Enzyme-catalyzed Polymerization Research Articles

Enzymatic synthesis of polyguaiacol and its thermal antioxidant behavior in polypropylene

Polyguaiacol was successfully synthesized by enzyme-catalyzed polymerization in an aqueous micelle system with horseradish peroxidase (HRP) as the catalyst. As-synthesized polyguaiacol was characterized by infrared spectrometry and gel permeation chromatography, and its thermal stability was evaluated by thermogravimetric analysis. Besides, the thermal antioxidant behavior of the as-synthesized phenolic polymer as an antioxidant in polypropylene was evaluated by differential scanning calorimetry and accelerated aging test, and the apparent activation energies of thermal decomposition of polypropylene (PP) were calculated by Friedman method. Results show that the as-synthesized polyguaiacol as an antioxidant is able to significantly increase the oxidation induction time of virgin PP, and it exhibits better antioxidative performance than corresponding monomer. Moreover, polyguaiacol, the as-synthesized phenolic polymer, exhibits good resistance against aging of polypropylene.

Enzymatic dehydrogenative polymerization of monolignol dimers

The structure and biosynthesis of lignin are not yet fully understood, especially the step following the initial dimerization of monolignol. Liquid chromatograph mass spectrometer (LC–MS) was used to analyze the consumption rates of monolignol dimers formed by β-O-4, β-5, and β-β couplings between coniferyl alcohols in efforts to understand the activity of monolignol dimers in enzymatic dehydrogenative polymerization. We investigated the reaction kinetics in single-component and mixed-component reaction systems containing one and two species of the dimers, respectively. A difference was observed between the consumption rates of the three dimers we tested, and the consumption rate of one dimer in the single-component reaction was different from that in a mixed-component reaction. In qualitative LC–MS analyses, coniferyl alcohol oligomers were detected in the reaction products. Some monolignol tetramers were formed by 5-5 and 5-O-4 coupling between the dimers. The results of this work suggested that monolignol dimers with β-5 and β-β linkages could function as radical mediators in enzyme-catalyzed polymerization.

Ultrasensitive electrochemical detection of breast cancer cells based on DNA-rolling-circle-amplification-directed enzyme-catalyzed polymerization.

An ultrasensitive cytosensor based on DNA-rolling-circle-amplification-directed enzyme-catalyzed polymerization is demonstrated. As a proof of concept, the cytosensor shows excellent sensitivity for MCF-7 cell detection with a lower detection limit of 12 cells per mL.

Enzymatic synthesis of a polymeric antioxidant for efficient stabilization of polypropylene

Enzyme-catalyzed polymerization of pyrogallic acid is reported as a new method for preparing effective antioxidant for polypropylene (PP). Besides, this polymerization was performed in aqueous micelle, an environmentally benign system, using horseradish peroxidase (HRP) as catalyst. Poly(pyrogallic acid) exhibits better thermal stability than pyrogallic acid. Data from radical scavenging and ferric reducing antioxidant potential assay suggest that poly(pyrogallic acid) has excellent radical scavenging capacity and ferric ion reducing ability. Characterization of PP by thermal methods tests indicate that poly(pyrogallic acid) possesses better antioxidant ability than pyrogallic acid and many commercial antioxidants. Thus, poly(pyrogallic acid) is proposed as a high efficiency, eco-friendly, and easily accessible antioxidant to inhibit thermo-oxidative degradation of PP.

Effects of laccase-catalyzed rutin polymer fraction on adipogenesis inhibition in 3T3-L1 adipocytes

Rutin was enzymatically polymerized by laccase and the rutin polymer fraction (RPF) was collected by ultrafiltration with a molecular cut off of 3kDa. The RPF contained a range of oligomer size varying from hexamer (m/z 3650) to octamer (m/z 4866). The Fourier transform infrared spectrum indicated that various linkages such as hydrogen bonds (OH), aromatic groups (CC), and ether bonds (CO) were involved in rutin polymer formation. RPF more effectively suppressed triglyceride accumulation compared to the rutin monomer in 3T3-L1 adipocytes at all tested concentrations (p<0.05). The anti-adipogenic effect of RPF was mediated by down-regulation of the expression of adipogenic transcriptional factors including peroxisome proliferator-activated receptor (PPARγ), CCATT/enhancer binding protein (C/EBPα), sterol regulatory binding protein (SREBP1C) and fatty acid synthase (FAS) while up-regulating lipolysis and adiponectin expression in 3T3-L1 adipocytes. These results suggest that RPF has a potential protective effect in the development of obesity and that enzyme-catalyzed polymerization could be useful in improving flavonoids bioactivity.

