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Hydrogels were prepared by Steglich esterification and by crosslinking pre-synthesized poly(sorbitol adipate)-graft-poly(ethylene glycol) mono methyl ether (PSA-g-mPEG) using different-chain-length-based disuccinyl PEG. PSA and PSA-g-mPEG were investigated for polymer degradation as a function of time at different temperatures. PSA-g-mPEG hydrogels were then evaluated for their most crucial properties of swelling that rendered them suitable for many pharmaceutical and biomedical applications. Hydrogels were also examined for their Sol-Gel content in order to investigate the degree of cross-linking. Physical structural parameters of the hydrogels were theoretically estimated using the modified Flory–Rehner theory to obtain approximate values of polymer volume fraction, the molecular weight between two crosslinks, and the mesh size of the hydrogels. X-ray diffraction was conducted to detect the presence or absence of crystalline regions in the hydrogels. PSA-g-mPEG hydrogels were then extensively examined for higher and lower molecular weight solute release through analysis by fluorescence spectroscopy. Finally, the cytotoxicity of the hydrogels was also investigated using a resazurin reduction assay. Experimental results show that PSA-g-mPEG provides an option as a biocompatible polymer to be used for pharmaceutical applications. [ABSTRACT FROM AUTHOR]

This review article presents the biomimetic helical inclusion of amylose toward hydrophobic polyesters as guests through a vine-twining polymerization process, which has been performed in the glucan phosphorylase (GP)-catalyzed enzymatic polymerization field to fabricate supramolecules and other nanostructured materials. Amylose, which is a representative abundant glucose polymer (polysaccharide) with left-handed helical conformation, is well known to include a number of hydrophobic guest molecules with suitable geometry and size in its cavity to construct helical inclusion complexes. Pure amylose is prepared through enzymatic polymerization of α-d-glucose 1-phosphate as a monomer using a maltooligosaccharide as a primer, catalyzed by GP. It is reported that the elongated amylosic chain at the nonreducing end in enzymatic polymerization twines around guest polymers with suitable structures and moderate hydrophobicity, which is dispersed in aqueous polymerization media, to form amylosic nanostructured inclusion complexes. As the image of this system is similar to how vines of a plant grow around a support rod, this polymerization has been named 'vine-twining polymerization'. In particular, the helical inclusion behavior of the enzymatically produced amylose toward hydrophobic polyesters depending on their structures, e.g., chain lengths and substituents, has been systematically investigated in the vine-twining polymerization field. Furthermore, amylosic supramolecular network materials, such as hydrogels, are fabricated through vine-twining polymerization by using copolymers, where hydrophobic polyester guests or maltooligosaccharide primers are covalently modified on hydrophilic main-chain polymers. The vine-twining polymerization using such copolymers in the appropriate systems induces the formation of amylosic nanostructured inclusion complexes among them, which act as cross-linking points, giving rise to supramolecular networks at the nanoscale. The resulting materials form supramolecular hydrogels, films, and microparticles. [ABSTRACT FROM AUTHOR]

This study investigates inclusion behavior of amylose towards, poly(β-propiolactone) (PPL), that is a hydrophobic polyester, via the vine-twining process in glucan phosphorylase (GP, isolated from thermophilic bacteria, Aquifex aeolicus VF5)-catalyzed enzymatic polymerization. As a result of poor dispersibility of PPL in sodium acetate buffer, the enzymatically produced amylose by GP catalysis incompletely included PPL in the buffer media under the general vine-twining polymerization conditions. Alternatively, we employed an ethyl acetate–sodium acetate buffer emulsion system with dispersing PPL as the media for vine-twining polymerization. Accordingly, the GP (from thermophilic bacteria)-catalyzed enzymatic polymerization of an α-d-glucose 1-phosphate monomer from a maltoheptaose primer was performed at 50 °C for 48 h in the prepared emulsion to efficiently form the inclusion complex. The powder X-ray diffraction profile of the precipitated product suggested that the amylose-PPL inclusion complex was mostly produced in the above system. The 1H NMR spectrum of the product also supported the inclusion complex structure, where a calculation based on an integrated ratio of signals indicated an almost perfect inclusion of PPL in the amylosic cavity. The prevention of crystallization of PPL in the product was suggested by IR analysis, because it was surrounded by the amylosic chains due to the inclusion complex structure. [ABSTRACT FROM AUTHOR]

