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.     

A high sensitivity amperometric biosensor using a monomolecular layer of laccase as biorecognition element

Laccases from various sources were tested, and laccase from Rigidoporus lignosus was found to be the most active towards syringaldazine and ABTS, which are typical substrates of this class of enzymes, and towards the phenols found in olive oil mill wastewaters. This laccase was covalently immobilised by carbodiimide chemistry, on a self-assembled monolayer of 3-mercaptopropionic acid deposited on a gold surface. A flow biosensor, using the monolayer of laccase as bioelement and a glassy carbon electrode as amperometric transduction system, was developed. Although the amount of the immobilised enzyme (about 140 ng/cm2 effective surface area) was tiny, the biosensor showed a sensitivity of 3 nA/microM when 1,4-hydroquinone was used as substrate, and a half-life of 35 days. The proposed device permits detection of phenols in aqueous solutions at concentrations in the low micromolar range, i.e. below European Community limits. The biosensor was successfully used to detect phenols in wastewaters from an olive oil mill after minimal sample preparation (incubation of the aqueous sample with sodium borohydride for a few minutes) to suppress the current due to oxidised compounds present in the wastewaters.     

Synthesis and properties of new photoactivable derivatives of tetrodotoxin

Eight lots of reagent-grade phenol from four companies were tested for capacity to interact with Cu2+ to produce an inactivator or inactivators of the transfective RNA obtained from poliovirions; such capacity to interact with Cu2+ is referred to as cofactor activity. Six of the lots showed cofactor activity; two did not. A review of the data on the phenol lots and of the properties of the impurity or impurities conferring cofactor activity suggested that the active impurity(ies) might be a dihydric or trihydric phenol. Commercial catechol, resorcinol, hydroquinone, orcinol and pyrogallol were tested and found active. The activity of hydroquinone was outstandingly high. Upon serial recrystallization, the activity of catechol, hydroquinone, orcinol and pyrogallol remained constant, but the activity of resorcinol decreased markedly, in stepwise fashion, showing the most of the activity of the commercial resorcinol was due to impurity(ies). Each of catechol, hydroquinone, orcinol, pyrogallol, and the commercial resorcinol was shown to react with Cu2+ to produce inactivator(s). The effective target for inactivator(s) was the RNA and not the transfection process. The kinetics of inactivator(s) production varied for the different phenols, and the inactivator activity of the incubated mixture of pyrogallol and Cu2+ was notably labile.     

Purification,characterization, and identification of a novel bifunctional catalase-phenol oxidase from <Emphasis Type="Italic">Scytalidium thermophilum</Emphasis>

A novel bifunctional catalase with an additional phenol oxidase activity was isolated from a thermophilic fungus, Scytalidium thermophilum. This extracellular enzyme was purified ca. 10-fold with 46% yield and was biochemically characterized. The enzyme contains heme and has a molecular weight of 320 kDa with four 80 kDa subunits and an isoelectric point of 5.0. Catalase and phenol oxidase activities were most stable at pH 7.0. The activation energies of catalase and phenol oxidase activities of the enzyme were found to be 2.7 +/- 0.2 and 10.1 +/- 0.4 kcal/mol, respectively. The pure enzyme can oxidize o-diphenols such as catechol, caffeic acid, and L-DOPA in the absence of hydrogen peroxide and the highest oxidase activity is observed against catechol. No activity is detected against tyrosine and common laccase substrates such as ABTS and syringaldazine with the exception of weak activity with p-hydroquinone. Common catechol oxidase inhibitors, salicylhydroxamic acid and p-coumaric acid, inhibit the oxidase activity. Catechol oxidation activity was also detected in three other catalases tested, from Aspergillus niger, human erythrocyte, and bovine liver, suggesting that this dual catalase-phenol oxidase activity may be a common feature of catalases.     

