Chemo-enzymatic synthesis and characterization of graft copolymers from lignin and acrylic compounds

Chemo-enzymatic initiation of graft copolymerization of acrylic compounds onto different technical lignosulfonates (LS) was compared to a Fenton-like system (ferrous ion, t-BHP). The enzyme tested was a phenoloxidase laccase (EC 1.10.3.2) from the white rot basidomycete Trametes versicolor. Most applied lignins were successfully grafted, resulting in a polymer yield of more than 90%. The effect of initiator concentration and the lignin/monomer ratio on the yield and M(w) of enzymatically grafted polymers were studied. The homopolymer proportion in the enzymatically produced grafts of Ca-LS and acrylic acid was 5 to 6x lower than those initiated by the Fenton-like reagent; no such differences were observed for Na-LS.     

The effect of ions on the enzymatically induced synthesis of lignin graft copolymers

The effect of different ions which are constituents of technical lignin sulfonates (LS) on chemo-enzymatic graft co-polymerization was determined. The application of the iron chelator desferrioxamine in the initial reaction mixture revealed that iron impurities of LS which catalyzed a Fenton-like reaction were crucial for the initiation of grafting, whereas calcium or chloride ions showed no such effect. The addition of laccase (ATCC 11235) to the reaction mixture which contained desferrioxamine caused a significantly higher yield compared to the control; this indicates a crucial effect of laccase with regard to the initiation of copolymerization. The involvement of laccase in the initiation of the graft copolymerization was additionally confirmed by the application of low molecular weight phenolics instead of LS. In the presence of the lignin-like substrates, 3,4-dihydroxybenzoic acid and guaiacol, the rate of the decomposition of t-butylhydroperoxide was significantly enhanced by laccase. It can be assumed that the enzymatically generated phenoxy radicals mediate the production of oxygen centered radicals (alkoxy or peroxy) which initiate grafting.     

Laccase-mediated detoxification of phenolic compounds.

The ability of a polyphenoloxidase, the laccase of the fungus Rhizoctonia praticola, to detoxify phenolic pollutants was examined. The growth of the fungus could be inhibited by phenolic compounds, and the effective concentration was dependent on the substituents of the phenol. A toxic amount of a phenolic compound was added to a fungal growth medium in the presence or absence of a naturally occurring phenol, and half of the replicates also received laccase. The medium was then inoculated with R. praticola, and the levels of phenols in the medium were monitored by high-performance liquid chromatography analysis. The addition of the laccase reversed the inhibitory effect of 2,6-xylenol, 4-chloro-2-methylphenol, and p-cresol. Other compounds, e.g., o-cresol and 2,4-dichlorophenol, were detoxified only when laccase was used in conjunction with a natural phenol such as syringic acid. The toxicity of p-chlorophenol and 2,4,5-trichlorophenol could not be overcome by any additions. The ability of the laccase to alter the toxicity of the phenols appeared to be related to the capacity of the enzyme to decrease the levels of the parent compound by transformation or cross-coupling with another phenol.     

Laccase-mediated detoxification of phenolic compounds

The ability of a polyphenoloxidase, the laccase of the fungus Rhizoctonia praticola, to detoxify phenolic pollutants was examined. The growth of the fungus could be inhibited by phenolic compounds, and the effective concentration was dependent on the substituents of the phenol. A toxic amount of a phenolic compound was added to a fungal growth medium in the presence or absence of a naturally occurring phenol, and half of the replicates also received laccase. The medium was then inoculated with R. praticola, and the levels of phenols in the medium were monitored by high-performance liquid chromatography analysis. The addition of the laccase reversed the inhibitory effect of 2,6-xylenol, 4-chloro-2-methylphenol, and p-cresol. Other compounds, e.g., o-cresol and 2,4-dichlorophenol, were detoxified only when laccase was used in conjunction with a natural phenol such as syringic acid. The toxicity of p-chlorophenol and 2,4,5-trichlorophenol could not be overcome by any additions. The ability of the laccase to alter the toxicity of the phenols appeared to be related to the capacity of the enzyme to decrease the levels of the parent compound by transformation or cross-coupling with another phenol.     

