THE PRODUCTION OF PLASTICS Technical Field

The present invention relates to nano-biocatalysts that are synthesized from Cu ²⁺ ion and plant extracts such as green tea extract and viburnum opulus extract that are capable of being used in polymerization reactions by exhibiting peroxidase-like activity.

The nano-biocatalyst subject to the invention can be generally used in some oxidation reactions in the chemical industry, and in particular, can be used in oxidative polymerization reactions in the chemistry of industrial polymers. Additionally, it can be employed in biosensor and biomedical applications as well.

Prior Art Enzymes are biocatalysts and used in various fields of science and technology. Since enzymes exhibit the characteristics of high catalytic activity, high selectivity, low toxicity and water solubility, they are applicaple to many fields of chemistry, biochemistry, medicine etc [1-7] Many free enzymes can catalyse reactions under milder reaction conditions (neutral pH, room temperature and atmospheric pressure), and act against certain substrates and functional groups. Enzymes are not only used in biological reactions, they can also be used in various industrial areas. As they exhibit substrate specificity and low toxicity, and do not allow the formation of by-products, enzymes seem to be quite advantegous compared to other catalysts [4] _·

Peroxidase Peroxidase catalysts perform oxidation of a proton donor compound by using hydrogen peroxide and they reveal two moles of water after completion of the reaction. Horseradish peroxidase (HRP) and Soybean peroxidase (SBP) are the most commonly used peroxidase enzymes are and they contain iron (Fe) ion in their active sites [8], and initiate radicalic reaction/polymerization. Horseradish Peroxidase (HRP)

Horseradish is a plant that can be cultivated in milder climate regions and it is resistant to environmental conditions. Horseradish peroxidase (HRP) is an important enzyme and can be isolated from the root of this plant. HRP has been studied in a variety of technical fields and research groups. HRP enzyme contains a heme group. The heme group is a planar structure having an iron atom located in the centre of a porphyrin ring consisting of four pyrrole rings.

HRP-Mediated Polymerization of Phenol and Aniline Derivatives

Aniline and phenol derivatives can be polymerized with HRP enzyme catalyst and thus different types of environmentally-friendly polyaromatic structures can be synthesized with high yields. Additionally, various organic and inorganic electron-donating compounds; phenols, amines, indoles, phenolic acids and sulphonates can be oxidized with the peroxidase enzymes. The polymerization of phenols with an enzyme catalyst was first introduced in the literature by Dordick et al. [10] In this study, the enzymatic polymerization of phenols in an aqueous organic solvent using HRP enzyme was reported. The polymerization of phenol compounds with the peroxidase catalyst provides an alternative method for the synthesis of polyphenol resins without using toxic formaldehyde comonomers [11-13] Phenol derivatives containing different types of functional groups can also be polymerized chemo-selectively with an enzyme catalyst [14] For example, when a phenolic compound containing a methacryloyl group is subjected to the enzymatic polymerization, only the polymerization of phenol occurs while the methacryloyl group does not react [15] Using enzymatic polymerization, conductive polymers can also be synthesized with high yields [16,17] Additionally, this method is widely used in the processes of removing phenolic pollutants from waste water [18-20]

Enzyme Catalyst-Mediated Free Radical Polymerization of Vinyl Monomers In addition to phenol and aniline derivatives, some monomers containing vinyl groups can be polymerized by free radical polymerization catalyzed with the HRP enzyme polymerization of vinyl monomers using the HRP enzyme were first reported by Derango et al in 1992 [21] In the carried out research; acrylamide, methyl acrylate, hydroxyethyl methacrylate, acrylic acid and methyl methacrylate monomers were polymerized using various enzymes (peroxidases, oxidases, etc.). It was observed that the obtained polymers was in the forms of either precipitate or gel. According to the elemental analysis results of the polymers, it was deduced that composition of the obtained yields was consistent. In another important research; Uyama et al. [15] examined the polymerization behavior of the phenol compound containing a methacrylate group [2-(4-hidroksifenil)etilmetakrilat] with the HRP catalyst. According to the report; polymerization only occurred chemoselectively from the phenol ring and the methacrylate group did not participate the polymerization. As a next step in the same research, the polymerization of 2-Phenylethyl Methacrylate with inexistence of the phenolic -OH group was performed using HRP enzyme and the formation of polymethacrylate was observed. Thus, the chemo-selectivity of HRP towards phenolic -OH group was proved.