Purification and characterization of a novel laccase from the ascomycete Trichoderma atroviride: Application on bioremediation of phenolic compounds

The extracellular laccase produced by the ascomycete Trichoderma atroviride was purified and characterized and its ability to transform phenolic compounds was determined. The purified laccase had activity towards typical substrates of laccases including 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), dimethoxyphenol (2,6-DMP), syringaldazine and hydroquinone. The enzyme was a monomeric protein with an apparent molecular mass of 80 kDa and an isoelectric point of 3.5. The pH optima for the oxidation of ABTS and 2,6-DMP were 3 and 5, respectively, and the optimum temperature was 50 °C with 2,6-DMP. The laccase was stable at slightly acidic pH (4 and 5). It retained 80% of its activity after 4 h incubation at 40 °C. Under standard assay conditions, K_m values of the enzyme were 2.5 and 1.6 mM towards ABTS and 2,6-DMP, respectively. This enzyme was able to oxidize aromatic compounds present in industrial and agricultural wastewater, as catechol and o-cresol, although the transformation of chlorinated phenols required the presence of ABTS as mediator.     

Purification and characterization of peroxidase from cauliflower (Brassica oleracea L. var. botrytis) buds

Peroxidases (EC 1.11.1.7; donor: hydrogen peroxide oxidoreductase) are part of a large group of enzymes. In this study, peroxidase, a primer antioxidant enzyme, was purified with 19.3 fold and 0.2% efficiency from cauliflower (Brassica oleracea L.) by ammonium sulphate precipitation, dialysis, CM-Sephadex ion-exchange chromatography and Sephadex G-25 purification steps. The substrate specificity of peroxidase was investigated using 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulphonic acid) (ABTS), 2-methoxyphenol (guaiacol), 1,2-dihydroxybenzene (catechol), 1,2,3-trihyidroxybenzene (pyrogallol) and 4-methylcatechol. Also, optimum pH, optimum temperature, optimum ionic strength, stable pH, stable temperature, thermal inactivation conditions were determined for guaiacol/H(2)O(2), pyrogallol/H(2)O(2), ABTS/H(2)O(2), catechol/H(2)O(2) and 4-methyl catechol/H(2)O(2) substrate patterns. The molecular weight (M(w)) of this enzyme was found to be 44 kDa by gel filtration chromatography method. Native polyacrylamide gel electrophoresis (PAGE) was performed for isoenzyme determination and a single band was observed. K(m) and V(max) values were calculated from Lineweaver-Burk graph for each substrate patterns.     

Facile preparation of amperometric laccase biosensor with multifunction based on the matrix of carbon nanotubes-chitosan composite

The carbon nanotubes–chitosan (CNTs–CS) composite provides a suitable biosensing matrix due to its good conductivity, high stability, and good biocompatibility. Enzymes can be firmly incorporated into the matrix without the aid of other cross-linking reagents. The composite is easy to form insoluble film in solution above pH 6.3. Based on this, a facilely fabricated amperometric biosensor by entrapping laccase into the CNTs–CS composite film has been developed. At pH 6.0, the fungi laccase incorporated into the composite film remains better catalytic activity than that dissolved in solution. The system is in favor of the accessibility of substrate to the active site of laccase, thus the affinity to substrates is improved greatly, such as 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS), catechol, and O₂ with K_m values of 19.86 μM, 9.43 μM, and 3.22 mM, respectively. The major advantages of the as-prepared biosensor are: detecting different substrates (ABTS, catechol, and O₂), possessing high affinity and sensitivity, durable long-term stability, and facile preparation procedure. On the other hand, the system can be applied in fabrication of biofuel cells as the cathodic catalysts based on its good electrocatalysis for oxygen reduction. It can be extended to immobilize other enzymes and biomolecules, which will greatly facilitate the development of biosensors, biofuel cells, and other bioelectrochemical devices.     

Reduction of laccase type 1 copper by 3,4-dihydroxyphenylalanine and other catechol derivatives

3,4-Dihydroxyphenylalanine (DOPA) is not a preferred substrate of Rhus vernicifera laccase, as rate constants for the anaerobic reduction of the type 1 cupric atom by L-DOPA (6.3 X 10(1) M-1 s-1), D-DOPA (2.6 X 10(1) M-1 s-1), and L-DOPA methyl ester (2.6 X 10(1) M-1 s-1) are considerably smaller than k1 (catechol) (7 X 10(2) M-1 s-1) and rate constants characteristic of numerous other nonphysiological organic substrates (25 degrees C, pH 7.0, I = 0.5 M). The reactions of DOPA derivatives with laccase are unique, however, in that a two-term rate law pertains: kobsd = k0 + k1[phenol]; k0(L-DOPA) = 7 X 10(-2) s-1. The reactivities of other catechol derivatives (pyrogallol, gallic acid, and methyl gallate) with laccase type 1 copper were also examined.     

