A heterologous production of the <Emphasis Type="Italic">Trametes hirsuta</Emphasis> laccase in the fungus <Emphasis Type="Italic">Penicillium canescens</Emphasis>

A heterologous protein expression in the fungus Penicillium canescens is described for the first time. The fungal strains producing Trametes hirsuta 072 accase under control of a highly efficient promoter of the P. canescens gene bgaS has been constructed. These strains efficiently transcribe the T. hirsuta 072 laccase gene with a correct intron splicing. Activity of the secreted heterologous laccase in the culture liquid reaches 3 U/ml, accounting for 98% of the total laccase activity, which demonstrates a high efficiency of heterologous secretion. The synthesized P. canescens laccase has the same molecular weight as the enzyme produced by T. hirsuta 072.     

Laccase production and wood degradation by a white-rot fungus Daedalea flavida

A comparative study has been conducted on seven white rot fungi to investigate their abilities to produce laccase and selectively degrade lignin. Laccase was produced constitutively on the different media tested. Of the different lignins, phenolic compounds and sugars involved, the highest laccase yield was obtained on indulin AT. Salicylic acid inhibited enzyme activity. A temperature of 20°C and 0.2% of indulin AT were found to be optimum for enzyme activity. No correlation was found between the amount of enzyme and fungal mass produced. During semisolid degradation of angiospermic wood sawdust, Daedalea flavida caused a total weight loss of 11%, with a lignin loss of 15.77% during two months of decay. Lignin removal was comparatively selective during the first month, during which time laccase production was also higher, indicating its probable role in lignin degradation.     

Effect of biological pretreatment with Trametes hirsuta yj9 on enzymatic hydrolysis of corn stover

In this study, a newly isolated Trametes hirsuta yj9 was used to pretreat corn stover in order to enhance enzymatic digestibility. T. hirsuta yj9 preferentially degraded lignin to be as high as 71.49% after 42-day pretreatment. Laccase and xylanase was the major ligninolytic and hydrolytic enzyme, respectively and filter paper activity (FPA) increased gradually with prolonged pretreatment time. Sugar yields increased significantly after pretreatment with T. hirsuta yj9, reaching an enzymatic digestibility of 73.99% after 42 days of pretreatment. Scanning electron microscopy (SEM) showed significant structural changes in pretreated corn stover, the surface of pretreated corn stover became increasingly coarse, the gaps between cellulose fibers were visible, and many pores were developed. Correlation analysis showed that sugar yields were inversely proportional to the lignin contents, less related to cellulose and hemicellulose contents.     

Removal of acetaminophen and carbamazepine in single and binary systems with immobilized laccase from Trametes hirsuta

Laccase-based bioprocesses represent a fascinating prospective for the removal of contaminants of emerging concern in wastewaters. In this work, immobilized laccase from Trametes hirsuta was used to transform carbamazepine (CBZ) and acetaminophen (ACE) in spiked single and binary solutions. The effects of pH, time course and reaction pathways on the transformation were studied. T. hirsuta secreted only laccase. The immobilized laccase was able to degrade 40% and 70% of CBZ and ACE, respectively, in the binary system, while only 5% and 25% of transformation was achieved in the single system for ACE and CBZ, respectively. The maximum removal of acetaminophen was found at pH 7. These obtained results confirm that the acetaminophen is a good laccase mediator compound. The most probable pathway in the binary system involved the formation of acetaminophen dimers and ACE-ACE-CBZ oligomers.     

The effect of lignin model compound structure on the rate of oxidation catalyzed by two different fungal laccases