Following researches carried out to examine the polymerization behaviour of vinyl compounds with enzyme catalysts focused on the use of b-diketone compounds as a cocatalyst to enhance the yield of the polymerization.

In the research carried out by Teixeira et al. in 1998 [22]; The polymerization of acrylamide using b-diketone compounds with HRP enzyme catalyst and H ₂ 0 ₂ was demonstrated and it was shown that b-diketone compounds play a key role for these types of polymerization reactions. Due to their tendency to release their a- hydrogens, b-diketones can simply form enolic forms. It has been suggested that these enolic forms were key by-products for the biocatalytic cycle and thus polymerization occurs.

In the research carried out by Kalra and Gross in 2000 [23]; The polymerization behaviour of methyl methacrylate (MMA) monomer using H ₂ 0 ₂ , 2,4-pentadione and a water soluble cosolvent (dioxane, THF, acetone and DMF) with the HRP enzyme was examined. According to the findings, a significantly high yield of polymer was observed in the polymerization processes performed using dioxane and THF cosolvents.

In another reseach carried out by Kalra and Gross [24]; The polymerization behaviour of acrylamide and sodium acrylate at room temperature using HRP enzyme was examined. In the polymerization of acrylamide carried out by using H ₂ 0 ₂ oxidant, 2,4-pentadion as a b- diketone compound and a surfactant, polyacrylamide was obtained in a very short time (within 60-75 minutes) with the yield up to 94%. In the polymerization processes carried out using surfactants, obtained polymers had narrow polydispersity index. Polymerization of sodium acrylate was achieved without using surfactants. For polymerizing vinylic monomers, photoinitiators or redox polymerization systems are generally used in an inert atmospheric conditions. Chemical systems generally contains toxic materials and/or heavy metals, Therefore, it is important to establish environmentally-friendly conditions for polymerization reactions. Since the polymerization reaction with enzyme catalyst are known to be green and much safer, it is apparent that enzymatic free-radical polymerization reactions are environment-friendly.

Enzyme Catalyst-Mediated Atom Transfer Radical Polymerization (ATRP) enzyme catalyzed ATRP in 2010 was first reported by Ng et al. as the RAFT-type (Reversible Addition Fragmentation Chain-Transfer) polymerization due to its similarity with ATRP polymerization [25] In this research The polymerization behaviour of methacrylic monomers (poly(ethylene glycol)methyl methacrylate and methyl methacrylate) using an alkyl halide initiator (2-bromopropionitrile and ethyl-a-bromoisobutyrate), ascorbic acid reducing agent, and a suitable chain transfer agent (L-cysteine or 2-cyano-2-propyl dithiobenzoate) was demonstrated. The molecular weight of the obtained polymers were controlled through chain transfer agents and the polymers were synthesized with quite high molecular weights.

In another research carried out in 2011 by the same group [26], it was reported that a similar polymerization was achieved using the method of ARGET ATRP (activators regenerated by electron transfer atom transfer radical polymerization). The polymerization behaviour of poly(ethylene glycol)methyl methacrylate monomer using a metalloenzyme catalyst (HRP and laccase), an ascorbic acid reducing agent and an alkyl halide (2-bromopropionitrile or ethyl-2-bromoisobutyrate) initiator was demonstrated and it was discovered that the polymerization mechanism was similar to the method of ARGET ATRP. The number average molecular weight of the obtained polymers ranged between 8500-12000 g/mol and polydispersity index was found to be between in the range of 1.47-1.68.

Similar research was reported by Sigg et al. [27] According to the study; HRP enzyme was capable of polymerizing N-isopropyl acrylamide (NIPAAm) monomer using an organobromine initiator (2-hydroxyethyl-2-bromo-isobutyrate) and sodium ascorbate reducing agent (which is used to reinactivate HRP) under ARGET ATRP conditions, without the need for using peroxide. The polymers obtained with this method had relatively low polydispersity (PDI ~ 1.44) and the number average molecular weight of the polymers ranged between 50000 and 220000 g/mol.