Silica sol-gel composite film as an encapsulation matrix for the construction of an amperometric tyrosinase-based biosensor

An amperometric tyrosinase enzyme electrode for the determination of phenols was developed by a simple and effective immobilization method using sol-gel techniques. A grafting copolymer was introduced into sol-gel solution and the composition of the resultant organic-inorganic composite material was optimized, the tyrosinase retained its activity in the sol-gel thin film and its response to several phenol compounds was determined at 0 mV vs. Ag/AgCl (sat. KCl). The dependences of the current response on pH, oxygen level and temperature were studied, and the stability of the biosensor was also evaluated. The sensitivity of the biosensor for catechol, phenol and p-cresol was 59.6, 23.1 and 39.4 microA/mM, respectively. The enzyme electrode maintained 73% of its original activity after intermittent use for three weeks when storing in a dry state at 4 degrees C.     

Extradiol dioxygenase-SiO₂ sol-gel modified electrode for catechol and its derivatives detection

A feasible and sensitive biosensor for catechol and its derivatives using 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC)-modified glassy carbon electrode was successfully constructed by polyvinyl alcohol-modified SiO? sol-gel method. The as-prepared biosensor was characterized by electrochemical impedance spectroscopy, and the surface topography of the film was imaged by atomic force microscope. Liquid chromatography-tandem mass spectrometry was applied to reveal the catalytic mechanism. BphC embedded in SiO? gel maintained its bioactivity well and exhibited excellent eletrocatalytical response to both catechol and some of its derivatives (such as 3-methylcatechol and 4-methylcatechol). The biosensor showed a linear amperometric response range between 0.002 mM and 0.8 mM catechol. And the sensitivity was 1.268 mA/(mM cm2) with a detection limit of 0.428 μM for catechol (S/N = 3). Furthermore, the BphC biosensor exhibited perfect selectivity for catechol in the mixtures of catechol and phenol. It was suggested that this flexible protocol would open up a new avenue for designing other ring-cleavage enzyme biosensors, which could be widely used for monitoring various kinds of environmental pollutants.     

Laccase‐catalysed polymeric dye synthesis from plant‐derived phenols for potential application in hair dyeing: Enzymatic colourations driven by homo‐ or hetero‐polymer synthesis

Laccase efficiently catalyses polymerization of phenolic compounds. However, knowledge on applications of polymers synthesized in this manner remains scarce. Here, the potential of laccase-catalysed polymerization of natural phenols to form products useful in hair dyeing was investigated. All 15 tested phenols yielded coloured products after laccase treatment and colour diversity was attained by using mixtures of two phenolic monomers. After exploring colour differentiation pattern of 120 different reactions with statistical regression analysis, three monomer combinations, namely gallic acid and syringic acid, catechin and catechol, and ferulic acid and syringic acid, giving rise to brown, black, and red materials, respectively, were further characterized because such colours are commercially important for grey hair dyeing. Selected polymers could strongly absorb visible light and their hydrodynamic sizes ranged from 100 to 400 nm. Analyses of enzyme kinetic constants, liquid chromatography and electrospray ionization-mass spectrometry (ESI-MS) coupled with collision-induced dissociation MS/MS indicate that both monomers in reactions involving catechin and catechol, and ferulic acid and syringic acid, are coloured by heteropolymer synthesis, but the gallic acid/syringic acid combination is based on homopolymer mixture formation. Comparison of colour parameters from these three reactions with those of corresponding artificial homopolymer mixtures also supported the idea that laccase may catalyse either hetero- or homo-polymer synthesis. We finally used selected materials to dye grey hair. Each material coloured hair appropriately and the dyeing showed excellent resistance to conventional shampooing. Our study indicates that laccase-catalysed polymerization of natural phenols is applicable to the development of new cosmetic pigments.     