Naringenin is one of the flavonoids originating from citrus fruit. This polyphenol is mainly found in grapefruit, orange and lemon. The antioxidant and antimicrobial properties of flavonoids depend on their structure, including the polymeric form. The aim of this research was to achieve enzymatic polymerization of naringenin and to study the properties of poly(naringenin). The polymerization was performed by methods using two different enzymes, i.e., laccase and horseradish peroxidase (HRP). According to the literature data, naringenin had not been polymerized previously using the enzymatic polymerization method. Therefore, obtaining polymeric naringenin by reaction with enzymes is a scientific novelty. The research methodology included analysis of the structure of poly(naringenin) by NMR, GPC, FTIR and UV-Vis and its morphology by SEM, as well as analysis of its properties, i.e., thermal stability (DSC and TGA), antioxidant activity (ABTS, DPPH, FRAP and CUPRAC) and antimicrobial properties. Naringenin oligomers were obtained as a result of polymerization with two types of enzymes. The polymeric forms of naringenin were more resistant to thermo-oxidation; the final oxidation temperature To of naringenin catalyzed by laccase (poly(naringenin)-laccase) was 28.2 °C higher, and poly(naringenin)-HRP 23.6 °C higher than that of the basic flavonoid. Additionally, due to the higher molar mass and associated increase in OH groups in the structure, naringenin catalyzed by laccase (poly(naringenin)-laccase) showed better activity for scavenging ABTS+• radicals than naringenin catalyzed by HRP (poly(naringenin)-HRP) and naringenin. In addition, poly(naringenin)-laccase at a concentration of 5 mg/mL exhibited better microbial activity against E. coli than monomeric naringenin. [ABSTRACT FROM AUTHOR]

ε-caprolactone (CL) has been enzymatically polymerized using α-amino acids based on sulfur (methionine and cysteine) as (co-)initiators and immobilized lipase B of Candida antarctica (CALB) as biocatalyst. In-depth characterizations allowed determining the corresponding involved mechanisms and the polymers thermal properties. Two synthetic strategies were tested, a first one with direct polymerization of CL with the native amino acids and a second one involving the use of an amino acid with protected functional groups. The first route showed that mainly polycaprolactone (PCL) homopolymer could be obtained and highlighted the lack of reactivity of the unmodified amino acids due to poor solubility and affinity with the lipase active site. The second strategy based on protected cysteine showed higher monomer conversion, with the amino acids acting as (co-)initiators, but their insertion along the PCL chains remained limited to chain endcapping. These results thus showed the possibility to synthesize enzymatically polycaprolactone-based chains bearing amino acids units. Such cysteine endcapped PCL materials could then find application in the biomedical field. Indeed, subsequent functionalization of these polyesters with drugs or bioactive molecules can be obtained, by derivatization of the amino acids, after removal of the protecting group. [ABSTRACT FROM AUTHOR]

This review presents our researches on the preparation and material application of inclusion complexes that comprises an amylose host and polymeric guests through phosphorylase-catalyzed enzymatic polymerization. Amylose is a well-known polysaccharide and forms inclusion complexes with various hydrophobic small molecules. Pure amylose is produced by enzymatic polymerization by using α-D-glucose 1-phosphate as a monomer and maltooligosaccharide as a primer catalyzed by phosphorylase. We determined that a propagating chain of amylose during enzymatic polymerization wraps around hydrophobic polymers present in the reaction system to form inclusion complexes. We termed this polymerization "vine-twining polymerization" because it is similar to the way vines of a plant grow around a rod. Hierarchical structured amylosic materials, such as hydrogels and films, were fabricated by inclusion complexation through vine-twining polymerization by using copolymers covalently grafted with hydrophobic guest polymers. The enzymatically produced amyloses induced complexation with the guest polymers in the intermolecular graft copolymers, which acted as cross-linking points to form supramolecular hydrogels. By including a film-formable main-chain in the graft copolymer, a supramolecular film was obtained through hydrogelation. Supramolecular polymeric materials were successfully fabricated through vine-twining polymerization by using primer-guest conjugates. The products of vine-twining polymerization form polymeric continuums of inclusion complexes, where the enzymatically produced amylose chains elongate from the conjugates included in the guest segments of the other conjugates. [ABSTRACT FROM AUTHOR]

