Farnesol stimulates laccase production in Trametes versicolor

Farnesol, a quorum‐sensing molecule, was used successfully to improve laccase production in submerged cultures of Trametes versicolor. At the optimal farnesol concentration of 60 μM added at the beginning of the culture, the extracellular laccase activity reached 629.3 U L^?1 after 6 days of cultivation, which represented a 1.92‐fold increase relative to the control without farnesol treatment. The addition of farnesol resulted in an increase in the accumulation of H₂O₂ and an increased expression of the laccase (lac) gene and the RhoA gene. The RhoA gene correlated with hyperbranched mycelia, which facilitated the secretion of the intracellular laccase. This study provides a basis for understanding the induction mechanism of farnesol for enhancing laccase production.     

Optimization of laccase production from Trametes versicolor by solid fermentation

The regulation of culture conditions, especially the optimization of substrate constituents, is crucial for laccase production by solid fermentation. To develop an inexpensive optimized substrate formulation to produce high-activity laccase, a uniform design formulation experiment was devised. The solid fermentation of Trametes versicolor was performed with natural aeration, natural substrate pH (about 6.5), environmental humidity of 60% and two different temperature stages (at 37 degrees C for 3 days, and then at 30 degrees C for the next 17 days). From the experiment, a regression equation for laccase activity, in the form of a second-degree polynomial model, was constructed using multivariate regression analysis and solved with unconstrained optimization programming. The optimized substrate formulation for laccase production was then calculated. Tween 80 was found to have a negative effect on laccase production in solid fermentation; the optimized solid substrate formulation was 10.8% glucose, 27.7% wheat bran, 9.0% (NH4)2SO4, and 52.5% water. In a scaled-up verification of solid fermentation at a 10 kg scale, laccase activity from T. versicolor in the optimized substrate formulation reached 110.9 IU/g of dry mass.     

Enhanced production of laccase in the fungus Trametes versicolor by the addition of xenobiotics

Agrochemicals, industrial compounds and their transformation products have been assayed for their ability to enhance laccase production in liquid cultures of Trametes versicolor, when added at 0.5 mM. After 3 days of treatment, enzymatic activity in the culture medium was increased 14-fold by 4-n-nonylphenol and 24-fold by aniline. Laccase activity was enhanced 10-fold by oxidised derivatives of the herbicide diquat, 17-fold by N,N-dimethyl-N-(5-chloro,4-hydroxyphenyl)urea and 22-fold by 9-fluorenone.     

Phylogenetic and biochemical characterisation of a recombinant laccase from Trametes versicolor

Laccases are important enzymes for bioremediation and the best-characterised are from the fungus Trametes versicolor. Here, we describe the cloning and characterisation of a new variant of laccase from T. versicolor and its expression in Saccharomyces cerevisiae. We have performed a sequence-based analysis of Trametes laccases that leads to a proposal for a new nomenclature of this fungus laccases according to their phylogenetic relationships since their nomenclature based on IPs is ambiguous. We also describe the kinetic properties of the recombinant enzyme.     

New and different lignocellulosic materials from Turkey for laccase and manganese peroxidase production by Trametes versicolor

In the present study, the production of laccase (Lac) and manganese‐dependent peroxidase (MnP) by the white‐rot fungus Trametes versicolor grown in submerged cultures with different agricultural residues was investigated. The lignocellulosic materials studied were almond shells, hazelnut husks, sunflower stems, clover straw and hazelnut cobs, because they are common agricultural wastes in Turkey. Among the different lignocellulosic materials studied, hazelnut cobs provided the highest Lac and MnP activities (47.09 and 109.21 U/L, respectively). The optimum conditions were determined for Lac and MnP production in submerged cultures of T. versicolor by using hazelnut cobs as substrate. For Lac production, the optimum incubation time, hazelnut cob concentration, pH, and shaking rate were found as 4 days, 2% w/v, 6.0 and 130 rpm, respectively. For MnP production, the optimum incubation time, hazelnut cob concentration, pH and shaking rate were found as 5 days, 2% w/v, 6.0 and 90 rpm, respectively.     

