Reaction of substituted phenols with thermostable laccase bound to Bacillus subtilis spores

A strain of B. subtilis produced 1.8 times more laccase on sporulation medium than on non-sporulation medium. Spores oxidized mono- and di-methoxyphenols (0.1 mM) at 50 °C. The half-life of laccase bound to spores was about 2 d and the substrate was repeatedly removed by spores recovered from the reaction mixture.     

Evaluation of solid substrates for enzyme production by Coriolus versicolor, for use in bioremediation of chlorophenols in aqueous effluents

In the development of a system for the removal of chlorophenols from aqueous effluents, a range of solid substrates for the growth of Coriolus versicolor were investigated. Substrates included wood chips, cereal grain, wheat husk and wheat bran. Suitability for transformation of chlorophenols depended on laccase production by the fungus. The greatest amount of laccase (<25 Units g⁻¹ substrate) was produced on wheat husk and wheat bran over 30 days colonisation. Aqueous extracts of laccase from wheat husk and wheat bran cultures removed 100% of 2,4-dichlorophenol (50 ppm) from solution within 5 h and 75–80% of pentachlorophenol (50 ppm) within 24 h. Wheat bran was formulated into pellets with biscuit flour to provide a compact substrate for fungal immobilisation. Addition of 8–12% yeast extract to the pellets increased laccase production five-fold. Colonised pellets were added to chlorophenol solutions in 200–4000-ml bioreactors, resulting in >90% removal of chlorophenols within 100 min. Received: 10 April 2000 / Received revision: 4 July 2000 / Accepted: 10 July 2000     

Bioprocess optimization of laccase production through solid substrate fermentation using Perenniporia tephropora-L168 and its application in bioremediation of triaryl-methane dye

Laccases are multi copper oxidases that can oxidize both phenolic and nonphenolic lignin related compounds. Consequently, there has been continuous demand for laccases for the oxidative degradation of phenolic dyes in effluents. In view of this, the present work was focused on laccase production by solid substrate fermentation using a newly isolated fungus Perenniporia tephropora-L168. To intensify the laccase production, the process parameters pH, nitrogen, inducer, and substrate: water ratio were optimized by using statistical model. A set of optimal conditions noted were pH 3, nitrogen 0.001 g/L; inducer 0.5% and substrate: water ratio (1:10), which yielded laccase 1,160 U/g. The crude laccase exhibited noteworthy potential to degrade a triaryl-methane dye especially Malachite green. Also, during bioremediation studies, the statistical process optimization could achieve 81% decolourization within 180 min. The laccase treatment brought chemical transformation in malachite green as evident from UV–Visible spectra, FTIR, HPLC while toxicity against bacteria and fungi was also reduced. During phytotoxicity study, effect of treated and untreated dye on germination of seed was analyzed. Interestingly, the germination index for Vigna aconitifolia and Vigna radiata was increased by two and fourfold, respectively. Overall, this work demonstrates optimized production of laccase using Perenniporia tephropora-L168 and its efficient bioremediation potential for triaryl-methane dye.     

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     

Solid state fermentation of a Mycelia Sterilia laccase using steam-exploded wheat straw

Mycelia Sterilia YY-5, an endophytic fungus isolated from Rhus Chinensis Mill, was used in SSF for laccase production using steam-exploded wheat straw (SEWS). The fermentation period of YY-5 in solid state fermentation (SSF) shortened to 4 days compared with 5 days of submerged liquid fermentation (SmF) and the maximum laccase activity was 678.1 IU g⁻¹ substrate. The steam-explosion intensity (Log₁₀ R ₀) of SEWS had a significant effect on the growth of YY-5 and laccase activity, since SEWS could provide enough carbon source for YY-5 and inducers for laccase. The optimum SSF conditions using SEWS with Log₁₀ R ₀ = 3.597 as substrate were: inoculating with liquid inocula, keeping the solid-to-liquid ratio (S/L) for 1:4 and cultivating at 26°C. Under the optimum fermentation condition the laccase activity of YY-5 reached 849.5 ± 42.5 IU g⁻¹ substrate. The enzyme composition analysis indicated that laccase was the dominant enzyme of YY-5. Assayed with SDS-PAGE and active PAGE electrophoresis, the molecular weight of YY-5 laccase was approximately 45 kDa.     