Yarrowia lipolytica (YL) is a "non-conventional" yeast that is capable of producing important metabolites. One of the most important products that is secreted by this microorganism is lipase, a ubiquitous enzyme that has considerable industrial potential and can be used as a biocatalyst in the pharmaceutical, food, and environmental industries. In this work, Yarrowia lipolytica lipase (YLL) was immobilized on Lewatit and Amberlite beads and is used in the enzymatic ring-opening polymerization (ROP) of cyclic esters in the presence of different organic solvents. YLL immobilized on Amberlite XAD7HP had the higher protein adsorption (96%) and a lipolytic activity of 35 U/g. Lewatit VPOC K2629 has the higher lipolytic activity (805 U/g) and 92% of protein adsorption. The highest molecular weight (Mn 10,685 Da) was achieved at 90 °C using YLL that was immobilized on Lewatit 1026 with decane as solvent after 60 h and 100% of monomer conversion. [ABSTRACT FROM AUTHOR]

Nowadays, "green" is a hot topic almost everywhere, from retailers to universities to industries; and achieving a green status has become a universal aim. However, polymers are commonly considered not to be "green", being associated with massive energy consumption and severe pollution problems (for example, the "Plastic Soup") as a public stereotype. To achieve green polymers, three elements should be entailed: (1) green raw materials, catalysts and solvents; (2) eco-friendly synthesis processes; and (3) sustainable polymers with a low carbon footprint, for example, (bio)degradable polymers or polymers which can be recycled or disposed with a gentle environmental impact. By utilizing biobased monomers in enzymatic polymerizations, many advantageous green aspects can be fulfilled. For example, biobased monomers and enzyme catalysts are renewable materials that are derived from biomass feedstocks; enzymatic polymerizations are clean and energy saving processes; and no toxic residuals contaminate the final products. Therefore, synthesis of renewable polymers via enzymatic polymerizations of biobased monomers provides an opportunity for achieving green polymers and a future sustainable polymer industry, which will eventually play an essential role for realizing and maintaining a biobased and sustainable society. [ABSTRACT FROM AUTHOR]

Potato phosphorylase is able to synthesize linear polyglucans from maltoheptaose primers. By coupling maltoheptaose to butane diamine, tris(2-aminoethyl)amine and amine functionalized amine functionalized poly ethyleneglycol (PEG), new primer molecules became available. The resulting di-, tri- and macro-primers were incubated with potato phosphorylase and glycogen branching enzyme from Deinococcus geothermalis. Due to the action of both enzymes, hyperbranched polyglucan arms were grown from the maltoheptaose derivatives with a maximum degree of branching of 11%. The size of the synthesized hyperbranched polyglucans could be controlled by the ratio monomer over primer. About 60%-80% of the monomers were incorporated in the glycoconjugates. The resulting hyperbranched glycoconjugates were subjected to Dynamic Light Scattering (DLS) measurements in order to determine the hydrodynamic radius and it became obvious that the structures formed agglomerates in the range of 14-32 nm. [ABSTRACT FROM AUTHOR]

This paper reviews preparation and applications of amylose supramolecules by means of phosphorylase-catalyzed enzymatic polymerization. When the enzymatic polymerization of α-D-glucose 1-phosphate (G-1-P) as a monomer was carried out in the presence of poly(tetrahydrofuran) (PTHF) of a hydrophobic polyether as a guest polymer, the supramolecule, i.e., an amylose-PTHF inclusion complex, was formed in the process of polymerization. Because the representation of propagation in the polymerization is similar to the way that vines of plants grow twining around rods, this polymerization method for the preparation of amylose-polymer inclusion complexes was proposed to be named "vine-twining polymerization". Various hydrophobic polyethers, polyesters, poly(ester-ether), and polycarbonates were also employed as the guest polymer in the vine-twining polymerization to produce the corresponding inclusion complexes. To obtain the inclusion complex from a strongly hydrophobic guest polymer, the parallel enzymatic polymerization system was developed as an advanced extension of the vine-twining polymerization. In addition, it was found that amylose selectively includes one side of the guest polymer from a mixture of two resemblant guest polymers, as well as a specific range in molecular weights of the guest PTHF. Amylose also exhibited selective inclusion behavior toward stereoisomers of poly(lactide)s. Moreover, the preparation of hydrogels through the formation of inclusion complexes of amylose in vine-twining polymerization was achieved. [ABSTRACT FROM AUTHOR]

