Fungal laccase grafts acrylamide onto lignin in presence of peroxides

Laccase (EC 1.10.3.2) from the white-rot basidomycete Trametes versicolor in the presence of organic peroxides, particularly dioxane peroxide, tetrahydrofuran peroxide and t-butylhydroperoxide, initiated free-radical copolymerization of acrylamide and lignin. Hydrogen peroxide showed no such effect. Both the type of peroxide and the catalytic efficiency of the enzyme were important to ensure a significant yield of copolymerisate and a high rate of acrylamide incorporation into a lignin backbone. The mechanism of the enzymatic grafting is discussed. Received: 12 August 1998 / Received revision: 18 November 1998 / Accepted: 21 November 1998     

Solvent-free vapor-phase photografting of acrylamide onto poly(ethylene terephthalate)

Poly(ethylene terphthalate) (PET) films were photografted under reduced pressure in a solvent-free vapor of acrylamide and a co-initiator, benzophenone. Characterization of grafted samples by ESCA and contact angles showed that the grafting increased with grafting time and temperature. The amide groups obtained by the acrylamide grafting were converted into amine groups by the Hofmann rearrangement to be used in coupling reactions. The amine groups were confirmed by reaction with pentafluorobenzoyl chloride, which provides a fluorine label for ESCA. Surface grafting of polymeric substrates in the vapor phase induced by plasma or high energy and UV irradiation is reviewed.     

Study of the reaction of grafting acrylamide onto xanthan gum

The present study aimed to study the reaction conditions of grafting of acrylamide on xanthan gum. It was analyzed the influence of reaction conditions, mainly type of initiator activation, initiator concentration and initiator/acrylamide ratio, on graft parameters and copolymer properties. Potassium persulfate was employed as an initiator and heating or N,N,N',N'-tetramethylethylenediamine was used to activate the initiator. Reaction time and initiator concentration were varied and final values for grafting percentage and grafting efficiency were the same for both methods, whereas speed in reaching these values differs from one technique to another. We found that reaction time was inversely proportional to intrinsic viscosity, likely due to main chain degradation promoted by potassium persulfate (KPS); furthermore, the increasing in the KPS concentration lowers grafting percentage, acrylamide conversion and chain degradation, possibly as a result of O(2) formation at high KPS concentrations.     

Effects of amylose/amylopectin ratio on starch-based superabsorbent polymers

Biodegradable superabsorbent polymers (SAPs) were prepared by grafting acrylamide onto starches then crosslinking with N,N′-methylene-bisacrylamide. This work focused on the effects of the amylose/amylopectin ratio of starches from the same source (corn) on the grafting reactions and performance of the resultant starch-based SAPs. To characterise each SAP, the acrylamide groups grafted onto the starch were detected by FTIR; grafting ratio and grafting efficiency were evaluated by a gravimetric method; and graft position and the length of the grafted segment were investigated by NMR. The relationships between the microstructures of the starches, and the graft reactions and performance of the SAPs were studied based on the amylose content in the starches. It was found that under the same reaction conditions, the grafting ratio and efficiency increased with increasing amylose content, which corresponds with water absorption ratio. NMR results indicated that the acrylamide group mainly grafted onto C6, and that the length of the grafted segment decreased with increasing amylopectin content in general, and in particular for waxy starch. The high molecular weight and branched structure of amylopectin reduced the mobility of the polymer chains and increased viscosity, which could explain the graft reactions and performance of the starch-based SAPs.     

Enzymatic co-polymerization of lignin with low-molecular mass compounds

The oxidoreductive enzyme laccase (E.C.1.10.3.2.) isolated from a culture medium of white-rot fungus Trametes versicolor transformed lignin preparations solubilized in a dioxane-H₂O (7:3) mixture. The obvious net result of lignin transformation was an increase in molecular mass. A superoxide radical was found in the reaction mixture during lignin incubation with laccase. It appeared that a change in the reaction medium or in the lignin molecule instigated by laccase could lead to polymerization after the lignin molecules had crossed a dialysis membrane and were separated from the enzyme. Two possible mechanisms are suggested, either diffusion of an activated oxygen species or diffusion of primed lignin molecules. Laccase was able to co-polymerize lignin with low-molecular-mass compounds of different origins, particularly with aromatics containing either carboxyl or isocyanate groups, as well as acrylamide — an aliphatic monomer containing a vinyl group. Correspondence to: O. Milstein     