Enzyme-Catalyzed Polymerization of &amp;#946;-alanine Esters, A Sustainable Route Towards the Formation of Poly-&amp;#946;-alanine

The synthesis of poly-β-alanine by a lipase catalyzed polycondensation reaction between β-alanine esters is reported. The effect of different solvents, reaction temperatures and substrate/enzyme concentrations on polymer yield and degree of polymerization (DP) was determined. Also the effect of methyl substituents at the α and β position of β-alanine was studied. Cross-Linked Enzyme Aggregates (CLEA) of Candida antarctica lipase B converted β-alanine esters into polymers in high yield (up to 90%) and with high (DP) (up to 53 units). Optimum results were obtained when methyl esters are used in methyl tert-butyl ether (MTBE) as the solvent, at 50°C for 16 h. Keywords: Biobased, biocatalysis, CLEA, enzymatic catalysis, lipases, Novozyme 435, nylon 3, polyamides.

Synthesis of poly(4-methoxyphenol) by enzyme-catalyzed polymerization and evaluation of its antioxidant activity

The efficient polymerization of 4-methoxyphenol in an aqueous micelle system has been realized with horseradish peroxidase as an enzymatic catalyst and sodium dodecyl sulfate as a surfactant. The obtained polymer is a kind of light yellow powder, which is completely soluble in acetone, DMF, DMSO and THF. Particle size distribution and 1H NMR analysis indicate that the polymerization is performed mainly in the palisade layer of micelles. The number-average molecular weight of poly(4-methoxyphenol) measured by gel permeation chromatography (GPC) is about 1000. IR analysis shows that both phenylene and oxyphenylene units are contained in the polymer structure. Results of antioxidant activity tests indicate that poly(4-methoxyphenol) possesses excellent antioxidant properties and may be used as a highly efficient antioxidant agent.

Enzyme-Catalyzed Polymerization of End-Functionalized Polymers in a Microreactor

An enzyme packed microreactor was compared with a batch reactor system to determine effects of reaction water content on immobilized Candida antarctica Lipase B (Novozyme 435, N435) reusability, efficiency of end-group functionalization, apparent monomer conversion rate constants (kapp), average molar mass (Mn), and leaching of Candida antarctica Lipase B (CALB) from Lewatit macroporous beads. Conversion of e-caprolactone to poly(e-caprolactone), PCL, using benzyl alcohol for end-functionalization, was the model system for studies conducted herein. The apparent rate constant in microreactor (kapp = 0.027 s–1) was 27 times larger than in batch reactor (kapp = 0.001 s–1). Furthermore, in microreactor, the Mn vs conversion plots for “dry” and “water saturated” conditions were similar. In contrast, under “water saturated” conditions in a batch reactor, Mn is much lower. Moreover, at both high and low water content, higher end-group functionalization was achieved for polymerizations in microreactor (0.75 to ≥0...

Oxygen-scavenging coatings and films based on lignosulfonates and laccase

Laccase and lignosulfonates were included in coating colors and embedded in latex-based or starch-based films and coatings on foil or board. After 6 days at 23 °C and 100% relative humidity, the oxygen content in airtight chambers decreased from 1.0% (synthetic gas consisting of 99% N(2) and 1% O(2)) to 0.3% in the presence of board coated with lignosulfonate and laccase, while the oxygen content remained unchanged in control experiments without enzyme. The water stability of lignosulfonate-containing latex-based coatings and starch-based films was improved after laccase-catalyzed oxidation of lignosulfonates, which indicates polymerization to products with lower solubility in water. Furthermore, the E' modulus of starch-based films increased with 30%, which indicates laccase-catalyzed polymerization of lignosulfonates resulting in increased stiffness of the film. The results suggest that laccases and lignosulfonates can be used as an oxygen-scavenging system in active packaging and that enzyme-catalyzed polymerization of lignosulfonates contributes to improved water stability and mechanical properties.

Increasing Molecular Mass in Enzymatic Lactone Polymerizations.