Enhanced removal of three phenols by laccase polymerization with MF/UF membranes

Guaiacol, catechol, m-cresol are common phenolic compounds presented in various industrial effluents but difficult to be removed by conventional wastewater treatment schemes. To elucidate mechanisms of enhanced membrane removal by laccase polymerization, different MF and UF membranes were employed in a cross-flow module for phenol concentration of 5mM. With 2.98 IU/l of laccase applied at room temperature, guaiacol, catechol and m-cresol were polymerized to products of averaged molecular weight of 9600, 8350 and 5400 Da (Dalton), respectively. Methoxy and hydroxyl-substituted phenols (guaiacol and catechol) were polymerized better than methyl-substituted phenol (m-cresol) due to more stable free-radical containing intermediate structure induced by oxygen-containing methoxy and hydroxyl functional groups. Removal efficiencies for the un-reacted phenols were dependent on the molecular sizes (length and width), but were dependent on the molecular weight for the polymerized phenolic compounds. Flux was declined initially but reached steady state after 180 min of filtration, indicating these MF/UF membranes can be used for removal of these polymerized phenols without significant fouling. In addition, pretreatments by the inactivated laccase only caused further flux reduction without additional removal of phenols.     

Immobilization of laccase from Streptomyces psammoticus and its application in phenol removal using packed bed reactor

Laccase was produced from Streptomyces psammoticus under solid-state fermentation. The enzyme was partially purified by ammonium sulphate precipitation and was immobilized in alginate beads by entrapment method. Calcium alginate beads retained 42.5% laccase activity, while copper alginate beads proved a better support for laccase immobilization by retaining 61% of the activity. Phenol and colour removal from a phenol model solution was carried out using immobilized laccase. Batch experiments were performed using packed bed bioreactor, containing immobilized beads. Reusability of the immobilized matrix was studied for up to 8 successive runs, each run with duration of 6 h. The system removed 72% of the colour and 69.9% of total phenolics from the phenol model solution after the initial run. The immobilized system maintained 50% of its efficiency after eight successive runs. The degradation of phenolic compounds by immobilized laccase was evaluated and confirmed by Thin layer chromatography and nuclear magnetic resonance spectroscopy.     

Phenolic removal in olive oil mill wastewater by strains of Pleurotus spp. in respect to their phenol oxidase (laccase) activity

The ability of several Pleurotus spp. strains to remove phenolic compounds from an olive oil mill wastewater (OMW) was studied. All strains tested in this work were able to grow in OMW without any addition of nutrients and any pre-treatment, except sterilization. High laccase activity was measured in the growth medium, while 69-76% of the initial phenolic compounds were removed. The black color of OMW became yellow-brown and brighter as the strains grew. The lowest phenolic concentrations were reached after 12/15 days. A decrease of the phytotoxicity, as described by the parameter Germination Index, was noticed in the OMW treated with some Pleurotus spp strains, although this decrease was not proportional to the phenolic removal. A new parameter, namely Phenol-toxicity Index, was considered in the present paper. Using this parameter it was found that the remaining phenolics and/or some of the oxidation products of the laccase reaction in the treated OMW were more toxic than the original phenolic compounds.     

Enhanced stability of laccase in the presence of phenolic compounds

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. Received: 25 April 2000 / Received revision: 16 June 2000 / Accepted: 18 June 2000     

Enzymatic modification of kraft lignin through oxidative coupling with water-soluble phenols

The aromatic polymer lignin can be modified through promotion of oxidative coupling between phenolic groups on lignin and various phenols. The reaction is initiated by an oxidation of both components, e.g., by using the oxidoreductases laccase or peroxidase. Coupling between phenolic monomers and lignin has previously been studied by the use of radio-labeled phenols. In this study, incorporation of water-soluble phenols into kraft lignin, using laccase as catalyst, was investigated. Several phenols with carboxylic or sulfonic acid groups were used as markers for the incorporation. The modified lignin was isolated and the amount of phenol incorporated was characterized by means of titration, quantitative 1H-NMR, and quantitative 31P-NMR after modification with 2-chloro-4,4,5,5-tetramethyl-1,2,3-dioxaphospholane. Only a few of the phenols studied were found to be incorporated into lignin. When the phenol guaiacol sulfonate was incorporated into kraft lignin, the lignin became water-soluble at pH 2.4 and a low ionic strength due to the introduction of sulfonic acid groups. The content of sulfonic acid groups in the product was 0.5-0.6 mmol/g lignin. A lower amount of 4-hydroxyphenylacetic acid was incorporated under similar conditions.     

First evidence of catalytic mediation by phenolic compounds in the laccase-induced oxidation of lignin models.