All of these aforementioned references were achieved by using Enzyme catalyst, and the invention comprises the biocatalysts obtained from Cu ²⁺ ions and plant extracts that we have suggested mimicking peroxidase activity.

Utilization of enzymes on an industrial scale is highly limited since they are expensive, unstable in aqueous solutions, undergo denaturation at high temperatures and in organic solvents, and not efficient to reuse, it is very important requirement to reuse enzyme catalysts in reactions for commercial production. Therefore, providing the new generation catalysts exhibiting similar catalytic activities that enzymes exhibit in polymerization reactions is of great importance for both commercial and scientific applications.

Brief Description and Aims of the Invention

On the contrary of horseradish peroxidase enzyme, obtained nano-biocatalysts do not undergo denaturation at high temperatures , and can react even at high temperatures like 60-70 °C without losing their catalytic activity. As enzymes are protein-structured, they undergo denaturation at high temperatures and thus lose their activities. However, since the catalysts subject to the invention do not contain proteins (in other words, enzymes), they do not undergo denaturation up to 70 °C and can exhibit quite high catalytic activities at high temperatures.

The nano-biocatalysts subject to the invention also have a quite high catalytic activity and the potential of being reused (they can be reused for 4-5 times) in reactions. Enzymes cannot be simply seperated from the reaction conditions and cannot be easily recycled. However, thanks to the invention, obtained nano-biocatalysts was successfully seperated from the product by centrifuging the mixture containing 1 M HC1 based on a report in the literature regarding the recovery of synthesized biocatalysts. Thus, the seperated catalyst is enabled to be reused.

Polystyrene and polymethylmethacrylate are the most commonly used polymers and have a high market share in the polymer industry. These polymers can be produced with the catalyst subject to the invention at low cost and environmentally friendly reaction conditions.

Definitions of the Drawings of the Invention

Figure 1: shows a schematic view of the obtaining flower-shaped nano-biocatalysts from plant extracts and Cu ²⁺ ions and the use thereof in polymerization reactions.

Figure 2:(a) and (b) show SEM images of the flower-shaped nano-biocatalysts obtained from viburnum opulus extracts.

Figure 3: (c) and (d) show SEM images of the flower-shaped nano-biocatalysts obtained from green tea extracts.

Detailed Description of the Invention

The invention is related to the production of nano-biocatalysts from plant extracts such as green tea extracts and viburnum opulus extracts and the use of them in polymerization reactions, comprising the following steps of the process;

• Preparing plant extracts,

• Producing flower-shaped nano-biocatalysts from the prepared plant extracts and Cu ²⁺ ion,

• Performing a polymerization reaction by using the obtained flower-shaped nano biocatalysts.

The Preparation of Plant Extracts

Dried green tea leaves (or viburnum opulus) are pulverized and extracted with methanol at room temperature. The extract is then filtered through a filter paper and evaporated until the dryness. The obtained extract is stored in a refrigerator at -20 °C. The plants to be used for obtaining extract within the invention are not limited to green tea and viburnum opulus. The plant extracts carrying amine, carboxyl, hydroxyl and thiol groups can also be used to obtain biocatalysts.

The Production of Flower-Shaped Nano-Structured Catalysts from Plant Extracts

The plant extract prepared in 0.1 and 0.5 mg mL ¹ concentrations is added into a mixture comprising of 10 mM 50 mL phosphate buffer (pH 7.4) and 0.8 mM Cu ²⁺ (or Fe ²⁺ ) ion (by using CuSC>4 or FeSCL). The mixture is treated in a vortex for 30 s and then incubated for 3 days at 4 °C. The obtained precipitate is collected, centrifuged and washed with water. The obtained product is dried at 50 °C and used for reactions.

The Achievement of the Polymerization Reaction by Using the Obtained Flower-Shaped Nano Catalysts

The monomers that are used in the polymerization reaction carried out by using the flower shaped nano biocatalysts obtained from plant extracts and in a polymerization process within the scope of the invention are; Phenol, phenol derivative monomers, acrylamide, N- isopropylacrylamide, styrene and methyl methacrylate. However, apart from the aforementioned monomers, polymerization reactions of different types of vinylic monomers can be achieved by using the same catalyst.