Using a model developed for the enzyme-catalyzed polymerization and degradation of poly(caprolactone), we illustrate a method and the kinetic mechanisms necessary to improve molecular mass by manipulating equilibrium reactions in the kinetic pathway. For these polymerization/degradation reactions, a water/linear chain equilibrium controls the number of chains in solution. Here, we control the equilibrium by adding water-trapping molecular sieves in the batch polymerization reactions of ε-caprolactone. While ring-opening rates were mostly unaffected, the molecular mass shifted to higher molecular masses after complete conversion was reached, and a good agreement between the experimental and modeling results was found. These results provide a framework to improve the molecular mass for enzyme-catalyzed ring-opening polymerization of lactone.

Synthesis of Polyhydroxyalkanoates by Polymerization of Methyl 3-Hydroxypropionate in the Catalysis of Lipase

The polymerization of methyl 3-hydroxypropionate as monomer catalyzed by lipase was selected as a model system in this study. The monomer with the purity around 95% could be polymerized to polyhydroxyalkanoates in the catalysis of Novozym 435. Decreasing the reaction pressure would result in the increase of product yield and high molecular weight. By choosing appropriate organic solvents, surfactants and reaction pressure, the molecular weight of polyhydroxy propionate polyester could be controlled from 1800 to 13000 ( M w value). The reusing ability of enzymatic catalyst was comparatively good. The relative activity could be maintained above 95% after 6 repeated batches reaction. 以 3-羟基丙酸甲酯为聚合单体, 建立了以固定化脂肪酶 Novozym 435 为催化剂的酶催化缩聚反应体系, 合成可完全降解的高分子聚酯聚羟基丙酸酯, 考察了反应条件和介质对反应性能的影响, 结果表明, 纯度大于 95% 的单体即可在温和条件下合成聚羟基羧酸酯; 降低反应压力可有效提升产物产率和分子量. 通过选择合适的有机溶剂介质和表面活性剂, 可使产物分子量提升至 13000 ( M w ) 以上. 脂肪酶催化剂重复利用能力优异, 经 6 批次反应后, 其相对活性保持在 95% 以上. A convenient enzyme-catalyzed polymerization route for polyhydroxypropionate synthesis was established, by which the polyester could be obtained directly by enzyme-catalyzed polycondensation using inexpensive chemical monomers.

Effect of additives on enzyme-catalyzed polymerization of phenols and aromatic amines

Among biological approaches to the removal of aromatic amines and phenols from wastewater, the so-called enzyme-catalyzed polymerization and precipitation (ECPP) process relies on the use of oxidoreductases acting via radical mechanisms and characterized by a rather relaxed substrate specificity, such as laccase, tyrosinase and peroxidases. The main technical constraints of ECPP processes are due to a variety of enzyme deactivation phenomena occurring during catalysis and to the incomplete removal of oxidation products from solution. In order to put ECPP into practice, these drawbacks have to be either counteracted or minimized. Although several approaches, such as enzyme immobilization and reaction engineering, have been proposed to limit these constraints, this review is intended to provide a wide survey on some chemical additives with either protective or coagulating effects that have been so far employed for these purposes.

In situ monitoring of enzyme-catalyzed (co)polymerizations by Raman spectroscopy

In situ, fiber optic-based Raman spectroscopy provided real time monitoring of enzyme-catalyzed ring-opening homo- and copolymerizations of e-caprolactone (e-CL) and δ-valerolactone (δ-VL). A custom designed reactor equipped with in situ fiber optic probe was used to measure monomer conversion as a function of time. The results from the in situ technique were in good agreement with those determined by offline 1H NMR analysis. Monomer reactivity ratios for the lipase-catalyzed copolymerization of e-CL and δ-VL were estimated using the Kelen-Tudos method as re-CL = 0.38 and rδ-VL = 0.29.

Enzyme-catalyzed polymerizations at higher temperatures: Synthetic methods to produce polyamides and new poly(amide-co-ester)s