Laccases (EC 1.10.3.2) are multicopper oxidases able to oxidize various substrates, such as phenolic subunits of lignin. The substrate range can be widened to non-phenolic units by the use of mediators. Since discovery of the laccase-mediator system, direct reactions of lignin and laccase without mediated electron-transfer have gained much less attention. The objective of this study was to investigate lignin as a substrate for fungal laccases by using lignin model compounds. These model compounds contained guaiacylic and syringylic moieties and also compounds of guaiacylic origin at a higher oxidation level. Some of these compounds are commercially available, but most of them were synthesized. The oxidation reaction rates of the lignin model compounds were studied by monitoring consumption of the co-substrate oxygen, in reactions catalyzed by laccases from two different fungi; Melanocarpus albomyces and Trametes hirsuta, possessing different molecular and catalytic properties. These reaction rate studies were compared to physicochemical properties of the lignin model compounds: relative redox potentials determined using cyclic voltammetry and pK_a-values. Docking of syringylic and biphenylic compounds to the active sites of both laccases was performed and the resulting model complex structures were used to further interpret the reaction rate results. Reaction rates of laccases are mainly affected by the ability of a lignin model compound to be oxidized and the pK_a-value of the substrate seems to be less important. As a consequence, syringylic compounds are oxidized with the highest rates and compounds at a higher oxidation level and redox-potential, such as vanillin, are oxidized at a much lower rate. Both guaiacylic and syringylic type compounds fit well in the active sites of both laccases. Only for a biphenylic compound, steric clashes were observed, and they are likely to have an effect on the reaction rate. When the oxidation rates on the selected model compounds with the two different laccases were compared, the redox-potential difference between laccases T1 copper and the lignin model compound (ΔE) was not the only property that determined the oxidation rate. In the case of lignin model substrates, also the selectivity of a specific laccase, reflected in the k_cat/K_m value, plays an important role.     

White‐rot fungi combined with lignite granules and lignitic xylite to decolorize textile industry wastewater

The feasibility of using immobilized fungi to decolorize textile industry wastewater containing dyes was examined in experiments with: two species of white‐rot fungi (a Marasmius species from Indonesia, which produces copious biomass, and Trametes hirsuta, which produces high levels of laccase); two types of lignite products as adsorbents and solid substrates (lignitic xylite and lignite granules); and four simulated wastewaters, each containing a different kinds of reactive textile azo dye. The growth, extracellular enzyme production, dye degradation and dye absorption parameters afforded by each permutation of fungus, substrate and dye were then measured. Both fungal species grew poorly on xylite, but much better on lignite granules. Marasmius sp. produced up to 67 U/L laccase on lignite granules, but just 10 U/L on xylite, and no other detectable extracellular enzymes. T. hirsuta produced 1343 U/L laccase and up to 12 U/L unspecific peroxidase when immobilized on lignite granules, and 898 U/L laccase with 14 U/L unspecific peroxidase when immobilized on xylite. The amount of color lost from the dye solutions depended on both the type of dye and the enzyme levels in the fermenter.     

Laccase of the lignolytic fungus Trametes hirsuta: Purification and characterization of the enzyme, and cloning and primary structure of the gene

The main physicochemical characteristics of the major isoform of the laccase secreted by the fungus Trametes hirsuta 072 were studied. The enzyme belongs to the group of high redox potential laccases (E _T1 ⁰ 790 ± 5), and it oxidizes with high efficiency various substrates of phenolic nature. The gene of this isoform was cloned, and its nucleotide sequence was determined. The length of the complete gene is 2134 bp. It comprises 11 exons and 10 introns. Analysis of the amino acid sequence of T. hirsuta 072 laccase demonstrated a high homology to the other laccases secreted by fungi of the genus Trametes.     

Enzymatic oxidation of manganese ions catalysed by laccase

The principal possibility of enzymatic oxidation of manganese ions by fungal Trametes hirsuta laccase in the presence of oxalate and tartrate ions, whereas not for plant Rhus vernicifera laccase, was demonstrated. Detailed kinetic studies of the oxidation of different enzyme substrates along with oxygen reduction by the enzymes show that in air-saturated solutions the rate of oxygen reduction by the T2/T3 cluster of laccases is fast enough not to be a readily noticeable contribution to the overall turnover rate. Indeed, the limiting step of the oxidation of high-redox potential compounds, such as chelated manganese ions, is the electron transfer from the electron donor to the T1 site of the fungal laccase.     

