Enhanced production of laccase fromTrametes sp. by combination of various inducers

In this study, we have attempted to determine the optimum concentration of inducers responsible for efficient laccase production by the white-rot fungus,Trametes sp. Variations in laccase activity were investigated with changing concentrations of 2,5-xylidine, syringaldazine, ABTS, and guaiacol. Enhancement of peak laccase activity was achieved via the combination of 2,5-xylidine with ABTS, syringaldazine, or guaiacol, resulting in increases of up to 359, 313, and 340%, respectively, as compared to control values. Among the tested inducers, the addition of 0.1 mM of ABTS coupled with 1.0 mM of 2,5-xylidine in the medium after 24 h of cultivation proved optimal with regard to laccase enzyme production.     

Demethylation and delignification of kraft pulp by Trametes versicolor laccase in the presence of 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate)

Summary Bleaching of hardwood kraft pulp by Trametes versicolor was accompanied by release and accumulation of methanol, which was produced by demethylation of the pulp. A partial demethylation of the pulp was observed with isolated laccase I from T. versicolor. The extent of demethylation by laccase was increased to the level released by the fungus by addition of 2,2-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS). Methanol release by the laccase/ABTS combination was followed by slower kappa reduction. Both methanol release and kappa reduction were dependent on laccase and ABTS concentrations. The fungus did not produce a stable equivalent of ABTS during bleaching, because extracellular culture fluid from bleaching cultures gave only the same methanol release from pulp as laccase I. Pulp viscosity, an indicator of cellulose chain length, was decreased only slightly by laccase. Thus the enzyme in the presence of ABTS, unlike the fungus, specifically attacks lignin.Offprint requests to: R. Bourbonnais     

Synergistic effect of laccase mediators on pentachlorophenol removal by Ganoderma lucidum laccase

Laccases have low redox potentials limiting their environmental and industrial applications. The use of laccase mediators has proven to be an effective approach for overcoming the low redox potentials. However, knowledge about the role played by the mediator cocktails in such a laccase-mediator system (LMS) is scarce. Here, we assembled different dual-agent mediator cocktails containing 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), vanillin, and/or acetovanillone, and compared their mediating capabilities with those of each individual mediator alone in oxidation of pentachlorophenol (PCP) by Ganoderma lucidum laccase. Cocktails containing ABTS and either vanillin or acetovanillone strongly promoted PCP removal compared to the use of each mediator alone. The removal enhancement was correlated with mediator molar ratios of the cocktails and incubation times. Analysis of the kinetic constants for each mediator compound showed that G. lucidum laccase was very prone to react with ABTS rather than vanillin and acetovanillone in the cocktails. Moreover, the presence of the ABTS radical (ABTS^+•) and vanillin or acetovanillone significantly enhanced PCP removal concomitant with electron transfer from vanillin or acetovanillone to ABTS^+•. These results strongly suggest that vanillin and acetovanillone mediate the reaction between ABTS and PCP via multiple sequential electron transfers among laccase and its mediators.     

Oxidation of aromatic alcohols by laccase from Trametes versicolor mediated by the 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) cation radical and dication

Oxidation of aromatic alcohols, such as non-phenolic lignin model compounds, by oxidised species of 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) has been investigated. The cation radical and dication formed from ABTS were both capable of oxidising aromatic alcohols to aldehydes. The reactions terminated at the level of the aldehyde and no acids were formed. The cation radical and dication worked in a cycle as an electron-transfer compound between an oxidant and alcohol. In addition to the oxidation of the primary benzyl-hydroxyl group, an oxidation of the secondary α-hydroxyl group to the ketone by the dication was possible. All distinguishing features of these reactions corresponded to the results of the oxidation performed by the laccase of Trametes versicolor in the presence of ABTS. The decomposition products from the dication alone and ABTS with laccase confirmed the supposition that the dication was involved in the laccase mediator system. A reaction mechanism based on deprotonation of the alcohol cation radical was predicted to play a key role in the irreversible followup reaction and to be the driving force of the process. Received: 8 June 1998 / Received revision: 23 September 1998 / Accepted: 2 October 1998     

Enhancement of laccase production by Pleurotus ostreatus and its use for the decolorization of anthraquinone dye