Novozyme-435 (N-435) catalyzed polycondensation reaction between different aliphatic (oligo)esters and diamines by precipitation polymerization at elevated temperatures yields various particulate polyamides and poly(amide-co-ester)s. The reaction between diethylsebacate (DES) and 1,8-diaminooctane (DAO) at 60 °C in dried toluene leads to a low Mn polyamide (NMR: Mn = 520 g mol−1 GPC: Mn = 2000 g mol−1 with Mw/Mn = 1.3). An improved three-step procedure with an adjusted temperature profile and an optimized amount of enzyme in dried diphenyl ether, yields 97% amide bond formation and a polymer with increased molecular mass (NMR: Mn = 5380 g mol−1, GPC: Mn = 4960 g mol−1 with Mw/Mn = 3.5). Three step ring-opening and polycondensation reactions between a cyclic ester (ethylene tridecanedioate) with three different diamines were also performed in dried toluene to obtain the corresponding polyamides. Here, diamine with higher alkyl chain length, i.e. 1,12-diaminododecane shows higher activity towards N-435 catalyzed amide bond formation and therefore the produced nylon-12,13 has higher molecular weight (GPC: Mn = 8250 g mol−1 and Mw/Mn = 5.8) compared to the other nylons. By adopting the three step synthetic protocol, a new series of poly(amide-co-ester)s with Mn up to 17,550 g mol−1 were produced. Melting and thermal degradation behaviors of these copolymers are compared with pure nylon-8,10 and polyester by DSC and TGA. The microstructure of the synthesized polyamides with different end groups was investigated by MALDI-TOF MS analysis while the particles’ morphology was studied by TEM analysis.

Modeling Enzymatic Kinetic Pathways for Ring-Opening Lactone Polymerization

A unified kinetic pathway for the enzyme-catalyzed polymerization and degradation of poly(ε-caprolactone) was developed. This model tracks the complete distribution of individual chain lengths, both enzyme-bound and in solution, and successfully predicts monomer conversion and the molecular mass distribution as a function of reaction time. As compared to reported experimental data for polymerization reactions, modeled kinetics generate similar trends, with ring-opening rates and water concentration as key factors to controlling molecular mass distributions. Water is critically important by dictating the number of linear chains in solution, shifting the molecular mass distribution at which propagation and degradation equilibrate. For the enzymatic degradation of poly(ε-caprolactone), the final reaction product is also consistent with the equilibrium dictated by the propagation and degradation rates. When the modeling framework described here is used, further experiments can be designed to isolate key reaction steps and provide methods for improving the efficiency of enzyme polymerization.

Continuous Flow Enzyme-Catalyzed Polymerization in a Microreactor

Enzymes immobilized on solid supports are increasingly used for greener, more sustainable chemical transformation processes. Here, we used microreactors to study enzyme-catalyzed ring-opening polymerization of ε-caprolactone to polycaprolactone. A novel microreactor design enabled us to perform these heterogeneous reactions in continuous mode, in organic media, and at elevated temperatures. Using microreactors, we achieved faster polymerization and higher molecular mass compared to using batch reactors. While this study focused on polymerization reactions, it is evident that similar microreactor based platforms can readily be extended to other enzyme-based systems, for example, high-throughput screening of new enzymes and to precision measurements of new processes where continuous flow mode is preferred. This is the first reported demonstration of a solid supported enzyme-catalyzed polymerization reaction in continuous mode.

Application of Polyaniline to an Enzyme-Amplified Electrochemical Immunosensor as an Electroactive Report Molecule

Conducting polymers (CPs) are widely used as matrixes for the entrapment of enzymes in analytical chemistry and biosensing devices. However, enzyme-catalyzed polymerization of CPs is rarely used for immunosensing due to the difficulties involved in the quantitative analysis of colloidal CPs in solution phase. In this study, an enzyme-amplified electrocatalytic immunosensor employing a CP as a redox marker has been developed. A polyanionic polymer matrix, α-amino-ω-thiol terminated poly(acrylic acid), was employed for precipitation of CP. The acrylic acid group acts as a polyanionic template. The thiol terminus of the polymer was used to produce self-assembled monolayers (SAMs) on Au electrodes and the amine terminus was employed for immobilization of biomolecules. In an enzyme- amplified sandwich type immunosensor, the polyaniline (PANI) produced enzymatically is attracted by the electro- static force of the matrix polymer. The precipitated PANI was characterized by electrochemical methods.