This study reports the first enzymatic synthesis leading to several oligomer analogues of poly[3-(3,4-dihydroxyphenyl)glyceric acid]. This biopolymer, extracted from plants of the Boraginaceae family has shown a wide spectrum of pharmacological properties, including antimicrobial activity. Enzymatic ring opening polymerization of 2-methoxycarbonyl-3-(3,4-dibenzyloxyphenyl)oxirane (MDBPO) using lipase from Candida rugosa leads to formation of poly[2-methoxycarbonyl-3-(3,4-dibenzyloxyphenyl)oxirane] (PMDBPO), with a degree of polymerization up to 5. Catalytic debenzylation of PMDBPO using H2 on Pd/C yields poly[2-methoxycarbonyl-3-(3,4-dihydroxyphenyl)oxirane] (PMDHPO) without loss in molecular mass. Antibacterial assessment of natural polyethers from different species of Boraginaceae family Symhytum asperum, S. caucasicum,S. grandiflorum, Anchusa italica, Cynoglossum officinale, and synthetic polymers, poly[2-methoxycarbonyl-3-(3,4-dimethoxyphenyl)oxirane (PMDMPO) and PMDHPO, reveals that only the synthetic analogue produced in this study (PMDHPO) exhibits a promising antimicrobial activity against pathogenic strains S.aureus ATCC 25923 and E.coli ATCC 25922 the minimum inhibitory concentration (MIC) being 100 µg/mL. [ABSTRACT FROM AUTHOR]

Enzymatic synthesis of aromatic biobased polyesters is a recent and rapidly expanding research field. However, the direct lipase-catalyzed synthesis of polyesters from ferulic acid has not yet been reported. In this work, various ferulic-based monomers were considered for their capability to undergo CALB-catalyzed polymerization. After conversion into diesters of different lengths, the CALB-catalyzed polymerization of these monomers with 1,4-butanediol resulted in short oligomers with a DPn up to 5. Hydrogenation of the double bond resulted in monomers allowing obtaining polyesters of higher molar masses with DPn up to 58 and Mw up to 33,100 g·mol−1. These polyesters presented good thermal resistance up to 350 °C and Tg up to 7 °C. Reduction of the ferulic-based diesters into diols allowed preserving the double bond and synthesizing polyesters with a DPn up to 19 and Mw up to 15,500 g·mol−1 and higher Tg (up to 21 °C). Thus, this study has shown that the monomer hydrogenation strategy proved to be the most promising route to achieve ferulic-based polyester chains of high DPn. This study also demonstrates for the first time that ferulic-based diols allow the synthesis of high Tg polyesters. Therefore, this is an important first step toward the synthesis of competitive biobased aromatic polyesters by enzymatic catalysis. [ABSTRACT FROM AUTHOR]

Trends in the dynamically developing application of biocatalysis for the synthesis and modification of polymers over the past 5 years are considered, with an emphasis on the production of biodegradable, biocompatible and functional polymeric materials oriented to medical applications. The possibilities of using enzymes not only as catalysts for polymerization but also for the preparation of monomers for polymerization or oligomers for block copolymerization are considered. Special attention is paid to the prospects and existing limitations of biocatalytic production of new synthetic biopolymers based on natural compounds and monomers from biomass, which can lead to a huge variety of functional biomaterials. The existing experience and perspectives for the integration of bio- and chemocatalysis in this area are discussed. [ABSTRACT FROM AUTHOR]

In this study, we attempted to prepare an amylose-oligo[(R)-3-hydroxybutyrate] (ORHB) inclusion complex using a vine-twining polymerization approach. Our previous studies indicated that glucan phosphorylase (GP)-catalyzed enzymatic polymerization in the presence of appropriate hydrophobic guest polymers produces the corresponding amylose–polymer inclusion complexes, a process named vine-twining polymerization. When vine-twining polymerization was conducted in the presence of ORHB under general enzymatic polymerization conditions (45 °C), the enzymatically produced amylose did not undergo complexation with ORHB. However, using a maltotriose primer in the same polymerization system at 70 °C for 48 h to obtain water-soluble amylose, called single amylose, followed by cooling the system over 7 h to 45 °C, successfully induced the formation of the inclusion complex. Furthermore, enzymatic polymerization initiated from a longer primer under the same conditions induced the partial formation of the inclusion complex. The structures of the different products were analyzed by X-ray diffraction, 1H-NMR, and IR measurements. The mechanism of formation of the inclusion complexes discussed in the study is proposed based on the additional experimental results. [ABSTRACT FROM AUTHOR]