The sulfonephthalein indicator, phenol red, exhibits an unusually slow rate of oxidation by laccase from Poliporus pinsitus, in spite of the fact that it is a phenol and therefore a natural substrate for this phenoloxidase enzyme. Nevertheless, after prolonged exposure to laccase (24 h) phenol red is oxidized by more than 90%. We found that phenol red, which can be oxidatively converted into a resonance-stabilized phenoxy radical, performs as a mediator in the laccase-catalyzed oxidation of a nonphenolic substrate (4-methoxybenzyl alcohol) and also of a hindered phenol (2,4,6-tri-tert-butylphenol). In particular, phenol red was found to be at least 10 times more efficient than 3-hydroxyanthranilate (a reported natural phenolic mediator of laccase) in the oxidation of 4-methoxybenzyl alcohol. Other phenols, which do not bear structural analogies to phenol red, underwent rapid degradation and did not perform as laccase mediators. On the other hand, several variously substituted sulfonephthaleins, of different pK2 values, mediated the laccase catalysis, the most efficient being dichlorophenol red, which has the lowest pK2 of the series. The mediating efficiency of phenol red and dichlorophenol red was found to be pH dependent, as was their oxidation Ep value (determined by cyclic voltammetry). We argue that the relative abundance of the phenoxy anion, which is easier to oxidize than the protonated phenol, may be one of the factors determining the efficiency of a phenolic mediator, together with its ability to form relatively stable oxidized intermediates that react with the desired substrate before being depleted in undesired routes.     

Characterization of alkaliphilic laccase activity in the culture supernatant of Myrothecium verrucaria 24G-4 in comparison with bilirubin oxidase

An enzyme showing alkaliphilic laccase activity was purified from the culture supernatant of Myrothecium verrucaria 24G-4. The enzyme was highly stable under alkaline conditions, showed an optimum reaction pH of 9.0 for 4-aminoantipyrine/phenol coupling, and decolorized synthetic dyes under alkaline conditions. It showed structural and catalytic similarities with bilirubin oxidase, but preferably oxidized phenolic compounds. The enzyme catalyzed veratryl alcohol oxidation at pH 9.0 with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as a mediator, suggesting that the laccase mediator system functioned well under alkaline conditions.     

Laccase induction in fungi and laccase/N-OH mediator systems applied in paper mill effluent

There has been increasing interest in extracellular enzymes from white rot fungi, such as lignin and manganese peroxidases, and laccases, due to their potential to degrade both highly toxic phenolic compounds and lignin. The optimum cultivation conditions for laccase production in semi-solid and liquid medium by Trametes versicolor, Trametes villosa, Lentinula edodes and Botrytis cinerea and the effects of laccase mediator system in E1 effluent were studied. The higher laccase activity (12756 U) was obtained in a liquid culture of T. versicolor in the presence of 1 mM of 2,5-xylidine and 0.4 mM copper salt as inducers. The effluent biotreatments were not efficient in decolorization with any fungal laccases studied. Maximum phenol reduction was approximately 23% in the absence of mediators from T. versicolor. The presence of 1-hydroxybenzotriazole did not increase phenol reduction. However, acetohydroxamic acid, which was not degraded by laccase, acted very efficiently on E1 effluent, reducing 70% and 73% of the total phenol and total organic carbon, respectively. Therefore, acetohydroxamic acid could be applied as a mediator for laccase bioremediation in E1 effluent.     

Enzymatic and spectroscopic studies on the activation or inhibition effects by substituted phenolic compounds in the oxidation of aryldiamines and catechols catalyzed by Rhus vernicifera laccase

The effect of various phenolic compounds on the activity of Rhus vernicifera laccase (Lc) has been evaluated using two different substrates, N,N-dimethyl-p-phenylenediamine and p-tert-butylcatechol. The observed effect strongly depends on the phenol employed and involves either a moderate activation, by halophenols, or inhibition, by acidic phenols. The collective data are consistent with an open active site in Lc, which is capable of accommodating more than one substrate or phenol molecule. According to NMR relaxation experiments, a phenol molecule binds at an average distance from type 1 Cu of about 6 Å, while evidence from electron paramagnetic resonance (EPR) experiments shows that binding of another phenol molecule induces a change, and probably occurs close to, the type 2/type 3 cluster. The effect of phenolic compounds on Lc reactivity is related to a modification of the substrate affinity for the enzyme. This affinity can either be increased, probably through π-stacking or other types of interactions, or decreased, due to competition for the same site. In addition, the alteration induced in the trinuclear copper cluster has a marked effect on the enzyme reactivity. The inhibition observed with acidic phenols is probably due to the protonation of an enzyme intermediate produced at the trinuclear site, e.g. the peroxy intermediate, that causes the release of hydrogen peroxide and prevents the reaction of this intermediate with the substrate.     