Decolorization and Detoxification of Textile Dyes with a Laccase from Trametes hirsuta

Trametes hirsuta and a purified laccase from this organism were able to degrade triarylmethane, indigoid, azo, and anthraquinonic dyes. Initial decolorization velocities depended on the substituents on the phenolic rings of the dyes. Immobilization of the T. hirsuta laccase on alumina enhanced the thermal stabilities of the enzyme and its tolerance against some enzyme inhibitors, such as halides, copper chelators, and dyeing additives. The laccase lost 50% of its activity at 50 mM NaCl while the 50% inhibitory concentration (IC₅₀) of the immobilized enzyme was 85 mM. Treatment of dyes with the immobilized laccase reduced their toxicities (based on the oxygen consumption rate of Pseudomonas putida) by up to 80% (anthraquinonic dyes). Textile effluents decolorized with T. hirsuta or the laccase were used for dyeing. Metabolites and/or enzyme protein strongly interacted with the dyeing process indicated by lower staining levels (K/S) values than obtained with a blank using water. However, when the effluents were decolorized with immobilized laccase, they could be used for dyeing and acceptable color differences (ΔE*) below 1.1 were measured for most dyes.     

Degradation of lignocellulosic residues by polyporus versicolor and the effect of moisture contents and phenolic compounds

Polyporus versicolor was selected to find out its ability to degrade four different lignocellulosic residues (angiospermic wood sawdust, sugarcane bagasse, paddy and wheat straw) under semisolid conditions. The production of laccase was also studied on these substrates. Sawdust suffered a maximum lignin loss though overall reduction in weight was maximum in paddy straw. Addition of malt extract and certain phenolic compounds (gallic acid, tannic acid and orcinol) favoured ligninolysis in sawdust. A moisture level of 5 ml/g of sawdust was found to be the most suitable for degradation whereas laccase yield increased with further rise in moisture content.     

The laccase-catalyzed modification of lignin for enzymatic hydrolysis

The efficient use of cellulases in the hydrolysis of pretreated lignocellulosic biomass is limited due to the presence of lignin. Lignin is known to bind hydrolytic enzymes nonspecifically, thereby reducing their action on carbohydrate substrates. The composition and location of residual lignin therefore seem to be important for optimizing the enzymatic hydrolysis of lignocellulosic substrates. The use of lignin-modifying enzymes such as laccase may have potential in the modification or partial removal of lignin from the biomass. In this study, the effect of lignin modification by laccase on the hydrolysis of pretreated spruce (Picea abies) and giant reed (Arundo donax) was evaluated. The substrates were first treated with laccase and then hydrolyzed with commercial cellulases. Laccase modification improved the hydrolysis yield of spruce by 12%, but surprisingly had an adverse effect on giant reed, reducing the hydrolysis yield by 17%. The binding properties of cellulases on the untreated and laccase-treated lignins were further studied using isolated lignins. The laccase treatment reduced the binding of enzymes on modified spruce lignin, whereas with giant reed, the amount of bound proteins increased after laccase treatment. Further understanding of the reactions of laccase on lignin will help to control the unspecific-binding of cellulases on lignocellulosic substrates.     

Solid-state fermentation of sugarcane bagasse with Flammulina velutipes and Trametes versicolor

The mushroom Flammulina velutipes and the white-rot fungus Trametes versicolor were cultivated separately on sugarcane bagasse for 40 days. Trametes versicolor produced laccase and manganese-peroxidase activities, showing a simultaneous degradation of lignin and holocellulose. However, only phenoloxidase activity was found with Flammulina velutipes. A preferential degradation of lignin was detected in F. velutipes, which exhibited a greater reduction in the ratio of weight loss to lignin loss than T. versicolor. A decrease in the syringyl/guaiacyl ratio observed with both fungi indicated the preferential degradation of non-condensed (syringyl-type) lignin units. An increase in the relative abundance of aromatic carboxylic acids suggested that the oxidative transformation of lignin unit side-chains was occurring. This was more noticeable with Flammulina velutipes than with T. versicolor.     