西瓜嫁接体发育中木质素合成及代谢相关酶活性的变化

研究了西瓜/葫芦嫁接体发育过程中砧木和接穗部分木质素含量及其代谢相关酶的活性变化.结果表明,嫁接体发育过程中木质素生物合成加快,砧木及接穗部分的过氧化物酶(POD)、过氧化氢酶(CAT)、苯丙氨酸解胺酶(PAL)、肉桂醇脱氢酶(CAD)等的活性均明显高于对照,接穗和砧木中POD活性在15 d内持续升高,H2O2含量和CAT活性于嫁接后9~12 d出现高峰,砧木中PAL于9 d时有活性高峰而接穗一直保持较高活性而没有活性高峰,木质素含量和CAD活性持续增长,葫芦砧木木质素的代谢水平高于西瓜接穗.     

Studying the effects of laccase-catalysed grafting of ferulic acid on sisal pulp fibers

Functionalization of sisal specialty pulp fibers by laccase-catalysed grafting of ferulic acid (FRC) was investigated. To this end, the extent of phenol coupling to fibers under different reaction conditions (laccase and FRC rates, and time) was evaluated in terms of pulp properties including kappa number (expressed as the combined contributions of lignin and hexenuronic acids), brightness, Klason lignin and surface anionic charge after Soxhlet extraction of acetone-treated pulp. The specific treatment resulting in the highest degree of grafting was then used in a comparative study of the effects of applying the laccase-FRC system to refined and unrefined pulp with a view to confirming whether the increased surface area obtained by effect of fibrillation would lead to enhanced grafting. Based on the results, refining the pulp prior to the enzyme treatment resulted in increased grafting which in turn led to handsheets with improved strength-related properties (particularly wet tensile strength) relative to control samples.     

Modification of Okra mucilage with acrylamide: Synthesis, characterization and swelling behavior

In the present communication, the synthesis and characterization of Okra mucilage, a food grade and water-soluble polysaccharide, based-materials are described. Okra mucilage has been modified by grafting acrylamide (AAm) for developing the new green polymeric materials of specialty applications. Grafting has been done under N₂ atmosphere using redox initiator and hydrogels were prepared by using N,N-methylenebisacrylamide (NN-MBAAm) as crosslinker. The effect of monomer concentration, initiator concentration, reaction time and temperature in terms of grafting efficiency (%GE), percent grafting (PG) and percent gel (%G) has been investigated. The grafted polymers and hydrogels were characterized by SEM, XRD and FTIR techniques to study various structural aspects. The swelling behavior of the crosslinked polymeric material has also been studied as a function of time, temperature and pH. The application area of these polymers is varied from biomaterials to the wastewater treatment.     

Xanthan-g-poly(acrylamide): Microwave-assisted synthesis,characterization and in vitro release behavior

Xanthan-g-poly(acrylamide) was synthesized employing microwave-assisted and ceric-induced graft copolymerization, and was characterized by FT-IR, DSC, XRD and SEM studies. Matrix tablets of diclofenac sodium were formulated using graft copolymer as the matrix by direct compression technique. Release behavior of the graft copolymer was evaluated using USP type-II dissolution apparatus in 900 ml of phosphate buffer (pH 6.8), maintained at 37 °C and at 50 rpm. Microwave-assisted grafting provided graft copolymer with higher % grafting in a shorter time in comparison to the ceric-induced grafting. The % grafting was found to increase with the increase in the power of microwave and/or time of exposure. The matrix tablets were found to release the drug by zero-order kinetics, and the faster release of drug was observed from the graft copolymer matrix as compared to the xanthan gum matrix. It was observed that grafting reduces the swelling, but increases the erosion of xanthan gum.     