Improving the bioremediation of phenolic wastewaters by Trametes versicolor

The successful bioremediation of a phenolic wastewater by Trametes versicolor was found to be dependent on a range of factors including: fungal growth, culture age and activity and enzyme (laccase) production. These aspects were enhanced by the optimisation of the growth medium used and time of addition of the pollutant to the fungal cultures. Different media containing 'high' (20 g/L), 'low' (2 g/L) and 'sufficient' (10 g/L) concentrations of carbon and nitrogen sources were investigated. The medium containing both glucose and peptone at 10 g/L resulted in the highest Growth Related Productivity (the product of specific yield and micro) of laccase (1.46 Units of laccase activity)/gram biomass/day and was used in all further experiments. The use of the guaiacol as an inducer further increased laccase activity 780% without inhibiting growth; similarly the phenolic effluent studied boosted activity almost 5 times. The timing of the addition of the phenolic effluent was found to have important consequences in its removal and at least 8 days of prior growth was required. Under these conditions, 0.125 g phenol/g biomass and 0.231 g o-cresol/g biomass were removed from solution per day.     

Enzymatic synthesis of Tinuvin

Coupling of 3-(3-tert-butyl-4-hydroxyphenyl) propionic acid methylester to 1H-benzotriazole using a laccase from Trametes hirsuta was studied. The potentially resulting coupling product Tinuvin 1130 is an important UV-absorber used in polymer based materials. Oxidation of the phenol by the laccase led to homomolecular coupling reactions while the laccase did not attack 1H-benzotriazole. Due to the homomolecular reaction of the phenol in the presence of laccase coupling of phenol and 1H-benzotriazole was only observed when 1H-benzotriazole was applied in four-fold molar excess. The reaction was monitored by UV/vis spectroscopy, TLC and MS (ion trap) analysis. Coupling of 1H-benzotriazole took place in ortho position according to the postulated mechanism.     

Phenols removal in musts: Strategy for wine stabilization by laccase

The potential of laccase from Trametes versicolor for phenolic removal in must for wine stabilization was evaluated through a combination of an analytical methodology (capillary zone electrophoresis) and kinetics of phenols removal as the total antioxidant potential variation. Total phenolic content, total antioxidant potential and polyphenols were monitored from 0 to 3 h of must treatment. The results indicated that the treatment of a red must with laccase affect mainly the phenolic compounds responsible for the must antioxidant properties. The treatment of white musts with laccase showed higher reduction in total phenol than in the total antioxidant potential. Phenol degradation by laccase was very fast for catechins, and slowly for stilbenes (cis- and trans-resveratrol) and derivatives of cinnamic (ferulic and caffeic) and benzoic (syringic, vanillic, and gallic) acids. It is possible to conclude in this case that the use of laccase in white wines is perfectly feasible. This would allow softer and ecologically correct treatments, which would diminish the cost of processing and avoid deterioration of wines for long storage times.     

A critical analysis of techniques for measuring tannins in ecological studies

Summary A series of seventeen plant extracts rich in phenolic materials, including condensed and hydrolysable tannins, have been subjected to a series of chemical analyses in an attempt to gather ecologically significant information about their structure. Procedures investigated were (i) the Folin-Denis and Hagerman and Butler methods for quantifying total phenolics, (ii) the vanillin and proanthocyanidin methods for quantifying condensed tannins, (iii) the iodate and nitrous acid methods for hydrolysable tannins. It was found that the techniques for total phenolics correlated well, the Hagerman and Butler method giving higher estimates where solutions were particularly phenol rich. By contrast there was considerable discrepancy between the methods examined for condensed tannins. This is probably due primarily to the very different chemical reactions that form the basis of these procedures and also to the fact that the extract dependent products of the proanthocyanidin method vary in their A ₁ ¹ values. During the study of condensed tannins methods for estimating their contribution to total phenolics and for measuring their average polymer length were examined. In both cases different procedures produced very variable results. Available methods for hydrolysable tannins were found not to be generally applicable across all extracts thought to contain this type of tannin on the basis of chromatographic analysis. An attempt to produce a quantitative spectrophotometric assay for hydrolysable tannins based on changes in reactivity to ferric chloride due to hydrolysis is described. This proved to be of limited sensitivity but may have some merit for estimating levels in hydrolysable tannins in phenol-rich plant extracts that also contain condensed tannins. It is concluded that whilst the overall level of phenolics in extracts can be estimated with some confidence the information imparted by more specific assays is very dependent on the procedures employed, particularly when dealing with extracts from taxonomically highly diverse sources.     