Screening of a fungus capable of powerful and selective delignification on wheat straw

Aims: To screen and characterize a novel fungus with powerful and selective delignification capability on wheat straw. Methods and Results: A fungus capable of efficient delignification under solid‐state fermentation (SSF) conditions on wheat straw was screened. After 5 days of incubation, 13·07% of the lignin was removed by fungal degradation, and 7·62% of the holocellulose was lost. Furthermore, 46·53% of the alkali lignin was removed after 2 days of liquid fermentation. The fungus was identified as Fusarium concolor based on its morphology and an analysis of its 18S rDNA gene sequence. The molecular weight distribution of lignin was evaluated by gel permeation chromatography. Enzyme assay indicated that the fungus produced laccase, cellobiose dehydrogenase, xylanase and cellulase during the incubation period. Intracellular lignin peroxidase, manganese peroxidase and laccase were produced during liquid fermentation. Conclusions: We have successfully screened a fungus, F. concolor, which can efficiently degrade the lignin of wheat straw, with slight damage to the cellulose, after 5 days of SSF. Significance and Impact of the Study: The newly isolated strain could be used in pretreatment of lignocellulose materials prior to biopulping, bioconversion into fuel and substrates for the chemical industry.     

Decolorization of simulated textile dye baths by crude laccases from Trametes hirsuta and Cerrena unicolor

In this study crude laccases from the white‐rot fungi Cerrena unicolor and Trametes hirsuta were tested for their ability to decolorize simulated textile dye baths. The dyes used were Remazol Brilliant Blue R (RBBR) (100 mg/L), Congo Red (12.5 mg/L), Lanaset Grey (75 mg/L) and Poly R‐478 (50 mg/L). The effect of redox mediators on dye decolorization by laccases was also assessed. C. unicolor laccase was able to decolorize all the dyes tested. It was especially effective towards Congo Red and RBBR with 91 and 80% of color removal in 19.5 h despite the fact that simulated textile dye baths were used. Also Poly R‐478 and Lanaset Grey were partially decolorized (69 and 48%, respectively). C. unicolor laccase did not need any mediators for removing the dyes. However, T. hirsuta laccase was only able to decolorize simulated Congo Red and RBBR dye baths (91 and 45%, respectively) in 19.5 h without mediators. When using mediators the decolorization capability was enhanced substantially, e.g. Poly R‐478 was decolorized by 78% in 25.5 h. On the whole, both laccases showed potential to be used in industrial applications.     

Enhanced saccharification of alkali-treated rice straw by cellulase from Trametes hirsuta and statistical optimization of hydrolysis conditions by RSM

A white rot fungus, identified as Trametes hirsuta based on morphological and phylogenetic analysis, was found to contain efficient cellulose degrading enzymes. The strain showed maximum endoglucanase (EG), cellobiohydrolase (CBH) and ß-glucosidase (BGL) activities of 55, 0.28 and 5.0 U/mg-protein, respectively. Rice straw was found to be a potentially good substrate for growth of T. hirsuta for cellulase production. Statistical experimental design was used to optimize hydrolysis parameters such as pH, temperature, and concentrations of substrates and enzymes to achieve the highest saccharification yield. Enzyme concentration was identified as the limiting factor for saccharification of rice straw. A maximum saccharification rate of 88% was obtained at an enzyme concentration of 37.5 FPU/g-substrate after optimization of the hydrolysis parameters. The results of a confirmation experiment under the optimum conditions agreed well with model predictions. T. hirsuta may be a good choice for the production of reducing sugars from cellulosic biomass.     

Increasing <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> laccase production by culture medium optimization and copper/lignin synergistic induction

Laccases have great biotechnological potential in diverse industries as they catalyze the oxidation of a broad variety of chemical compounds. Production of laccases by basidiomycetes has been broadly studied as they secrete the enzymes, grow on cheap substrates, and they generally produce more than one isoenzyme (constitutive and/or inducible). Laccase production and isoenzyme profile can be modified through medium composition and the use of inducers. The objective of this work was to increase laccase production by Pleurotus ostreatus CP-50 through culture medium optimization and the simultaneous use of copper and lignin as inducers. Increased fungal growth was obtained through the use of a factorial fractional experimental design 2^6–2 where the influence of the nature and concentration of carbon and nitrogen sources was assessed. Although specific laccase production (U/mg biomass) decreased when malt extract medium was supplemented with carbon and nitrogen sources, fungal growth and laccase volumetric activity increased four and sixfold, respectively. The effect of media supplementation with copper and/or lignin on laccase production by P. ostreatus CP-50 was studied. A positive synergistic effect between copper and lignin was observed on laccase production. Overall, the use of an optimized medium and the simultaneous addition of copper and lignin improved growth, laccase volumetric activity, and process productivity by 4-, 60-, and 10-fold, respectively.     