Rapid synthesis of antimicrobial paper under microwave irradiation

The silver-nanoparticle (AgNP) containing paper was successfully prepared. The AgNP is deposited by the in situ reduction of silver nitrate on the acrylamide grafted bagasse paper sheets in the presence of citrate molecules as stabilizing agent. In the present paper, grafting of acrylamide onto bagasse paper sheets using potassium persulfate was carried out under the influence of microwave radiations (MWR). The modified paper sheets were characterized by Fourier transform infrared spectroscopy (FTIR), UV-spectroscopy, and scanning electron microscopy (SEM). Antimicrobial activities of the prepared paper sheets were also investigated against G+ve bacterium Staphylococcus aureus, G-ve bacterium Pseudomomas aeruginosa, and yeast Candida albicans, which are model microorganisms for testing bactericidal properties. The AgNP containing paper sheets exhibited antibacterial activity.     

Studies on preparing and adsorption property of grafting terpolymer microbeads of PEI-GMA/AM/MBA for bilirubin

Crosslinking copolymer microbeads with a diameter range of 100-150 microm were synthesized by suspension copolymerization of glycidyl methacrylate (GMA), acrylamide (AM) and N,N'-methylene bisacrylamide (MBA). Subsequently, polyethyleneimine (PEI) was grafted on the surfaces of the terpolymer microbeads GMA/AM/MBA via the ring-opening reaction of the epoxy groups, and the grafting microbeads PEI-GMA/AM/MBA were prepared. In this paper, the adsorption property of the grafting microbeads for bilirubin was mainly investigated, and the effects of various factors, such as pH value, ionic strength and grafting degree of PEI on the surface of grafting microbeads and the adsorption capacity of the grafting microbeads for bilirubin were examined. The batch adsorption experiment results show that by right of the action of grafted polyamine macromolecules PEI, the grafting microbeads PEI-GMA/AM/MBA have quite strong adsorption ability for bilirubin; the isotherm adsorption conforms to Freundlich equation. The pH value of the medium affects the adsorption capacity greatly, As in the nearly neutral solutions with pH 6, the grafting microbeads have the strongest adsorption ability for bilirubin, whereas in acidic and basic solutions their adsorption ability is weak. The ionic strength hardly affects the adsorption ability of the grafting microbeads. The grafting degree of PEI on the surfaces of the grafting microbeads also has a great effect on the adsorption capacity, and higher the grafting degree of PEI on the surface of the microbead PEI-GMA/AM/MBA, the stronger is the adsorption ability of the microbeads.     

A chitosan-based flocculant prepared with gamma-irradiation-induced grafting

A natural polymer, chitosan, was modified to prepare an efficient flocculant using grafting method initiated by gamma ray in acid-water solution. A vinyl monomer, acrylamide, was used as the grafted monomer. The graft copolymer obtained was characterized using Fourier transform infrared spectroscopy, X-ray diffraction and thermogravimetric analysis. Effects of acetic acid concentration, total irradiation dose, dose rate and monomer concentration on the grafting percentage were investigated. Flocculation experiment results demonstrated that the graft copolymer produced was significantly superior to chitosan and polyacrylamide (PAM).     

Free radical grafting kinetics of acrylamide onto a blend of starch/chitosan/alginate

Grafting of monomer onto polymer backbone is one of the effective and accessible methods for the chemical modification of polysaccharides. Grafting of acrylamide (AAm) onto polysaccharides blend (PsB) composed of starch, chitosan and alginate has been carried out using potassium persulfate (KPS) as an initiator. The kinetics of the grafting polymerization also has been studied. The grafting parameters have been evaluated by changing the initial concentrations of AAm from 8 to 16 g, PsB from 6 to 14 g and KPS from 0.2 to 1 g. Evidence of grafting has been obtained from FTIR, XRD and TGA. The kinetics of the grafting polymerization also has been studied. The grafting rate equation of the produced hydrogel (PsB-g-AAm) hydrogel has been expressed by: Rg = k[AAm] [PsB]^0.5 [KPS]^0.5. The grafting rate is a first order dependence to [AAm] initial concentration and square root to [PsB] and [KPS] initial concentrations in the used concentrations range.     