The white-rot fungus Pleurotus ostreatus strain 32 is an excellent producer of the industrially important enzyme laccase. Laccase was the only ligninolytic activity detected in the supernatant when the fungus was grown in liquid culture with or without shaking. Growth and laccase production in static cultivation were superior to that in agitated cultivation, and N-limited culture is of benefit to laccase production. When using cellobiose and peptone as carbon and nitrogen source, a higher activity level was obtained. 2,2′-Azino-di-(3-ethylbenzothialozin-6-sulfonic acid) (ABTS) (1 mM) was shown to be the best inducer of laccase production, reaching maximum values of about 400 U/ml. Cu²⁺ (1 mM) also had a positive effect on laccase production, activity being enhanced to 360 U/ml. In addition, anthraquinone dye SN4R can be effectively decolorized by crude laccase (30 U/ml), the rate of which was 66%. The decolorization rate was increased by 90% with ABTS (0.16%) addition as a mediator of laccase.     

Laccase activities of Penicillium chrysogenum in relation to lignin degradation

 An extracellular laccase capable of oxidizing ABTS (the diammonium salt of 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) was detected in ligninolytic cultures of Penicillium chrysogenum. By contrast, no lignin peroxidase, manganese-dependent peroxidase or aryl-alcohol oxidase was detected at any time during culturing. Both ABTS laccase activity and mineralization of dehydrogenative polymerizate of coniferyl alcohol were regulated by the C/N ratio in the medium and partially inhibited in the presence of thioglycolic acid, suggesting that both events are associated. In the presence of several known laccase inducers neither ABTS laccase activity nor mineralization rates were enhanced. However, a new laccase was detected in P. chrysogenum, able to oxidize 2,6-dimethoxyphenol but not involved in lignin mineralization. Studies with the known ligninolytic basidiomycete Trametes villosa suggest that lignin degradation by this fungus also involves the action of laccase. Received: 6 July 1995/Received revision: 28 October 1995/Accepted: 6 November 1995     

Cloning of a Laccase Gene from a Novel Basidiomycete <Emphasis Type="Italic">Trametes</Emphasis> sp. 420 and Its Heterologous Expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The laccase gene lacD, cloned from a novel laccase-producing basidiomycete Trametes sp. 420, contained 2,052 base pairs (bp) interrupted by 8 introns. lacD displayed a relatively high homology with laccase genes from other white rot fungi, whereas the homology between lacD and laccase genes from plants, insects, or bacteria was less than 25%. A 498–amino acid peptide encoded by the lacD cDNA was heterologously expressed in the Pichia pastoris strain GS115, resulting in the highest yield of laccase (8.3 × 10⁴ U/l) as determined with ABTS (2,2′-azinobis [3-ethylbenzothia-zoline-6-sulfonic acid]) as the substrate. Additionally, the enzyme activity of recombinant laccase on decolorization of some industrial dyes was assessed.     

Effect of pretreatment of hydrothermally processed rice straw with laccase-displaying yeast on ethanol fermentation

A gene encoding laccase I was identified and cloned from the white-rot fungus Trametes sp. Ha1. Laccase I contained 10 introns and an original secretion signal sequence. After laccase I without introns was prepared by overlapping polymerase chain reaction, it was inserted into expression vector pULD1 for yeast cell surface display. The oxidation activity of a laccase-I-displaying yeast as a whole-cell biocatalyst was examined with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), and the constructed yeast showed a high oxidation activity. After the pretreatment of hydrothermally processed rice straw (HPRS) with laccase-I-displaying yeast with ABTS, fermentation was conducted with yeast codisplaying endoglucanase, cellobiohydrolase, and β-glucosidase with HPRS. Fermentation of HPRS treated with laccase-I-displaying yeast was performed with 1.21-fold higher activities than those of HPRS treated with control yeast. The results indicated that pretreatment with laccase-I-displaying yeast with ABTS was effective for direct fermentation of cellulosic materials by yeast codisplaying endoglucanase, cellobiohydrolase, and β-glucosidase.     

Catalytic behavior and detoxifying ability of a laccase from the fungal strain Cerrena unicolor

The kinetics and stability of a laccase isolated and purified from the fungal strain Cerrena unicolor were studied. The enzyme was produced in a great yield without inducers. Kinetic parameters were determined by using 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as substrate. At high ABTS concentrations (> 10 mM) a substrate inhibition phenomenon appeared and an inhibition constant K_i of 24 mM was determined. The pH- and temperature-profiles as well as the sensitivity of the enzyme to several deactivation agents were almost similar to those observed with laccase from different origins. Freezing-thawing treatment, high temperature, acidic pH (< 3.0) and acetonitrile strongly affected laccase activity. The laccase showed a good ability to oxidize different phenolic substances; a significant enhancing effect was showed by ABTS acting as co-substrate. These results seem to suggest that this new laccase preparation may be suitable for environmental purposes.     