Differences in gill raker morphology between two local populations of a benthophagous filter-feeding fish, Goniistius zonatus (Cheilodactylidae)

Gill raker morphology of a benthophagous fish Goniistius zonatus (Cheilodactylidae) (10.9–29.2 cm SL), using a filter-feeding mode, was compared between two locations (Morode and Arakashi) in southern Japan. Although gill raker number and gill raker length at the two locations did not differ, gill raker spacing was narrower relative to overall fish size at Morode than at Arakashi, mainly because gill raker width was greater at Morode. The difference of gill raker spacing is unlikely to have a genetic or physiochemical explanation. Small invertebrates (≤1.0 mm) were dominant on the substrate at Morode but were less common at Arakashi. Such small animals were consumed by many fish at Morode but were rarely exploited at Arakashi. At Morode, the narrow gill raker spacing would be effective in retaining small prey, which should be an important energy resource in this population. The difference of interraker spacing at the two locations seems to be related to available prey size at each location. Received: November 14, 2000 / Revised: February 13, 2001 / Accepted: February 28, 2001     

Differential expression of manganese peroxidase and laccase in white-rot fungi in the presence of manganese or aromatic compounds

White-rot fungi (basidiomycetes) play an important role in the degradation of lignin which is, beside cellulose, the major compound of wood. This process is catalyzed by ligninolytic enzymes, which are able to cleave oxidatively aromatic rings in lignin structure. Manganese peroxidase and laccase of white-rot-fungi are the most important of these among the ligninolytic enzymes. In addition, they are able to degrade xenobiotic aromatic polymers, persisting as environmental pollutants. Manganese and aromatic compounds have often been discussed as being inducers, enhancers or mediators of these ligninolytic enzymes. It is known that supplementing the growth medium with either Mn²⁺, veratryl alcohol or coal-derived humic acids leads to significantly enhanced extracellular ligninolytic activities. Measuring the amount of expressed mRNA of the two enzymes by quantitative RT-PCR provided evidence that the expression of manganese peroxidase was induced in the three tested white-rot fungi, Clitocybula dusenii b11, Nematoloma frowardii b19, and a straw-degrading strain designated i63–2. Laccase, on the other hand, was expressed in all three fungi with a significant basic activity even without inducer added. However, since the level of laccase mRNA was higher in cultures supplemented with any one of the tested inducers, we conclude that both manganese and the aromatic substances also increase the expression of laccase. Received: 4 February 2000 / Received revision: 11 May 2000 / Accepted: 12 May 2000     

Removal of phenols from mixtures by co-immobilized laccase/tyrosinase and Polyclar adsorption

An enzymatic method for removal of phenols from their mixtures was investigated. Phenols in an aqueous solution were removed after a two-step treatment with co-immobilized laccase and tyrosinase and Polyclar (polyvinylpolypyrrolidone). A laccase from Pyricularia oryzae and mushroom tyrosinase were co-immobilized on Mikroperl in a fixed-bed tubular bioreactor by a rapid and simple method. The support immobilized 95% of the total laccase units and 35% of the total tyrosinase units. Different mixtures of phenols were passed through the column with co-immobilized laccase and tyrosinase. This method removed 42–90% of different phenolic substances by a single passage through the bioreactor. The second step employed Polyclar for additional removal of phenolic substances from mixtures. The degree of removal depends on the nature of the phenols. Complete removal was achieved for a-naphthol, 2,4-dichlorophenol, 4-methoxyphenol, b-naphthol, 4-chloro-3-methylphenol and catehin. The operational stability of the immobilized system was 10–90 h depending on the substrate. The biocatalyst was capable of continuous transformation of different phenols in mixtures. Journal of Industrial Microbiology & Biotechnology (2000) 24, 383–388. Received 12 August 1999/ Accepted in revised form 18 February 2000     

Characterisation of the laccase-encoding gene abr2 of the dihydroxynaphthalene-like melanin gene cluster of Aspergillus fumigatus

Aspergillus fumigatus is an important pathogen of the immunocompromised host. Previously, it was shown that the polyketide synthase encoded by the pksP (alb1) gene represents a virulence determinant. pksP is part of a gene cluster involved in dihydroxynaphthalene (DHN)-like melanin biosynthesis. Because a putative laccase-encoding gene (abr2) is also part of the cluster and a laccase was found to represent a virulence factor in Cryptococcus neoformans, here, the Abr2 laccase was characterised. Deletion of the abr2 gene changed the gray-green conidial pigment to a brown color and the ornamentation of conidia was reduced compared with wild-type conidia. In contrast to the white pksP mutant, the susceptibility of the Δabr2 mutant against reactive oxygen species (ROS) was not increased, suggesting that the intermediate of DHN-like melanin produced up to the step catalysed by Abr2 already possesses ROS scavenging activity. In an intranasal mouse infection model, the Δabr2 mutant strain showed no reduction in virulence compared with the wild type. In the Δabr2 mutant, overall laccase activity was reduced only during sporulation, but not during vegetative growth. An abr2p-lacZ gene fusion was expressed during sporulation, but not during vegetative growth confirming the pattern of laccase activity due to Abr2.     