不同苗龄接穗的西瓜嫁接体愈合过程中的3种酶活性变化

用贴接法嫁接西瓜品种早佳(8424)和砧木葫芦品种将军的结果表明:接穗苗龄越大,嫁接成活率越低.多酚氧化酶(PPO)活性嫁接初期较高,以后变化平稳;大苗龄接穗嫁接体的PPO活性较高.嫁接初期过氧化物酶(POD)和苯丙氨酸解氨酶(PAL)活性升高,以后呈下降趋势,木质素合成和维管组织分化阶段再度升高.     

Incorporation of p-cresol into lignins via peroxidase-catalysed copolymerization in nonaqueous media

Lignin, the second most abundant biopolymer after cellulose, is a low value by-product of agricultural and wood conversion processes, including wood pulp manufacture. Copolymerization with phenols has the potential to convert by-product lignins to higher-value phenolic resins. In this initial investigation, we have studied the use of horseradish peroxidase (HRP) in aqueous dioxane to catalyse the grafting of p-cresol (p-methylphenol) onto milled wood lignin, kraft lignin, and a lignin selectively o-demethylated by a brown-rot fungus. Advantages of this system are (1) the mild reaction conditions employed and (2) the ability of HRP to function in the dioxane: water solutions which solubilize lignin. The reaction is monitored by gel permeation chromatography using a reaction system of [¹⁴C]-p-cresol with unlabeled lignins. We have found that optimal incorporation of cresol into high-molecular-weight polymer occurs at 50–70% dioxane in water under the conditions used; a maximum incorporation of ca. 4 mol% (e.g., p-cresol incorporated per C₉ lignin unit) was obtained. Blocking the phenolic hydroxyl groups of the lignin inhibits copolymerization, consistent with the proposed mechanism of phenoxy radical copolymerization for this reaction.     

Laccase-induced HBT-grafting to milled beech wood reduces unspecific protein adsorption

Lignocellulosic biomass is a ubiquitous and renewable feedstock for the production of platform chemicals and biofuels. Typically, this recalcitrant biomass is pretreated by physico-chemical techniques causing disintegration and delignification. An additional treatment with laccase-mediator-systems (LMS) has been found to further improve the subsequent enzymatic cellulose hydrolysis. The aim of this study was to investigate the impact of different LMS on the glucose yield of a subsequent hydrolysis of treated beech wood and to elucidate the underlying effect of LMS treatment. The mediators 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 1-hydroxybenzotriazol (HBT) and syringaldehyde were evaluated, but an enhancing effect of LMS treatment on beech wood hydrolysis was only found for HBT. In mass spectrometry analysis of the acid hydrolysate of LMS-treated samples, the mediator HBT could be found in the lignin samples, suggesting a grafting reaction. The fluorescent protein mCherry was used as a reporter for unspecific protein adsorption to biomass samples. LMS treatment with HBT reduced the unspecific adsorption of mCherry to raw beech wood by about 50%, suggesting that the HBT grafting to beech wood lignin decreased the unproductive cellulase binding. In summary, the reduction of unspecific protein adsorption by biomass surface modification with laccase-HBT treatment is proposed to be the underlying mechanism for increased cellulose conversion.     

Study of polyacrylamide grafted starch based algal flocculation towards applications in algal biomass harvesting

Microalgae may be the source of high amount of lipid and protein. It has the property for carbon dioxide sequestration, recycling and also can remove pollutants from wastewater. Using traditional methods, collection of algal biomass is either cost effective, time consuming or may be toxic due to use of chemical salts. The aim of this study is to harvest freshwater microalgae (Chlorella sp. CB4) biomass by using polymer. Polyacrylamide grafted starch (St-g-PAM) has been synthesized by microwave assisted method involving a synergism of microwave radiation and ceric ammonium nitrate (CAN) to initiate the grafting reaction. The synthesis was optimized in terms of CAN and monomer (acrylamide) concentration. The algal flocculation efficacy of all the grades of this graft copolymer was studied through standard 'Jar test' procedure. Effects of percentage grafting, pH and zeta potential on percentage recovery of algal biomass were thoroughly investigated.     

