sub\u003e. About 40% of weight loss happened between 330\u0026ndash;470 \u0026ordm;C removal of H\u003csub\u003e2\u003c/sub\u003eO and CO\u003csub\u003e2\u003c/sub\u003e from the remaining product (Figure S8, supplementary information). The PNIPAM only showed decomposition temperature at 400 \u0026ordm;C from DSC thermogram result, and no glass transition temperature was detected (Figure S12, supplementary information).\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eHRP-Cu\u003csup\u003e2+\u003c/sup\u003e mediated polymerization of four different vinyl monomers including styrene, acrylamide, methylmethacrylate and \u003cem\u003eN\u003c/em\u003e-isopropylacrylamide was successfully investigated in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 2,4-pentanedione mediators under emulsion media. The obtained results showed that HRP-Cu\u003csup\u003e2+\u003c/sup\u003e mediated catalyst systems can be efficiently used to polymerize a variety of vinyl monomers in emulsion media with very high yields and molecular weights. Acrylamide monomer was polymerized surfactant-free media with very high yield and molecular weight due to solubility of polyacrylamide in water Non-ionic tween 40 surfactant provides better polymerization system for the HRP-Cu\u003csup\u003e2+\u003c/sup\u003e mediated polymerization of monomers compared to the anionic sodiumdodecyl sulfate and cationic cetyltrimethylammonium bromide surfactants. According to the structural characterization results, the synthesized polymers are mainly obtained as atactic structures. While free-HRP enzyme loses its catalytic activity at 60 \u0026ordm;C, HRP-Cu\u003csup\u003e2+\u003c/sup\u003e hybrid nanoflowers showed very high catalytic activity and stability even at 70 \u0026ordm;C. Increasing catalytic activity and stability of hybrid nanoflowers provide significant advantages for both scientific and industrial applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgement\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by Hatay Mustafa Kemal University Coordinatorship of Scientific Research Projects (project # 21.GAP.058).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGO contributed to methodology, formal analysis and investigation. MM contributed to methodology, formal analysis and investigation. SD contributed to methodology, formal analysis, investigation,\u0026nbsp;review and editing of manuscript. IO contributed to conceptualization, writing\u0026mdash;review and editing. EG contributed to conceptualization, project administration, funding acquisition, writing\u0026mdash;review and editing, and supervision.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe following analysis results are included as supporting information: FT-IR (Figures S1-S4), \u003csup\u003e1\u003c/sup\u003eH NMR (Figures S30-S33) and \u003csup\u003e13\u003c/sup\u003eC NMR (Figures S34-S37) spectra results; TGA (Figures S5-S8) and DSC (Figures S9-S12) thermograms; and GPC chromatograms (Figures S13-S29) \u0026nbsp;of the synthesized polymers.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMiletić N, Nastasović A, Loos K (2012) Immobilization of biocatalysts for enzymatic polymerizations: Possibilities, advantages, applications. 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Green Chemistry 4:174-178; b) Teixeira D, Lalot T, Brigodiot M, Mar\u0026eacute;chal E (1999) \u0026beta;-Diketones as Key Compounds in Free-Radical Polymerization by Enzyme-Mediated Initiation. Macromolecules 32:70-72.\u003c/li\u003e\n\u003cli\u003eOcsoy I, Dogru E, Usta S (2015) A new generation of flowerlike horseradish peroxides as a nanobiocatalyst for superior enzymatic activity. Enzyme Microb Tech 75-76:25-29.\u003c/li\u003e\n\u003cli\u003eTsujimoto T, Uyama H, Kobayashi S (2001) Polymerization of Vinyl Monomers Using Oxidase Catalysts. Macromolecular Bioscience\u003cem\u003e \u003c/em\u003e1:228-232.\u003c/li\u003e\n\u003cli\u003eHollmann F, Arends IWCE (2012) Enzyme Initiated Radical Polymerizations. Polymers\u003cem\u003e \u003c/em\u003e4:759-793.\u003c/li\u003e\n\u003cli\u003eKohri M (2014) Development of HRP-mediated enzymatic polymerization under heterogeneous conditions for the preparation of functional particles. Polymer Journal 46:373-380.\u003c/li\u003e\n\u003cli\u003eShan J, Kitamura Y, Yoshizawa H (2005) Emulsion polymerization of styrene by horseradish peroxidase-mediated initiation. Colloid and Polymer Science\u003cem\u003e \u003c/em\u003e284:108-111.\u003c/li\u003e\n\u003cli\u003eLeung WM, Axelson DE, Van Dyke JD (1987) Thermal degradation of polyacrylamide and poly(acrylamide-co-acrylate). \u003cem\u003eJournal of Polymer Science Part A: \u003c/em\u003ePolymer Chemistry 25:1825-1846.\u003c/li\u003e\n\u003cli\u003eNising P, Meyer T, Carloff R, Wicker M (2005) Thermal Initiation of MMA in High Temperature Radical Polymerizations. Macromolecular Materials and Engineering 290:311-318.\u003c/li\u003e\n\u003cli\u003eNikolaidis AK, Achilias DS (2018) Thermal Degradation Kinetics and Viscoelastic Behavior of Poly(Methyl Methacrylate)/Organomodified Montmorillonite Nanocomposites Prepared via In Situ Bulk Radical Polymerization Polymers 10(5):491.