Laccase, cellulase and xylanase activities during growth ofPleurotus sajor-caju on sagohampas

Sagohampas, the fibrous pith residue left after starch extraction from sago palm, is abundant at sago-processing factories and can be used as a substrate for the production of laccase by solid substrate fermentation (SSF) withPleurotus sajorcaju, an edible mushroom. The fungus grown onhampas with an adjusted carbon : nitrogen ratio of 35:1, exhibited high laccase activity together with variable cellulase (0.3-2.8 U/g) and xylanase (0.9-10.1 U/g) activity. The maximum amount of laccase produced was approximately 17.7 U/g after 6 days of SSF using 4-week-old inoculum at a density of 10%. With the mature four-week inoculum, laccase activity increased 12-fold compared to that achieved with two-week-old inoculum. The optimum pH and temperature of the crude laccase were 6.0 and 50‡C, respectively. The apparent K_m and V_max values obtained were 0.073 mM and 0.962 U/min, respectively. The maximum laccase activity could be almost doubled after 6 days of fermentation by addition of 0.2 mM vanillin or ferulic acid; the cellulose to lignin ratio increased significantly during the 12 days of SSF, from 2.74 in the control to 3.3, when 0.2 mM of either vanillin or ferulic acid was added to the substrate.     

红平菇产漆酶条件优化及对孔雀绿染料脱色的研究

采用LNAS(低氮天冬酰胺-琥珀酸)培养基添加方式,对红平菇Pleurotus djamor HP1进行培养,检测不同时间培养液对不同底物的氧化作用,进而得到光密度值的变化情况,作为漆酶的产生及活性测定的主要依据。结果表明:在含Cu2+的培养液中漆酶最大酶活为235.4 U/L。含Cu2+的培养液添加底物木屑后漆酶最大酶活为458.8 U/L。提取经优化筛选后的培养基培养出的漆酶粗酶液,对4种具有不同化学结构的染料进行了脱色试验。结果表明:三苯基甲烷类的孔雀绿在6 h时脱色率为87.5%,蒽醌类的SN4R在24 h时脱色率为49.4%,偶氮类的甲基橙在24 h时脱色率为45%,杂环类的中性红在24 h时脱色率为23.6%。因此,显示出红平菇漆酶对孔雀绿染料脱色具有较大的应用潜力,进而对废水处理具有更好的应用前景。     

The effects of aqueous ammonia-pretreated rice straw as solid substrate on laccase production by solid-state fermentation

Chemical composition and physical structure of solid substrate have significantly impacts on fermentation performance. The aqueous ammonia was used to pretreat rice straw. Furthermore, the feasibility of pretreatment to improve laccase production was also evaluated in terms of the enzymatic digestibility, chemical structure, physical structure, and laccase production. The results showed that aqueous ammonia pretreatment could modify chemical compositions, destroy rigid structure of the lignocellulosic substrate, increase enzymatic digestibility and change water state, which were beneficial to facilitate the fungus growth and nutrition utilization. Pretreatment of lignocellulosic substrate with aqueous ammonia at 80 °C gave the best effect on laccase production, yielding 172.74 U/g laccase at 14 days, which was 3.4 times higher than that of the control. The aqueous ammonia pretreatment could alternate the physicochemical characteristics of lignocellulosic substrate, resulting in the improved laccase production, which was a promising method that might be explored in solid-state fermentation.