Biocatalysis has developed in the last decades as a major tool for green polymer synthesis. The particular ability of lipases to catalyze the synthesis of novel polymeric materials has been demonstrated for a large range of substrates. In this work, novel functional oligoesters were synthesized from ε-caprolactone and D,L/L-malic acid by a green and sustainable route, using two commercially available immobilized lipases as catalysts. The reactions were carried out at different molar ratios of the comonomers in organic solvents, but the best results were obtained in solvent-free systems. Linear and cyclic oligomeric products with average molecular weights of about 1500 Da were synthesized, and the formed oligoesters were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The oligoester synthesis was not enantioselective in the studied reaction conditions. The operational stability of both biocatalysts (Novozyme 435 and GF-CalB-IM) was excellent after reutilization in 13 batch reaction cycles. The thermal properties of the reaction products were investigated by thermogravimetric (TG) and differential scanning calorimetry (DSC) analysis. The presence of polar pendant groups in the structure of these oligomers could widen the possible applications compared to the oligomers of ε-caprolactone or allow the conversion to other functional materials. [ABSTRACT FROM AUTHOR]

Among the various catalysts that can be used for polycondensation reactions, enzymes have been gaining interest for three decades, offering a green and eco-friendly platform towards the sustainable design of renewable polyesters. However, limitations imposed by their delicate nature, render them less addressed. As a case study, we compare herein bulk and solution polycondensation of 1,6-hexanediol and diethyl adipate catalyzed by an immobilized lipase from Candida antarctica. The influence of various parameters including time, temperature, enzyme loading, and vacuum was assessed in the frame of a two-step polymerization with the help of response surface methodology, a statistical technique that investigates relations between input and output variables. Results in solution (diphenyl ether) and bulk conditions showed that a two-hour reaction time was enough to allow adequate oligomer growth for the first step conducted under atmospheric pressure at 100 °C. The number-average molecular weight (Mn) achieved varied between 5000 and 12,000 g·mol−1 after a 24 h reaction and up to 18,500 g∙mol−1 after 48 h. The statistical analysis showed that vacuum was the most influential factor affecting the Mn in diphenyl ether. In sharp contrast, enzyme loading was found to be the most influential parameter in bulk conditions. Recyclability in bulk conditions showed a constant Mn of the polyester over three cycles, while a 17% decrease was noticed in solution. The following work finally introduced a statistical approach that can adequately predict the Mn of poly(hexylene adipate) based on the choice of parameter levels, providing a handy tool in the synthesis of polyesters where the control of molecular weight is of importance. [ABSTRACT FROM AUTHOR]

In recent years, developing potent antioxidants has been a very active area of research. In this context, phenolic compounds have been evaluated for their antioxidant activity. However, the use of phenolic compounds has also been limited by poor antioxidant activity in several in vivo studies. Polymeric phenols have received much attention owing to their potent antioxidant properties and increased stability in aqueous systems. To be truly effective in biological applications, it is important that these polymers be synthesized using benign methods. In this context, enzyme catalyzed synthesis of polymeric phenols has been explored as an environmentally friendly and safer approach. This review summarizes work in enzymatic syntheses of polymers of phenols. Several assays have been developed to determine the antioxidant potency of these polymeric phenols. These assays are discussed in detail along with structure-property relationships. A deeper understanding of factors affecting antioxidant activity would provide an opportunity for the design of versatile, high performing polymers with enhanced antioxidant activity. [ABSTRACT FROM AUTHOR]

Enzyme catalyzed synthesis of renewable polyamides was investigated using Candida antarctica lipase B. A fatty acid-derived AB-type functional monomer, having one amine and one methyl ester functionality, was homopolymerized at 80 and 140 °C. Additionally, the organobase 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) was used as a catalyst. The results from the two catalysts were comparable. However, the amount of lipase added was 1.2 × 103 times lower, showing that the lipase was a more efficient catalyst for this system as compared to TBD. Moreover, the AB-type monomer was copolymerized with 1,12-diaminododecane to synthesize oligoamides of two different lengths. [ABSTRACT FROM AUTHOR]

In this work the polymerization of catechin, epicatechin, and resveratrol was carried out through a peroxidase oxidation process in order to improve the biological activity of these phenolic compounds. The antioxidant activity of the oligomers was evaluated by their ability to scavenge reactive oxygen species (ROS) and their capacity to chelate metal ions Fe2+ and Cu2+. The antitumor effect of the oligomers was determined by their ability to induce toxicity in the T24 human bladder cancer cell line. By enzymatic peroxidase oxidation, it was possible to produce oligomers of catechin, epicatechin, and resveratrol with antioxidant capacity significantly higher than their preceding monomers. The ROS scavenging capacity of the oligomers was 20 times higher than that of the monomers, while the ability of the oligomers to chelate metal ions increased up to about 1000 times. Our data show the antitumor effect of the oligomers of catechin, epicatechin, and resveratrol in the T24 cell line, which was similar to that observed with cisplatin. Oligomers of catechin, epicatechin, and resveratrol have great potential to be used as therapeutic agents for the treatment of oxidative stress-related diseases and bladder cancer. [ABSTRACT FROM AUTHOR]