Purification and Characterization of Extracellular Laccase Secreted by Pleurotus sajor-caju MTCC 141

The effect of lignin containing natural substrates corn-cob, coir-dust, saw-dust, wheat straw and bagasse particles on the extracellular secretion of laccase in the liquid culture growth medium of Pleurotus sajor-caju MTCC 141 has been studied. The culture conditions for maximum secretion of laccase by Pleurotus sajor-caju MTCC 141 have been optimized. Homogeneous preparation of laccase from the culture filtrate of the fungus has been achieved using ammonium sulphate precipitation, anion exchange chromatography on DEAE and gel filtration chromatography on Sephadex G-100. The purified enzyme preparation gave a single protein band in SDS-PAGE analysis indicating a molecular weight of 90 kD. The enzymatic characteristics Km, kcat, pH and temperature optima of the purified laccase have been determined using 2, 6-dimethoxyphenol as the substrate and have been found to be 35μmol/L, 0.30 min-1, 4.5 and 37℃ respectively. The Km values for the other substrate like catechol, m-cresol, pyrogallol and syringaldazine have also been determined which were found to be 216 μmol/L, 380 μmol/L, 370 μmol/L and 260 μmol/L respectively.     

Polyphenoloxidase from six mature grape varieties and their activities towards various phenols

Juices were prepared from three white and three red grape varieties harvested at full maturity and comparative studies on their oxygen-uptake, absorbance at 420 nm (degree of browning), polyphenoloxidase (EC 1.10.3.1; PPO) activity, and their phenol compositions were done. There was no correlation among the amounts of oxygen-uptake and oxidizable phenols in the juices and their degree of browning. However, there was similarity among the PPO from the six grape varieties in their general enzymatic properties and substrate specificity towards twenty-five phenols. A partially purified PPO fraction from Koshu juice, which did not contain free phenols, showed strong activity towards (+)-catechin, (−)-epicatechin, caffeic acid, catechol, pyrogallol, and protocatechuic acid (oxidizable phenols), but had no activity towards the other fifiteen phenols. The oxidizable substrates were not always the only limiting factor in the oxidation and browning of phenols by the PPO. Some unoxidizable phenols such as gallic acid, p-cresol, and tannic acid which were not substrates for PPO inhibited the oxidation of the oxidizable phenols except pyrogallol which was not inhibited by gallic acid. On the other hand, hydroquinone promoted the oxidation of the oxidizable phenols except protocatechuic acid. These showed that there were competitive reactions and synergism during the enzymatic oxidation of phenols.     

Evidence for a halotolerant-alkaline laccase in Streptomyces psammoticus: Purification and characterization

An unusual halotolerant-alkaline laccase from Streptomyces psammoticus has been purified to homogeneity through anion exchange and gel filtration chromatography steps with an overall purification fold of 12.1. The final recovery of the enzyme was 22.1%. The molecular mass of the purified laccase was about 43 kDa. The enzyme was active in the alkaline pH range with pH optima at 8.5 and 97% activity retention at pH 9.0. The optimum temperature was 45 °C. The enzyme was stable in the pH range 6.5–9.5 and up to 50 °C for 90 min. The enzyme was tolerant to NaCl concentrations up to 1.2 M. It was inhibited by all the putative laccase inhibitors while the enzyme was activated by metal ions like Fe, Zn, Cu, Na and Mg. Fe enhanced the enzyme activity by twofold (204%). The enzyme showed lowest K_m value with pyrogallol (0.25 mM) followed by ABTS (0.39 mM). The purified enzyme was a typical blue laccase with an absorption peak at 600 nm.     

Natural mediators in the oxidation of polycyclic aromatic hydrocarbons by laccase mediator systems

The oxidation of polycyclic aromatic compounds was studied in systems consisting of laccase from Trametes versicolor and so-called mediator compounds. The enzymatic oxidation of acenaphthene, acenaphthylene, anthracene, and fluorene was mediated by various laccase substrates (phenols and aromatic amines) or compounds produced and secreted by white rot fungi. The best natural mediators, such as phenol, aniline, 4-hydroxybenzoic acid, and 4-hydroxybenzyl alcohol were as efficient as the previously described synthetic compounds ABTS [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] and 1-hydroxybenzotriazole. The oxidation efficiency increased proportionally with the redox potentials of the phenolic mediators up to a maximum value of 0.9 V and decreased thereafter with redox potentials exceeding this value. Natural compounds such as methionine, cysteine, and reduced glutathione, containing sulfhydryl groups, were also active as mediator compounds.     