In the polymerization reactions according to the invention, instead of iron and copper catalysts used in the prior art, the polymerization reaction are carried out over a mechanism that is similar to the fenton reaction, however, the catalytic activity that they exhibit in the polymerization reactions is greater compared to enzymes. Free iron and copper ions release the oxidation reaction of proton donor compounds following the fenton reaction, however, their free activity is quite low. Because the most important problem encountered in the fenton reactions carried out with free iron and copper ions is to enable the control of the reactivity and the selectivity of the reaction to be improved. Additionally, in the fenton reactions carried out with free iron and copper ions, pH, solvent, counter ion, chelation of iron/copper, UV and microwave radiations have a direct effect on the oxidation reactivity and the regeneration abilty of Fe or Cu . However, in the present invention, Fe or Cu ions are coordinated with plant extracts and thus their activity is highly enhanced and provide oxidation products at quite high yields.

The experimental method of the invention is the same with the method of the fenton reaction. It is known that copper (Cu ²⁺ ) or iron (Fe ²⁺ ) ions exhibit a peroxidase-type activity in the presence of hydrogen peroxide (H2O2). It is known that the mechanism for this reaction which is similar to the fenton reaction is performed as follows (Diagram 1). The nano-biocatalyst mentioned in the invention also contains Cu ²⁺ (or iron Fe ²⁺ ) ions. These ions react with hydrogen peroxide and produce Cu ¹⁺ ions. As a result of the reaction between the Cu ¹⁺ produced and ¾(¾ a higly reactive hydroxyl radical is produced. This free hydroxyl radical initiates the oxidation of the monomer to be polymerized and the polymerization is carried out over this mechanism. Peroxidase-like activity of nano biocatalysts is enhanced with an increase in the concentration of H2O2 and the catalyst.

Cu ²⁺ + H ₂ 0 ₂ - - Cu ⁺ + HOO' + H ⁺

Cu ⁺ + H ₂ 0 ₂ - ^► Cu ²⁺ + HO" + OH-

Diagram l:The mechanism of the fenton reaction

A Representative Polymerization Procedure for Phenol Derivative Monomers

100 mg Phenol compound and the catalyst to be applied according to the invention (10 mg) are placed in a buffer solution (5 mL). If the phenol derivative compound does not dissolve, an amount (0.5 mL) of organic solvent (water soluble, such as methanol, ethanol, acetone) is added to the obtained mixture, and the reaction is adjusted to the optimum temperature (50 °C). Next, the polymerization is initiated by adding 70 pL hydrogen peroxide to the mixture for 10 times in 10 minute intervals. At the end of the polymerization process, the precipitated product is filtered, washed with water and methanol, and dried at 60 °C (Diagram 2).

Diagram 2: The polymerization of Phenol with the flower-shaped nano biocatalyst and the structure of the expected polymer to be formed. (NFs = nanoflowers). *NFs= The nano biocatalyst obtained from green tea or viburnum opulus

Guaiacol, salicylic acid, -methoxy phenol and -hydroxy benzoic acid monomers were used as phenol derivative compounds.

Table 1. The polymerization of Phenol with the nano biocatalyst obtained from green tea

7 _p : polymerization temperature .

Table 2. The polymerization of Phenol with the nano biocatalyst obtained from viburnum opulus

G _r : polymerization temperature .

Table 3. The polymerization of other phenol derivatives with the nano biocatalyst obtained from green tea aAll of the polymerizations were carried out in pH 7.4 PBS buffer and at 50 °C, b0.5 mL methanol was added to the reaction medium to increase solubility.

Table 4. The polymerization of other Phenol derivatives with the nano biocatalyst obtained from viburnum opulus aAll of the polymerizations were carried out in pH 7.4 PBS buffer and at 50 °C, b0.5 mL methanol was added to the reaction medium to increase solubility.