     

Laccase isoforms with unusual properties from the basidiomycete Steccherinum ochraceum strain 1833

Aims: To isolate and characterize the laccase isoforms from S. ochraceum 1833 – a new active producer of high extracellular laccase activity. Methods and Results: Three laccase isoforms (laccases I, II and III) with 57·5, 59·5 and 63 kDa molecular masses respectively were purified from S. ochraceum 1833 and in contrast to the known laccases had strongly pronounced absorption at 611 nm with molar extinction coefficients ranging from 7170 to 7830 mol^?1 l cm^?1. All isoforms showed maximal activity with ABTS at low pH (≤2) and temperatures in the range 70–80°C, were stable for long time of incubation at high temperature (60–80°C) and at pH values ranging from 2 to 6. Laccase II showed a higher activity and wider substrate specificity. N‐terminal amino acid sequence analysis of the purified laccase II (VQIGPVTDLH) showed 80% identity with the N‐terminal amino acid sequence of laccase from Lentinula edodes [Appl Microbiol Biotechnol 60 (2002) 327]. Conclusions: Elevated temperature optima, high thermo‐ and pH‐stabilities, the broad substrate specificity of the isoforms make the laccases from S. ochraceum 1833 a suitable model for biotechnological processes proceeding at high temperatures. Significance and Impact of the Study: For the first time, new basidiomycete strain S. ochraceum was reported as a producer of novel thermostable, pH stable, acidophilic laccases with unusual spectral properties.     

Efficient production of Al(OH)3‐immobilized laccase with a Heterobasidion annosum strain selected by microplate screening

Aims: Wild‐type white rot fungi are the most important production organisms for laccase, a promising oxidative biocatalyst with numerous applications. This study aimed at identifying novel highly productive strains, finding optimal cultivation conditions for laccase production and establishing a simple immobilization procedure. Methods and Results: By using a newly developed 96‐well microplate cultivation method, 23 species of white rot fungi, represented by 29 strains, were directly compared with regard to the amount of secreted laccase. Both, with glucose and spruce saw dust as growth substrate a Heterobasidion annosum strain and a Physisporinus vitreus strain were the most productive (730–2200 U l^?1 of secreted laccase). Cultivation conditions for laccase production with H. annosum were optimized in larger‐scale liquid cultures. Aeration with a sparger lead to a 3·8‐fold increase in laccase activity when compared to nonaerated flask cultures. More than 3000 U l^?1 laccase was produced in glucose medium supplemented with yeast extract and the inducer veratryl alcohol. Culture supernatant was incubated with short‐range ordered Al(OH)₃ particles to directly immobilize and concentrate laccase by adsorption. Active laccase was recovered in 40% yield and the Al(OH)₃‐adsorbed laccase was suitable for repeated decolourization of indigo carmine. Conclusions: Microplate cultivation allowed a large‐scale comparison of the capacity of different fungal species for laccase production. Laccase secretion of a highly productive H. annosum strain was found to vary strongly with different cultivation conditions. Adsorption to Al(OH)₃ proved to be suitable as direct immobilization technique. Significance and Impact of the Study: The microplate screening method simplifies strain and medium development for laccase production. Two novel fungal strains suitable for laccase production were identified. Procedures for simple and efficient production of immobilized H. annosum laccase were established.     

Docking Simulation and Competitive Experiments Validate the Interaction Between the 2,5-Xylidine Inhibitor and Rigidoporus lignosus Laccase

Abstract

Laccases are polyphenol oxidases which oxidize a broad range of reducing substrates, preferably phenolic compounds, and their use in biotechnological applications is increasing.

Recently, the first X-ray structure of active laccase from white rot fungus Rigidoporus lignosus has been reported containing a full complement of copper ions. Comparison among selected fungal laccases of known 3D structure has shown that the Rigidoporus lignosus laccase has a very high similarity with the Trametes versicolor laccase that, being co-crystallized with 2,5-xylidine, shows a well defined binding pocket for the substrate. Global sequence alignment between Rigidoporus lignosus and Trametes versicolor laccases shows 73% of identity but, surprisingly, there is no identity and neither conservative substitutions between the residues composing the loops directly contacting the 2,5-xylidine. Moreover the structural alignment of these two enzymes identifies in these loops a striking structural similarity proposing the question if 2,5-xylidine may bind in same enzyme pocket.

Here we report the protein-ligand docking simulation of 3D structure of Rigidoporus lignosus laccase and 2,5-xylidine. Docking simulation analyses show that spatial conformation of the two 2,5-xylidine binding pockets, despite differences in the residues directly contacting the ligand, may arrange a similar pocket that allows a comparable accommodation of the inhibitor. To validate these results the binding of 2,5-xylidine in the substrate cavity has been confirmed by kinetic competitive experiments.     