Degradation of phenanthrene by Trametes versicolor and its laccase

Phenanthrene is a three-ring polycyclic aromatic hydrocarbon and commonly found as a pollutant in various environments. Degradation of phenanthrene by white rot fungus Trametes versicolor 951022 and its laccase, isolated in Korea, was investigated. After 36 h of incubation, about 46% and 65% of 100 mg/l of phenanthrene added in shaken and static fungal cultures were removed, respectively. Phenanthrene degradation was maximal at pH 6 and the optimal temperature for phenanthrene removal was 30 degrees C. Although the removal percentage of phenanthrene was highest (76.7%) at 10 mg/l of phenanthrene concentration, the transformation rate was maximal (0.82 mg/h) at 100 mg/L of phenanthrene concentration in the fungal culture. When the purified laccase of T versicolor 951022 reacted with phenanthrene, phenanthrene was not transformed. The addition of redox mediator, 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) or 1-hydroxybenzotriazole (HBT) to the reaction mixture increased oxidation of phenanthrene by laccase about 40% and 30%, respectively.     

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

Summary Laccase 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.     

Purification and characterization of laccase from the white rot fungus Trametes versicolor

Laccase is one of the ligninolytic enzymes of white rot fungus Trametes versicolor 951022, a strain first isolated in Korea. This laccase was purified 209-fold from culture fluid with a yield of 6.2% using ethanol precipitation, DEAE-Sepharose, Phenyl-Sepharose, and Sephadex G-100 chromatography. T. versicolor 951022 excretes a single monomeric laccase showing a high specific activity of 91,443 U/mg for 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as a substrate. The enzyme has a molecular mass of approximately 97 kDa as determined by SDS-PAGE, which is larger than those of other laccases reported. It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 3.0 and a temperature of 50 degrees C. The Km value of the enzyme for substrate ABTS is 12.8 micrometer and its corresponding Vmax value is 8125.4 U/mg. The specific activity and substrate affinity of this laccase are higher than those of other white rot fungi, therefore, it may be potentially useful for industrial purposes.     

Oxidation of phenyl compounds using strongly stable immobilized-stabilized laccase from Trametes versicolor

The hydrolysis of phenolic compounds using an immobilized and highly active and stable derivative of laccase from Trametes versicolor is presented. The enzyme was immobilized on aldehyde supports. For this, the enzyme was enriched in amino groups by chemical modification of its carboxyl groups. The aminated enzyme was immobilized with a high recovered activity (over 60%). Aldehyde derivatives were more stable than soluble or aminated-soluble enzyme and the reference derivatives after incubation in different inactivating conditions (high temperatures, different pH values or presence of organic cosolvents). The most stable derivative was obtained immobilizing the chemically aminated enzyme at pH 10 on aldehyde supports with a stabilization factor approximately 280 fold after incubation at pH 7 and 55 °C. In addition, it was possible to prepare immobilized derivatives with a maximal enzyme loading of 60 mg g^?1 of support. This derivative could be reused for 10 reaction cycles with negligible lost of activity.     

Improving laccase production by employing different lignocellulosic wastes in submerged cultures of Trametes versicolor

Laccase production by the white-rot fungus Trametes versicolor (CBS100.29) grown in submerged cultures was studied. Addition of different insoluble lignocellulosic materials into the culture medium in order to enhance laccase production was investigated. The lignocellulosic materials were grape seeds, grape stalks and barley bran, selected because of their availability and low cost, since they are agro-industrial wastes abundant in most countries. Barley bran gave the highest activities, a maximum value of 639U/l, which was 10 times the value attained in the cultures without lignocellulosics addition. The decolourisation of a model dye, Phenol Red, by the ligninolytic fluids obtained in the above-mentioned cultures was investigated. Grape stalk and barley bran cultures showed the highest ability to decolourise the dye, attaining a percentage of decolourisation of around 60% in 72 h.     