\u003c/li\u003e\n\u003cli\u003eBauri K, Roy SG, Arora S, Dey RK, Goswami A, Madras G, De P (2013) Thermal degradation kinetics of thermoresponsive poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) copolymers prepared via RAFT polymerization. Journal of Thermal Analysis and Calorimetry\u003cem\u003e \u003c/em\u003e111:753-761.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"polymer-bulletin","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pobu","sideBox":"Learn more about [Polymer Bulletin](http://link.springer.com/journal/289)","snPcode":"289","submissionUrl":"https://submission.nature.com/new-submission/289/3","title":"Polymer Bulletin","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Horseradish peroxidase, polymerization, styrene, methylmethacrylate, acrylamide, N-isopropylacrylamide","lastPublishedDoi":"10.21203/rs.3.rs-4914498/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4914498/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, we report the production of flower-shaped HRP-Cu\u003csup\u003e2+\u003c/sup\u003e hybrid nano biocatalyst from the complexation between horseradish peroxidase (HRP) enzyme and Cu\u003csup\u003e2+\u003c/sup\u003e ions, and investigate catalytic activity and stability of the obtained nanoflowers on the polymerization of some vinyl monomers (styrene, methylmethacrylate, acrylamide and \u003cem\u003eN\u003c/em\u003e-isopropylacrylamide). Polymerizations of these monomers, except water soluble acrylamide, were accomplished under emulsion conditions using cationic, anionic and non-ionic surfactants in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 2,4-pentanedione mediator. Optimum polymerizations were achieved under the conditions of non-ionic surfactant (tween 40) used. HRP-Cu\u003csup\u003e2+\u003c/sup\u003e mediated polymerizations resulted in very high yields and molecular weights (\u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e) of the polymers. Optimum polymerization of styrene with 84% of yield (\u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e= 319 kDa) was accomplished at room temperature. However, the highest polymerization yields for acrylamide (96%, \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e= 171 kDa) and \u003cem\u003eN\u003c/em\u003e-isopropylacrylamide (85%, \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e= 185 kDa) was achieved at 70 \u0026ordm;C. Similarly, optimum polymerization of methylmethacrylate was accomplished with 84% of yield (\u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e= 190 kDa) at 60 \u0026ordm;C. While free-HRP loses its catalytic activity at 60 \u0026ordm;C and above temperatures, HRP-Cu\u003csup\u003e2+\u003c/sup\u003e showed very high catalytic activity and stability even at 70 \u0026ordm;C. Increasing activity and stability of hybrid nanoflowers provide significant advantages for both scientific and industrial applications.\u003c/p\u003e","manuscriptTitle":"Investigation of the Free-Radical Polymerization of Vinyl Monomers Using Horseradish Peroxidase (HRP) Nanoflowers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-16 07:46:08","doi":"10.21203/rs.3.rs-4914498/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-02T05:57:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-26T17:34:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"140168088804915655567628398828661127164","date":"2024-12-16T18:27:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"174278750696758025572793458019446182643","date":"2024-09-18T13:37:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-17T02:53:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-19T07:56:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-16T14:18:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Polymer Bulletin","date":"2024-08-14T14:49:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"polymer-bulletin","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pobu","sideBox":"Learn more about [Polymer Bulletin](http://link.springer.com/journal/289)","snPcode":"289","submissionUrl":"https://submission.nature.com/new-submission/289/3","title":"Polymer Bulletin","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"249f949e-8afb-4221-a661-26191cfb7be4","owner":[],"postedDate":"September 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-27T16:05:25+00:00","versionOfRecord":{"articleIdentity":"rs-4914498","link":"https://doi.org/10.1007/s00289-025-05664-z","journal":{"identity":"polymer-bulletin","isVorOnly":false,"title":"Polymer Bulletin"},"publishedOn":"2025-01-24 15:58:25","publishedOnDateReadable":"January 24th, 2025"},"versionCreatedAt":"2024-09-16 07:46:08","video":"","vorDoi":"10.1007/s00289-025-05664-z","vorDoiUrl":"https://doi.org/10.1007/s00289-025-05664-z","workflowStages":[]},"version":"v1","identity":"rs-4914498","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4914498","identity":"rs-4914498","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}