Enzymatic polymerization has been noted as a powerful method to precisely synthesize polymers with complicated structures, such as polysaccharides, which are not commonly prepared by conventional polymerization. Phosphorylase is one of the enzymes which have been used to practically synthesize well-defined polysaccharides. The phosphorylase-catalyzed enzymatic polymerization is conducted using α-d-glucose 1-phosphate as a monomer, and maltooligosaccharide as a primer, respectively, to obtain amylose. Amylose is known to form supramolecules owing to its helical conformation, that is, inclusion complex and double helix, in which the formation is depended on whether a guest molecule is present or not. In this paper, we would like to report the preparation of amylose-carboxymethyl cellulose (CMC) conjugated supramolecular networks, by the phosphorylase-catalyzed enzymatic polymerization, using maltoheptaose primer-grafted CMC. When the enzymatic polymerization was carried out using the graft copolymer, either in the presence or in the absence of a guest polymer poly (ε-caprolactone) (PCL), the enzymatically elongated amylose chains from the primers on the CMC main-chain formed double helixes or inclusion complexes, depending on the amounts of PCL, which acted as cross-linking points for the construction of network structures. Accordingly, the reaction mixtures totally turned into hydrogels, regardless of the structures of supramolecular cross-linking points. [ABSTRACT FROM AUTHOR]

The design of drug-loaded nanoparticles (NPs) appears to be a suitable strategy for the prolonged plasma concentration of therapeutic payloads, higher bioavailability, and the reduction of side effects compared with classical chemotherapies. In most cases, NPs are prepared from (co)polymers obtained through chemical polymerization. However, procedures have been developed to synthesize some polymers via enzymatic polymerization in the absence of chemical initiators. The aim of this work was to compare the acute in vitro cytotoxicities and cell uptake of NPs prepared from poly(benzyl malate) (PMLABe) synthesized by chemical and enzymatic polymerization. Herein, we report the synthesis and characterization of eight PMLABe-based polymers. Corresponding NPs were produced, their cytotoxicity was studied in hepatoma HepaRG cells, and their uptake by primary macrophages and HepaRG cells was measured. In vitro cell viability evidenced a mild toxicity of the NPs only at high concentrations/densities of NPs in culture media. These data did not evidence a higher biocompatibility of the NPs prepared from enzymatic polymerization, and further demonstrated that chemical polymerization and the nanoprecipitation procedure led to biocompatible PMLABe-based NPs. In contrast, NPs produced from enzymatically synthesized polymers were more efficiently internalized than NPs produced from chemically synthesized polymers. The efficient uptake, combined with low cytotoxicity, indicate that PMLABe-based NPs are suitable nanovectors for drug delivery, deserving further evaluation in vivo to target either hepatocytes or resident liver macrophages. [ABSTRACT FROM AUTHOR]

A detailed mechanistic and kinetic study of enzymatically initiated RAFT polymerization is performed by combining enzymatic assays and polymerization kinetics analysis. Horseradish peroxidase (HRP) initiated RAFT polymerization of dimethylacrylamide (DMAm) was studied. This polymerization was controlled by 2-(propionic acid)ylethyl trithiocarbonate (PAETC) in the presence of H2O2 as a substrate and acetylacetone (ACAC) as a mediator. In general, well controlled polymers with narrow molecular weight distributions and good agreement between theoretical and measured molecular weights are consistently obtained by this method. Kinetic and enzymatic assay analyses show that HRP loading accelerates the reaction, with a critical concentration of ACAC needed to effectively generate polymerization initiating radicals. The PAETC RAFT agent is required to control the reaction, although the RAFT agent also has an inhibitory effect on enzymatic performance and polymerization. Interestingly, although H2O2 is the substrate for HRP there is an optimal concentration near 1 mM, under the conditions studies, with higher or lower concentrations leading to lower polymerization rates and poorer enzymatic activity. This is explained through a competition between the H2O2 acting as a substrate, but also an inhibitor of HRP at high concentrations. [ABSTRACT FROM AUTHOR]