Evaluating laccase-facilitated coupling of phenolic acids to high-yield kraft pulps

In an effort to alter the physical properties of high-yield kraft, fibers were treated at high consistency (20%) with laccase and syringic, vanillic, or 4-hydroxybenzoic acid. Treatment with laccase and 4-hydroxybenzoic acid resulted in a 20-point increase in kappa number and a 100% increase in bulk acid groups. ESCA analysis of the treated and untreated pulp revealed that the laccase-grafted fibers had a two-fold enrichment in acid groups, strongly suggesting a laccase-facilitated coupling of 4-hydroxybenzoic acid to the fiber surface. A model system consisting of lignin-coated cellulosic fibers was developed to determine changes to the lignin structure during laccase grafting. ³¹P NMR analysis of lignin from the model system revealed an increase in acid groups with a concomitant decrease in phenolic hydroxyl groups.     

Modification of lignin for the production of new compounded materials

The cell walls of woody plants are compounded materials made by in situ polymerization of a polyphenolic matrix (lignin) into a web of fibers (cellulose), a process that is catalysed by polyphenoloxidases (laccases) or peroxidases. The first attempt to transform the basic strategy of this natural process for use in human craftsmanship was the ancient lacquer method. The sap of the lacquer tree (Rhus verniciflua) contains large amounts of a phenol (urushiol), a polysaccharide and the enzyme laccase. This oil-in-water emulsion solidifies in the presence of oxygen. The Chinese began using this phenomenon for the production of highly creative artwork more than 6,000 years ago. It was the first example of an isolated enzyme being used as a catalyst to create an artificial plastic compound. In order to apply this process to the production of products on an industrial scale, an inexpensive phenol must be used, which is transferred by an enzyme to active radicals that react with different components to form a compounded material. At present, the following approaches have been studied: (1) In situ polymerization of lignin for the production of particle boards. Adhesive cure is based on the oxidative polymerization of lignin using phenoloxidases (laccase) as radical donors. This lignin-based bio-adhesive can be applied under conventional pressing conditions. The resulting particle boards meet German performance standards. By this process, 80% of the petrochemical binders in the wood-composite industry can be replaced by materials from renewable resources. (2) Enzymatic copolymerization of lignin and alkenes. In the presence of organic hydroperoxides, laccase catalyses the reaction between lignin and olefins. Detailed studies on the reaction between lignin and acrylate monomers showed that chemo-enzymatic copolymerization offers the possibility to produce defined lignin-acrylate copolymers. The system allows control of the molecular weights of the products in a way that has not been possible with chemical catalysts. This is a novel attempt to enzymatically induce grafting of polymeric side chains onto the lignin backbone, and it enables the utilization of lignin as part of new engineering materials. (3) Enzymatic activation of the middle-lamella lignin of wood fibers for the production of wood composites. The incubation of wood fibers with a phenol oxidizing enzyme results in oxidative activation of the lignin crust on the fiber surface. When such fibers are pressed together, boards are obtained which meet the German standards for medium-density fiber boards (MDF). The fibers are bound together in a way that comes close to that by which wood fibers are bound together in naturally grown wood. This process will, for the first time, yield wood composites that are produced solely from naturally grown products without any addition of resins.     

Versatile grafting of polysaccharides in homogeneous mild conditions by using atom transfer radical polymerization

A versatile atom transfer radical polymerization (ATRP) method for polysaccharide grafting in homogeneous mild conditions without using protecting group chemistry is presented. Water/DMF mixtures with different compositions were used as the solvent. The "grafting-from" approach was used in order to prepare suitable pullulan and dextran ATRP macroinitiators with a well controlled degree of functionalization. Methacrylate and acrylamide monomers were grafted obtaining good control over the number, molecular weight and polydispersity of the grafted chains without homopolymer formation and polysaccharide degradation. The versatility of this method allowed us to prepare comblike derivatives with a wide range of properties (amphiphilic, ionic, and thermoresponsive) by simply changing the solvent composition and the catalyst. This could make possible the synthesis of new interesting biomaterials starting from a wide range of polysaccharides.