Die Phenoloxydasen des Ascomyceten Podospora anserina

Summary The ascomycete Podospora anserina forms two different phenoloxidases. According to their substrate specifity they can be classified as laccase and tyrosinase, respectively. While laccase is found in freshly prepared extracts in a highly active form, the tyrosinase needs to be activated by cold- or heat-treatment. Further characteristics of the two enzymes are: their different solubilities in ammoniumsulfate solution and their different heat inactivation rates. The half life times of partially purified extracts at 60° C are for laccase 2.5 min and for tyrosinase 23 min.     

Extracellular ligninolytic enzymes by Lentinus polychrous Lév. under solid-state fermentation of potential agro-industrial wastes and their effectiveness in decolorization of synthetic dyes

Six agro-industrial wastes were evaluated as a support for ligninolytic enzyme production by the white-rot fungus Lentinus polychrous Lév. under solid-state fermentation. Enzyme production was markedly different according to the substrate used. Rice bran (RB) yielded the highest laccase activity of 1,449 U/L (after 21 days of culture) with specific activity of 4.4 U/g substrate. Rice bran supplemented with rice husk (RH) (2:1 by wt) showed high laccase activity of 1,425 U/L with specific activity of 10.0 U/g substrate (after 17 days of culture). The crude enzyme of the RH-RB culture also contained manganese peroxidase (MnP) and manganese-independent peroxidase (MIP) activities in relative proportions of 1.9:1.4:1 of laccase:MnP:MIP, respectively. Zymogram studies showed the same isoenzyme pattern with these ligninolytic enzymes. The high enzyme production level and low substrate cost of SSF-L. polychrous Lév. suggest that it has potential for industrial applications. Our studies showed that the crude enzyme from this culture exhibited in vitro decolorization of Indigo Carmine. The highest efficiency of dye decolorization was observed under alkaline conditions (pH 9.0) at an initial dye concentration of 10 mg/L. The rather high pH conditions and high efficiency in Indigo Carmine decolorization make the enzyme further interest for the applications in treatment of waste water from the textile industry, which contains synthetic dyes.     

Induction and Characterization of Laccase in the Ligninolytic Fungus Pleurotus eryngii

Two protein bands with laccase activity were found after PAGE of culture liquid or mycelium extract of Pleurotus eryngii, grown on glucose–ammonium tartrate–yeast extract medium with and without inducers. A major and a minor laccase band were observed in the basal medium. The intensity of the major band (laccase I) did not change after the addition of inducers. However, the minor band (laccase II), characterized by higher electrophoretic mobility, was strongly induced by wheat–straw alkalilignin and vanillic and veratric acids. Laccase activity in the basal medium had an optimum pH of 4.5 and was stable from pH 3 to 10 during 24 h at room temperature. This enzyme had wide substrate specificity on hydroquinones, methoxy-substituted monophenols, and aromatic amines. In general, laccase activity was found only with compounds having a redox potential lower than 0.5 mV. The highest activity was obtained with methoxy- and methyl-substituted p-hydroquinones and aromatic diamines. Some activity also occurred with the aliphatic compound 3,5-cyclohexadiene-1,2-diol. Received: 22 April 1996 / Accepted: 29 June 1996     

Effect of Nutritional Parameters on Laccase Production by the Culinary and Medicinal Mushroom, <Emphasis Type="Italic">Grifola frondosa</Emphasis>

Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 μM Cu over the basal level (1.6 μM Cu) and peak levels observed at 300 μM Cu were 7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1–2 mM Cu²⁺. SDS-PAGE combined with activity staining revealed the presence of a single protein band (M _r 70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct laccase protein band (M _r 45 kDa) was observed in cultures supplemented with 1–2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. Using 2,2′-azino-bis(ethylbenzothiazoline-6-sulfonate) (ABTS) as substrate, the optimal temperature and pH values for laccase activity were 65°C and pH 2.2, respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development.     

Effect of nutritional parameters on laccase production by the culinary and medicinal mushroom, Grifola frondosa

Summary Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 (M Cu over the basal level (1.6 mM Cu) and peak levels observed at 300 mM Cu were ∼ ∼7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1–2 mM Cu²⁺. SDS-PAGE combined with activity staining revealed the presence of a single protein band (M _r ∼ ∼70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct second laccase protein band (M _r␣∼ ∼45 kDa) was observed in cultures supplemented with 1–2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. The optimal temperature and pH values for laccase activity were 65 °C and pH 2.2 (using 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) {ABTS} as substrate), respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development.