Purification of a new isoform of laccase from a Marasmius quercophilus strain isolated from a cork oak litter (Quercus suber L)

A new isoform of laccase from Marasmius quercophilus is described in this study. The strain of this white-rot fungus was isolated for the first time on a cork oak litter. This isoform exhibited certain common properties of laccases (a molecular weight of 65 Kda, an optimum pH of 6.2 with syringaldazine). But this laccase has also particularly novel features: the best activity measured was observed at high temperatures (80 C) and this isoform was not inhibited with EDTA. Furthermore, this induced laccase was able to transform most of the aromatic compounds tested without the addition of mediators to the reaction mixture, and the transformation of certain chlorophenols (2-chlorophenol and 2,4-dichlorophenol) by a laccase isoform from M. quercophilus is reported here for the first time. We also demonstrate the importance of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as a mediator since it allowed veratryl alcohol and p-hydroxybenzoic acid transformation. Moreover, new products of transformation were observed using the combination of ABTS with this isoform of laccase.     

Fabrication of a carbon fiber paper as the electrode and its application toward developing a sensitive unmediated amperometric biosensor

Carbon fiber paper (CFP), a material frequently used as the diffusion layer in fuel cells, was found recently to exhibit a potential as an electrode for the development of sensitive, unmediated biosensors. After nitrogen plasma treatment, the CFP exhibited a quasi-reversible behavior to the redox couple (e.g., ferricyanide) with an electron transfer rate constant of 7.2 × 10(-3)cms(-1). This rate constant is approximately double that of a Pt-electrode and is much higher than that of many carbon-based electrodes. The unmediated CFP-based tyrosinase biosensor fabricated for this study exhibited an optimal working potential and operating pH value of -0.2V and 6.5, respectively. Compared to other unmediated tyrosinase biosensors, the CFP-based tyrosinase biosensor offers a high sensitivity for the monitoring of phenolic compounds (17.8, 7.1, 5.2 and 3.7 μA μM(-1)cm(-2) for catechol, phenol, bisphenol and 3-aminophenol, respectively). The lowest detection limit for catechol, phenol, bisphenol and 3-aminophenol was 2, 5, 5 and 12 nM, respectively. Furthermore, this biosensor exhibited a good repeatability, a fast response time (around 10s), and a wide linear dynamic range of detection for phenolic compounds.     

Determination of laccase activity in mixed solvents: comparison between two chromogens in a spectrophotometric assay

Spectrophotometric determination of laccase activity with ABTS acting as chromogen yields exceedingly low values whenever conducted in a water-organic mixed solvent. Nevertheless, there is firm evidence that laccase is able to oxidize substrates such as phenols and amines quantitatively in these mixed solvents. We show that the apparently small rate of ABTS oxidation by laccase in a mixed solvent, such as buffered water-dioxane 1:1, is not amenable to the denaturation of laccase but rather to the decreased stability of ABTS(.+). We propose HAA as a more reliable chromogen for the determination of laccase activity in mixed solvents.     

Transglycosylation by Streptococcus mutans GS-5 glucosyltransferase-D: acceptor specificity and engineering of reaction conditions

The acceptor specificity of Streptococcus mutans GS-5 glucosyltransferase-D (GTF-D) was studied, particular the specificity toward non-saccharide compounds. Dihydroxy aromatic compounds like catechol, 4-methylcatechol, and 3-methoxycatechol were glycosylated by GTF-D with a high efficiency. Transglycosylation yields were 65%, 50%, and 75%, respectively, using 40 mM acceptor and 200 mM sucrose as glucosyl donor. 3-Methoxylcatchol was also glycosylated, though at a significantly lower rate. A number of other aromatic compounds such as phenol, 2-hydroxybenzaldehyde, 1,3-dihydroxybenzene, and 1, 2-phenylethanediol were not glycosylated by GTF-D. Consequently GTF-D aromatic acceptors appear to require two adjacent aromatic hydroxyl groups. In order to facilitate the transglycosylation of less water-soluble acceptors the use of various water miscible organic solvents (cosolvents) was studied. The flavonoid catechin was used as a model acceptor. Bis-2-methoxyethyl ether (MEE) was selected as a useful cosolvent. In the presence of 15% (v/v) MEE the specific catechin transglucosylation activity was increased 4-fold due to a 12-fold increase in catechin solubility. MEE (10-30% v/v) could also be used to allow the transglycosylation of catechol, 4-methylcatechol, and 3-methoxycatechol at concentrations (200 mM) otherwise inhibiting GTF-D transglycosylation activity.