O. Milstein

Chemoenzymatical grafting of acrylamide onto lignin

Laccase (E.C.1.10.3.2.) from white-rot basidiomycete Trametes versicolor and dioxane peroxides were essential in the copolymerization of acrylamide and a derivative with lignin in a dioxane-H(2)O (7:3) mixture. Both a solubility test and an elemental analysis of the eluted and separated fractions provided evidence of grafting. Gel permeation chromatography on Sephadex G-100 showed that the side chain and the lignin backbone migrate as one unit through the column. Copolymers obtained were characterized by UV-VIS-spectroscopy as well as FT-IR, and 13C-CPMAS-NMR spectroscopy. Freeze-dried copolymers of lignin and acrylamide appeared as homogeneous fibril-like particulate. The mechanism of the enzymatical grafting is discussed.

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.

Oxidation of aromatic compounds in organic solvents with laccase from Trametes versicolor

SummaryLaccase purified from Trametes versicolor oxidizes 2,6-dimethoxyphenol (2,6-DMP) and syringaldazine in hydrophobic solvents presaturated with water, and in hydrophilic organic solvents provided that a sufficient amount of water is added. Ease of performance of the laccase test in organic solvents is improved after immobilization of the enzyme by entrapping in Sepharose CL-6B during enzyme filtration through the gel beads. The gel-enzyme association has been shown to be stable in water-presaturated solvents. Efficiency of the immobilized laccase in organic solvents containing 7% water was 10%–20% of that in potassium-citrate buffer. Immobilized laccase in organic solvents showed good stability and high tolerance to elevated temperatures.

Fungal laccase grafts acrylamide onto lignin in presence of peroxides

Abstract 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.

Transformation of lignin-related compounds with laccase in organic solvents

Abstract The extracellular laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) of Trametes versicolor was isolated from culture medium and immobilized by entrapment of the enzyme in a solvent-resistant hydrophilic matrix like Sepharose-CL-6B. The gel-enzyme association has been shown to be stable in water containing organic solvents. The efficiency of the immobilized laccase in different organic solvents was comparable with the activity shown in a buffered aqueous system. The immobilized laccase in organic solvents showed a good stability and a high tolerance to elevated temperatures. Water-insoluble organosolv lignin (OL), dissolved in dioxane/water, was readily converted by immobilized laccase from Trametes versicolor . The transformed lignin showed an increase in phenolic groups, changes in the quantity of conjugated elements, and a significant modification of both the aliphatic and aromatic carbon moieties of the lignin molecule. The changes in the lignin molecule were analyzed by UV-, IR-spectroscopy, and 13 C-NMR solid-state spectroscopy. High-performance size exclusion chromatography (HPSEC) of the lignin transformed with the laccase—Sepharose complex revealed a pronounced increase in weight-average molecular weight. Polymerization of the lignin in the organic solvent proved to be 4 times more effective than polymerization of the same compound in an aqueous system. Water-insoluble organosolv lignin as well as a variety of lignin-related aromatics, solubilized in dioxane-H 2 O (7 : 3), was readily converted by laccase preparation either in batch or in continuous flow column. Reaction of laccase with the solubilized lignin generates in the reaction media reduced oxygen species able to reduce the cytochrome c. For the first time it is now possible to perform enzymatically catalyzed reactions with lignin in an organic solvent. This is a first step towards an enzymatically derivatization of lignin, the formation of polymer blends on the basis of lignin by an enzymatically catalyzed reaction.

Enhanced stability of laccase in the presence of phenolic compounds

Abstract The storage stability of laccase (EC 1.10.3.2) from the white-rot basidomycete Trametes versicolor in potassium-citrate buffer was enhanced by various phenolic compounds as well as by lignin sulfonate. The highest storage stability was obtained with phenolics, e.g. phloroglucin and 3,5-dihydroxybenzoic acid; these represent substrates of laccase which are oxidized slowly because of their relatively high redox potential and which did not precipitate from the solution within the tested period of time. Sterilization enhanced the stability of laccase but additional stabilization by phenolics was observed both under sterile and non-sterile conditions. We thus concluded that stabilization occurred not only through prevention of microbial degradation.

Infrared and nuclear magnetic resonance evidence of degradation in thermoplastics based on forest products

The degradation of lignin-(1-phenylethylene) graft copolymers (lignin-styrene graft copolymers) by white rot basidiomycete fungi was followed by monitoring aromatic absorption bands by Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. The FTIR of the graft copolymers shows a series of characteristic absorbance peaks from multi-substituted aromatic rings and a strong poly(1-phenylethylene) (polystyrene) absorbance peak from monosubstituted aromatic rings. Subtraction of copolymer spectra taken before incubation from spectra taken after 50 days of incubation with the four tested fungi shows the loss of functional groups from the copolymer. NMR spectra also show reduction of aromatic ring resonances from the copolymer and incorporation of peaks from fungi as a result of incubation with fungi. The biodegradation tests were run on lignin-(1-phenylethylene) graft copolymers which contained 10.3, 32.2, and 50.4% of lignin. The polymer samples were incubated with the white rot fungiPleurotus ostreatus, Phanerochaete chrysosporium, andTrametes versicolor, and the brown rot fungusGleophyllum trabeum. White rot fungi degraded the plastic samples at a rate that increased with increasing lignin content in the copolymer sample. Both poly(1-phenylethylene) and lignin components of the copolymer were readily degraded. Observation by scanning electron microscopy of incubated copolymers showed a deterioration of the plastic surface. The brown rot fungus did not affect any of these plastics, nor did any of the fungi degrade pure poly(1-phenylethylene).

Fungal biodegradation of lignin graft copolymers from ethene monomers

Abstract White rot Basidiomycetes were able to biodegrade styrene (1-phenylethene) or methyl methacrylate (4-methyl-2-oxy-3-oxopent-4-ene) graft copolymers of lignin containing different proportions of lignin and polystyrene [poly(1-phenylethylene)] or polymethyl methacrylate [poly(1-methyl-1-(1-oxo-2-oxypropyl)ethylene)]. The biodegradation tests were run on lignin/styrene copolymerization products which contained 10.3, 32.2, and 50.4 wt% lignin while biodegradation tests were run on lignin/methyl methacrylate copolymerization products which contained 11 to 18 wt% lignin. The styrene polymer samples were incubated with white rot Pleurotus ostreatus, Phanerochaete chrysosporium, Trametes versicolor, and brown rot Gloeophyllum trabeum. The methyl methacrylate polymer samples were incubated with white rot Pleurotus ostreatus, Trametes versicolor, and Phlebia radiata. White rot fungi degraded the plastic samples at a rate which increased with increasing lignin content in the copolymer sample. Both polystyren...

Methods for the Evaluation of Lignin Properties Suitable for Conversion

Lignin, a natural polymer composed of C6-C3-phenolics, in theory at least, seems to be a very attractive feedstock with many potential uses for the polymer industry. However file market potential of lignin is greatly limited at present because lignins derived from the pulp and paper industry have rather unattractive properties, notably i) a low content of hydroxyl groups, ii) unsuitable molecular weight, which is either too high (lignosulphonate and Kraft lignin) or too low (organosolv lignin) and iii) the wrong polarity, being again either too high (lignosulphonate) or too low (Kraft and organosolv lignin).