Biocatalytic Fabrication of Fast-Degradable, Water-Soluble Polycarbonate Functionalized with Tertiary Amine Groups in Backbone

Degradable polymers with specifically designed functionality have wide applications in biomedical fields. We reported herein the synthesis and characterization of a water-soluble and fast-degradable polycarbonate, functionalized with tertiary amine groups in the backbone. A novel cyclic carbonate monomer, namely, 6,14-dimethyl-1,3,9,11-tetraoxa-6,14-diaza-cyclohexadecane-2,10-dione (ADMC)(2), was synthesized and polymerized to provide the title polycarbonate [poly(ADMC)] via Novozym-435 lipase or tin(II) 2-ethylheaxanoate [Sn(Oct)(2)] catalyzed ring-opening polymerization (ROP). Novozym-435 lipase exhibited high activity toward the ROP in terms of molecular weight (M(n)) and monomer conversion, whereas the attempt with Sn(Oct)(2) failed. In the presence of molecular sieves-4 Å, the highest M(n) value of 1.2 × 10(4) g/mol was obtained in toluene with an initial monomer concentration of 0.58 M at 75 °C in the presence of 10 wt % of Novozym-435 to the monomer. Parameters that influence the polymerization, including reaction temperature, enzyme concentration, monomer concentration, and solvent composition, were investigated systematically. The resultant data suggested "living" characteristics for this enzyme-catalyzed polymerization, and the "living" feature seemed independent of the lipase concentration. The polymerization conducted in mixed solvents (toluene/isooctane) showed that product M(n)s were heavily dependent on the solvent composition. Poly(ADMC) was demonstrated to be amorphous by DSC technique. The obtained poly(ADMC) was found to be soluble in most of the organic solvents and interestingly in H(2)O as well. In vitro hydrolytic degradation of poly(ADMC) as monitored by GPC indicated the degradation was a relatively fast process. HPLC-ESI/MS and (1)H NMR analyses demonstrated that N-methyl diethanolamine was the main product after degradation. Poly(ADMC) presented low cytotoxicity toward human cervix carcinoma (HeLa) cells and hepatoblastoma cells (Hep G2), as demonstrated by MTT assay.

Enzyme-catalysis breathes new life into polyester condensation polymerizations

Traditional chemical catalysts for polyester synthesis have enabled the generation of important commercial products. Undesirable characteristics of chemically catalyzed condensation polymerizations include the need to conduct reactions at high temperatures (150-280 degrees C) with metal catalysts that are toxic and lack selectivity. The latter is limiting when aspiring towards synthesis of increasingly complex and well-defined polyesters. This review describes an exciting technology that makes use of immobilized enzyme-catalysts for condensation polyester synthesis. Unlike chemical catalysts, enzymes function under mild conditions (< or =100 degrees C), which enables structure retention when polymerizing unstable monomers, circumvents the introduction of metals, and also provides selectivity that avoids protection-deprotection steps and presents unique options for structural control. Examples are provided that describe the progress made in enzyme-catalyzed polymerizations, as well as current limitations and future prospects for developing more efficient enzyme-catalysts for industrial processes.

Polymeric Bionanocomposite Cast Thin Films with In Situ Laccase-Catalyzed Polymerization of Dopamine for Biosensing and Biofuel Cell Applications

We report here on the facile preparation of polymer-enzyme-multiwalled carbon nanotubes (MWCNTs) cast films accompanying in situ laccase (Lac)-catalyzed polymerization for electrochemical biosensing and biofuel cell applications. Lac-catalyzed polymerization of dopamine (DA) as a new substrate was examined in detail by UV-vis spectroscopy, cyclic voltammetry, quartz crystal microbalance, and scanning electron microscopy. Casting the aqueous mixture of DA, Lac and MWCNTs on a glassy carbon electrode (GCE) yielded a robust polydopamine (PDA)-Lac-MWCNTs/GCE that can sense hydroquinone with 643 microA mM(-1) cm(-2) sensitivity and 20-nM detection limit (S/N = 3). The DA substrate yielded the best biosensing performance, as compared with aniline, o-phenylenediamine, or o-aminophenol as the substrate for similar Lac-catalyzed polymerization. Casting the aqueous mixture of DA, glucose oxidase (GOx), Lac, and MWCNTs on a Pt electrode yielded a robust PDA-GOx-Lac-MWCNTs/Pt electrode that exhibits glucose-detection sensitivity of 68.6 microA mM(-1) cm(-2). In addition, 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS) was also coimmobilized to yield a PDA-Lac-MWCNTs-ABTS/GCE that can effectively catalyze the reduction of O(2), and it was successfully used as the biocathode of a membraneless glucose/O(2) biofuel cell (BFC) in pH 5.0 Britton-Robinson buffer. The proposed biomacromolecule-immobilization platform based on enzyme-catalyzed polymerization may be useful for preparing many other multifunctional polymeric bionanocomposites for wide applications.