毛栓菌产漆酶

<正>漆酶是一种环境友好型酶类,其催化底物单电子氧化形成自由基,同时将分子氧还原形成水,产生的自由基则耦合成二聚体或低聚物。漆酶可催化氧化多种底物,如酚类及其衍生物、芳胺及其衍生物、羟酸及其衍生物、一些金属有机化合物和甾类激素、生物色素等,在环境保护与修复、造纸工业、有机合成、食品工业、生物监测等领域有着广泛的应用前景。例如在染料脱色方面,以白腐真菌为主的一组丝状真菌对染料的降解脱色在近二十年来成为染料生物脱色研究的热点和主流。尽管漆酶在自然界中分布广泛,但     

Enhanced production of laccase in Trametes vesicolor by the addition of ethanol

In a medium containing 40 g ethanol l^–1, laccase production by Trametes versicolor was 2.6 unit per ml of the supernatant, which was over 20 times higher than that without ethanol. Laccase activity with ethanol was quite comparable to that with the well-known inducers such as veratryl alcohol, xylidine and guaiacol. With other white-rot fungi, Coriolus hirsutus and Grifola frondosa, ethanol had a similar stimulatory effect on laccase production.     

Enhancement of phenolic compounds oxidation using laccase from Trametes versicolor in a microreactor

Laccases catalyse the oxidation of a wide range of substrates by a radical-catalyzed reaction mechanism, with a corresponding reduction of oxygen to water in a four-electron transfer process. Due to that, laccases are considered environmentally friendly enzymes, and lately there has been great interest in their use for the transformation and degradation of phenolic compounds. In this work, enzymatic oxidation of catechol and L-DOPA using commercial laccase from Trametes versicolor was performed, in continuously operated microreactors. The main focus of this investigation was to develop a new process for phenolic compounds oxidation, by application of microreactors. For a residence time of 72 s and an inlet oxygen concentration of 0.271 mmol/dm³, catechol conversion of 41.3% was achieved, while approximately the same conversion of L-DOPA (45.0%) was achieved for an inlet oxygen concentration of 0.544 mmol/dm³. The efficiency of microreactor usage for phenolic compounds oxidation was confirmed by calculating the oxidation rates; in the case of catechol oxidation, oxidation rates were in the range from 76.101 to 703.935 g/dm³/d (18–167 fold higher, compared to the case in a macroreactor). To better describe the proposed process, kinetic parameters of catechol oxidation were estimated, using data collected from experiments performed in a microreactor. The maximum reaction rate estimated in microreactor experiments was two times higher than one estimated using the initial reaction rate method from experiments performed in a cuvette. A mathematical model of the process was developed, and validated, using data from independent experiments.     

Overproduction of Trametes versicolor laccase by making glucose starvation using yeast

In the present paper, overproduction of laccase by microbe interaction was studied. When Trametes versicolor was co-cultured with Candida sp. HSD07A in submerged fermentation, laccase activity could be improved significantly and reached 10500 ± 160 U/l, 11.8 times more than that of the contrast group. Fermentation tests of the yeast indicated that it could produce amylase and cellulase, but couldn’t excrete laccase and the overproductive laccase was produced by T. versicolor; the interaction mechanism between T. versicolor and Candida sp. HSD07A was investigated and the results showed that amylase and cellulose could hydrolyze cell walls of T. versicolor; however, the degree of hydrolysis was at a very low level, could not lead to overproduction of laccase; glucose starvation state made by the yeast was the real reason why T. versicolor could overproduce laccase; moreover, this study also proved that making glucose starvation using the yeast was a novel and effective method.     