Phenolic compounds protect HepG2 cells from oxidative damage: relevance of glutathione levels

In the present work, the potential hepatoprotective effects of five phenolic compounds against oxidative damages induced by tert-butyl hydroperoxide (t-BHP) were evaluated in HepG2 cells in order to relate in vitro antioxidant activity with cytoprotective effects. t-BHP induced considerable cell damage in HepG2 cells as shown by significant LDH leakage, increased lipid peroxidation, DNA damage as well as decreased levels of reduced glutathione (GSH). All tested phenolic compounds significantly decreased cell death induced by t-BHP (when in co-incubation). If the effects of quercetin are given the reference value 1, the compounds rank in the following order according to inhibition of cell death: luteolin (4.0) > quercetin (1.0) > rosmarinic acid (0.34) > luteolin-7-glucoside (0.30) > caffeic acid (0.21). The results underscore the importance of the compound's lipophilicity in addition to its antioxidant potential for its biological activity. All tested phenolic compounds were found to significantly decrease lipid peroxidation and prevent GSH depletion induced by t-BHP, but only luteolin and quercetin significantly decreased DNA damage. Therefore, the lipophilicity of the natural antioxidants tested appeared to be of even greater importance for DNA protection than for cell survival. The protective potential against cell death was probably achieved mainly by preventing intracellular GSH depletion. The phenolic compounds studied here showed protective potential against oxidative damage induced in HepG2 cells. This could be beneficial against liver diseases where it is known that oxidative stress plays a crucial role.     

Enzymatic and spectroscopic studies on the activation or inhibition effects by substituted phenolic compounds in the oxidation of aryldiamines and catechols catalyzed by Rhus vernicifera laccase

The effect of various phenolic compounds on the activity of Rhus vernicifera laccase (Lc) has been evaluated using two different substrates, N,N-dimethyl-p-phenylenediamine and p-tert-butylcatechol. The observed effect strongly depends on the phenol employed and involves either a moderate activation, by halophenols, or inhibition, by acidic phenols. The collective data are consistent with an open active site in Lc, which is capable of accommodating more than one substrate or phenol molecule. According to NMR relaxation experiments, a phenol molecule binds at an average distance from type 1 Cu of about 6 Å, while evidence from electron paramagnetic resonance (EPR) experiments shows that binding of another phenol molecule induces a change, and probably occurs close to, the type 2/type 3 cluster. The effect of phenolic compounds on Lc reactivity is related to a modification of the substrate affinity for the enzyme. This affinity can either be increased, probably through π-stacking or other types of interactions, or decreased, due to competition for the same site. In addition, the alteration induced in the trinuclear copper cluster has a marked effect on the enzyme reactivity. The inhibition observed with acidic phenols is probably due to the protonation of an enzyme intermediate produced at the trinuclear site, e.g. the peroxy intermediate, that causes the release of hydrogen peroxide and prevents the reaction of this intermediate with the substrate.     

Immobilization of pycnoporus coccineus laccase on Eupergit C: Stabilization and treatment of olive oil mill wastewaters

The use of olive oil mill wastewaters (OMW) as an organic fertilizer is limited by their phytotoxic effect, due to the high concentration of phenolic compounds. As an alternative to physico-chemical methods for OMW detoxification, the laccase from Pycnoporus coccineus, a white-rot fungus with the ability to decrease the chemical oxygen demand (COD) and color of the industrial effluent, is being studied. In this work, the P. coccineus laccase was immobilized on two acrylic epoxy-activated resins, Eupergit C and Eupergit C 250L. The highest activity was obtained with the macroporous Eupergit C 250L, reaching 110 U g^?1 biocatalyst. A substantial stabilization effect against pH and temperature was obtained upon immobilization. The soluble enzyme maintained ≥80% of its initial activity after 24 h at pH 7.0–10.0, whereas the immobilized laccase kept the activity in the pH range 3.0–10.0. The free enzyme was quickly inactivated at temperatures >50°C, whereas the immobilized enzyme was very stable up to 70°C. Gel filtration profiles of the OMW treated with the immobilized enzyme (for 8 h at room temperature) showed both degradation and polymerization of the phenolic compounds.