The Polymerization of Acrylamide 0,73 M Acrylamide solution is prepared and the mixture is purged with nitrogen gas to remove the dissolved oxygen. The nano biocatalyst of the invention (16 mg), H ₂ 0 ₂ (0.092 mmol) and 1-diketone (2,4-pentadione) (0.136 mmol) compound are added to the solution. The mixture is stirred for 3 hours at room temperature. Then, the mixture is precipitated and the obtained precipitate is filtered and washed with methanol and then dried (Diagram 3).

Diagram 3:The polymerization of Acrylamide with the flower-shaped nano biocatalyst (NFs = nanoflowers).

Table 5.The polymerization of Acrylamide with the nano biocatalyst obtained aThe polymerization was carried out with the nano biocatalyst obtained from viburnum opulus at room temperature, bThe polymerization was carried out with the nano biocatalyst obtained from green tea at room temperature.

The Polymerization of /V-Isopropylacrylamide through the ATRP Method 100 mM sodium phosphate buffer (pH 6.0) is prepared (2 mL) and the mixture is purged with argon gas (or nitrogen gas) for 20-30 minutes and thus the dissolved oxygen in the solution is removed. 2-hydroxyethyl-2-bromoisobutyrate (33 pmol) and A-isopropyl acrylamide (2 mmol) are put into a double necked flask. The biocatalyst of the invention (5 mg) and sodium ascorbate (45 pmol) are added into a vial. Certain amount of deoxygenated buffer solution is added into the flask and vial, and the solution obtained in the vial is added into the flask and thus the polymerization is initiated at 40 °C. After the reaction is completed, the mixture is passed through alumina and washed with water. Water is evaporated by applying lyophilisation, and the obtained solid sample is dissolved in THF and passed through alumina again (to remove salt and catalyst), and afterwards THF is evaporated and thus the product is obtained (Diagram 4).

Diagram 4:The polymerization of A sopropyl acrylamide through the ATRP method with the flower-shaped nano biocatalyst (NFs = nanoflowers). Table 6. The polymerization of N-i sopropy 1 aery 1 a i de with the obtained nano biocatalyst aThe polymerization was carried out with the nano biocatalyst obtained from viburnum opulus at room temperature, bThe polymerization was carried out with the nano biocatalyst obtained from green tea at room temperature.

The Polymerization of Styrene

0.7 mL distilled water and 0.3 mL tetrahydrofuran solvent is mixed, the mixture is purged with nitrogen gas to remove the dissolved oxygen. Styrene (0.687 mL) is added to the mixture and the mixture is purged again with nitrogen gas. The nano biocatalyst of the invention (16 mg), H2O2 (13.6 pL) and 2,4-pentadione (0.136 mmol) compound are added to the solution. The mixture is stirred for 24 hours at room temperature. Then, the mixture is poured into methanol and the precipitate is filtered, washed with methanol and dried (Diagram 5). Diagram 5: The polymerization of Styrene with the flower-shaped nano biocatalyst (NFs = nanoflowers).

Table 7.The polymerization of Styrene with the obtained nano biocatalyst ^a The polymerization was carried out with the nano biocatalyst obtained from viburnum opulus at room temperature, bThe polymerization was carried out with the nano biocatalyst obtained from green tea at room temperature.

The Polymerization of Methyl Methacrylate

0.7 mL distilled water and 0.3 mL tetrahydrofuran solvent is added to methyl methacrylate (5.6 mmol) and the mixture is purged with nitrogen gas to remove the dissolved oxygen. The nano biocatalyst of the invention (16 mg), ¾(¾ (0.092 mmol) and 2,4-pentadione (0.136 mmol) compound are added to the solution. The mixture is stirred for a while under nitrogen atmosphere at room temperature. Then, the mixture is poured into methanol and the precipitate is filtered, washed with methanol and dried (Diagram 6). Diagram 6:The polymerization of Methyl Methacrylate with the flower-shaped nano biocatalyst (NFs = nanoflowers).

Table 8.The polymerization of Methyl Methacrylate with the nano biocatalyst obtained aThe polymerization was carried out with the nano biocatalyst obtained from viburnum opulus at room temperature, ^b The polymerization was carried out with the nano biocatalyst obtained from green tea at room temperature.

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