云芝Trametes versicolor1126所产漆酶对靛蓝废水脱色的初步研究

考察了云芝Trametes versicolor1126发酵培养中漆酶酶活和pH的变化,同时研究了羧甲基纤维素钠及苯酚添加量对漆酶活力的影响。结果表明当培养基中加入0.8%羧甲基纤维素钠、100mg/L苯酚时,均能明显提高漆酶的活力。以漆酶/HBT介质体系对靛蓝废水进行脱色,反应100min后,脱色率达90.8%。使用漆酶处理靛蓝废水具有广阔的应用前景。     

Gallic acid interference on polyphenolic amperometric biosensing using Trametes versicolor laccase

The present work reports the gallic acid (GA) interference on polyphenolic amperometric biosensing using Trametes versicolor laccase (TvLac). GA′ inhibitory effect on TvLac activity was investigated on the oxidation of caffeic acid (CA) by free TvLac and its immobilised form on modified polyethersulfone membrane (PES/TvLac), using spectrophotometric and amperometric biosensor detection methods. The results have indicated that GA presents inhibitory behaviour on TvLac activity in a concentration-dependent manner. The GA concentration leading to 50% activity lost, IC₅₀, was determined to be 19.15 ± 0.11 μM and 5.11 ± 0.19 μM for free and immobilised enzyme, respectively. The results have also shown that GA exhibited a competitive and a mixed inhibition types on the TvLac activity for spectrophotometric and amperometric biosensor methods, respectively. Further GA′ and CA′ cyclic voltammetry studies have demonstrated that GA′ oxidation products interfered with CA′ redox reaction products. In fact, a decrease of the reduction current was observed at cyclic voltammograms of CA, when mixed with GA. Therefore, the GA′ interference on polyphenolic amperometric biosensing is the result of the combination of two factors: on one hand, we have the inhibitory enzymatic effect, and on the other, the reaction of GA′ oxidation products with the o-quinones obtained by the enzymatic oxidation of CA. Both gave rise to the amperometric signal decreasing effect.     

Immobilization of Trametes versicolor cultures for improving laccase production in bubble column reactor intensified by sonication

The mycelia of Trametes versicolor immobilized in alginate beads provided higher laccase production than that in pelleted form. An efficient ultrasonic treatment enhanced laccase production from the immobilized T. versicolor cultures. The optimized treatment process consisted of exposing 36-h-old bead cultures to 7-min ultrasonic treatments twice with a 12-h interval using a fixed ultrasonic power and frequency (120 W, 40 kHz). Using the intensification strategy with sonication, laccase production increased by more than 2.1-fold greater than the untreated control in both flasks and bubble column reactors. The enhancement of laccase production by ultrasonic treatment is related to the improved mass transfer of nutrients and product between the liquid medium and the gel matrix. These results provide a basis for the large-scale and highly-efficient production of laccase using sonobioreactors.     

Detoxification of wood hydrolysates with laccase and peroxidase from the white-rot fungus Trametes versicolor

Fermentation of wood hydrolysates to desirable products, such as fuel ethanol, is made difficult by the presence of inhibitory compounds in the hydrolysates. Here we present a novel method to increase the fermentability of lignocellulosic hydrolysates: enzymatic detoxification. Besides the detoxification effect, treatment with purified enzymes provides a new way to identify inhibitors by assaying the effect of enzymatic attack on specific compounds in the hydrolysate. Laccase, a phenol oxidase, and lignin peroxidase purified from the ligninolytic basidiomycete fungus Trametes versicolor were studied using a lignocellulosic hydrolysate from willow pretreated with steam and SO₂. Saccharomyces cerevisiae was employed for ethanolic fermentation of the hydrolysates. The results show more rapid consumption of glucose and increased ethanol productivity for samples treated with laccase. Treatment of the hydrolysate with lignin peroxidase also resulted in improved fermentability. Analyses by GC-MS indicated that the mechanism of laccase detoxification involves removal of monoaromatic phenolic compounds present in the hydrolysate. The results support the suggestion that phenolic compounds are important inhibitors of the fermentation process. Received: 3 November 1997 / Received revision: 4 February 1998 / Accepted: 6 February 1998