Laccase production by polyporus versicolor on different substrates

Extracellular production of laccase (E.C. 1.10.3.2) by Polyporus versicolor was studied on lignin, complex and defined media. Although the production of enzyme was more on lignin the specific activity was more on malt extract. Laccase was produced on all the media tested i.e. phenolic compounds, lignins and sugars alone, and in combination with malt extract, excepting salicylic acid. On single source media maximum yields of enzyme were obtained on polyfon followed by resorcinol, reax and lignin. As compared to phenolic compounds the enzyme production was low on sugars. Addition of malt extract enhanced the enzyme yield which was maximum in the case of lignin followed by gallic acid.     

Catalytic Efficiency of some Mediators in Laccase-Catalyzed Alcohol Oxidation

The enzyme laccase oxidises phenolic groups of lignin but not the non-phenolic ones. Redox mediators activate laccase towards the non-phenolic groups, particularly the benzyl alcohols. The oxidation step is performed by the oxidised form of the mediator, generated on its interaction with laccase. The oxidised mediator can follow an electron transfer, a radical hydrogen atom transfer or an ionic mechanism in the oxidation of the non-phenolic subunits. Support for these conclusions is provided by (i) investigating the product pattern with suitable probe substrates, (ii) measuring the intramolecular kinetic isotope effect. Determination of electrochemical properties and bond dissociation energies via semiempirical calculations enabled us to rationalise the origin of the different mechanistic behaviour of the mediators. Finally, a comparison of different laccase-mediator-systems (LMS), when applied to the delignification of wood pulp, indicates violuric acid as the most efficient mediator, in an oxidation that is selectively directed towards lignin only.     

Potential of ligninolytic enzymatic complex produced by white‐rot fungi from genus Trametes isolated from Bulgarian forest soil

Because of the crucial role of ligninolytic enzymes in a variety of industrial processes, the demand for a new effective producer has been constantly increasing. Furthermore, information on enzyme synthesis by autochthonous fungal strains is very seldom found. Two fungal strains producing ligninolytic enzymes were isolated from Bulgarian forest soil. They were identified as being Trametes trogii and T. hirsuta. These two strains were assessed for their enzyme activities, laccase (Lac), lignin peroxidase (LiP) and Mn‐dependent peroxidase (MnP) in culture filtrate depending on the temperature and the type of nutrient medium. T. trogii was selected as the better producer of ligninolytic enzymes. The production process was further improved by optimizing a number of parameters such as incubation time, type of cultivation, volume ratio of medium/air, inoculum size and the addition of inducers. The maximum activities of enzymes synthesized by T. trogii was detected as 11100 U/L for Lac, 2.5 U/L for LiP and 4.5 U/L for MnP after 14 days of incubation at 25°C under static conditions, volume ratio of medium/air 1:6, and 3 plugs as inoculum. Among the supplements tested, 5% glycerol increased Lac activity to a significant extent. The addition of 1% veratryl alcohol had a positive effect on MnP.     

Induction of laccase production inCyathus bulleri under shaking and static culture conditions

Laccase production byCyathus bulleri was lower in lignins and phenolic compounds as compared to malt extract medium (8 U/mL) which increased significantly on supplementing these compounds with malt extract. Of the different lignins and phenolic compounds, Reax, lignin and orcinol exhibited maximum laccase formation (12 and 68 U/mL, respectively) under static culture conditions, while sugars repressed it. Laccase activity inC. bulleri was higher under static than under shaking cultivation conditions. Moreover, agitation repressed laccase formation even in the presence of inducers.