Influence of very low doses of mediators on fungal laccase activity - nonlinearity beyond imagination

Laccase, an enzyme responsible for aerobic transformations of natural phenolics, in industrial applications requires the presence of low-molecular substances known as mediators, which accelerate oxidation processes. However, the use of mediators is limited by their toxicity and the high costs of exploitation. The activation of extracellular laccase in growing fungal culture with highly diluted mediators, ABTS and HBT is described. Two high laccase-producing fungal strains, Trametes versicolor and Cerrena unicolor, were used in this study as a source of enzyme. Selected dilutions of the mediators significantly increased the activity of extracellular laccase during 14 days of cultivation what was distinctly visible in PAGE technique and in colorimetric tests. The same mediator dilutions increased demethylation properties of laccase, which was demonstrated during incubation of enzyme with veratric acid. It was established that the activation effect was assigned to specific dilutions of mediators. Our dose-response dilution process smoothly passes into the range of action of homeopathic dilutions and is of interest for homeopaths.     

Studies on homoveratric acid transformation by the ligninolytic fungus Pleurotus eryngii

Homoveratric acid (HVA) degradation was observed in cultures of Pleurotus eryngii lacking lignin peroxidase (LiP) activity. Extracellular enzymes seemed responsible for this transformation, and the lack of activity after ultrafiltration of the culture liquid suggests that the presence of some low-molecular-size compounds is required. This hypothesis is supported by rapid HVA transformation after addition of the synthetic laccase substrate 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) to the ultrafiltered liquid. HVA transformation by the extracellular enzymes from P. eryngii takes place via C-C breakdown and formation of veratryl alcohol, which is further transformed into veratraldehyde. The same major compounds were found during HVA transformation by LiP from Phanerochaete chrysosporium, but this reaction was not stimulated by ABTS. Although the involvement of other enzymes cannot be ruled out, purified laccase from Pleurotus eryngii caused the same HVA transformation pattern in presence of ABTS. Moreover, veratryl alcohol oxidation by P. eryngii laccase was demonstrated in the presence of ABTS. These results suggest that enzymatic systems lacking LiP could be responsible for natural degradation of lignin.     

A newly isolated <Emphasis Type="Italic">Paecilomyces</Emphasis> sp. WSH-L07 for laccase production: isolation,identification, and production enhancement by complex inducement

Laccase can catalyze the oxidation of a wide range of organic and inorganic substrates. In this study, an easily detectable method was employed for screening laccase-producing microorganisms by using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as laccase-secretion indicator. A novel laccase-producing strain was isolated and identified as Paecilomyces sp. WSH-L07 according to the morphological characteristics and the comparison of internal transcribed spacer (ITS) ribosomal DNA (rDNA) gene sequences. In further investigation, the production of laccase by Paecilomyces sp. WSH-L07 was greatly enhanced by the nontoxic inducers of copper sulphate and methylene blue. Under the induction of 50 μM copper sulphate and 20 μM methylene blue, the maximum laccase production was obtained. When these inducers were added into cultivation medium at 24 h and 12 h, respectively, an increment of about 100 times of laccase activity compared with that of in inducer-free medium and about two times of that of in single copper-supplemented medium was observed. Compared with other Paecilomyces species, Paecilomyces sp. WSH-L07 exhibit the better laccase-producing characteristics with an activity of 1,650 U/l on the eighth day, suggesting its potential ability for industrial application.     

Purification and characterization of laccase produced by a white rot fungus Pleurotus sajor-caju under submerged culture condition and its potential in decolorization of azo dyes

An extracellular laccase was isolated and purified from Pleurotus sajor-caju grown in submerged culture in a bioreactor, and used to investigate its ability to decolorize three azo dyes. The extracellular laccase production was enhanced up to 2.5-fold in the medium amended with xylidine (1 mM). Purification was carried out using ammonium sulfate (70% w/v), DEAE-cellulose, and Sephadex G-100 column chromatography. The enzyme was purified up to 10.3-fold from the initial protein preparation with an overall yield of 53%. The purified laccase was monomeric with an apparent molecular mass of 61.0 kDa. The purified enzyme exerted its optimal activity with 2,2-azino–bis(3-ethylbenzo-thiazoline-6-sulfonate (ABTS) and oxidized various lignin-related phenols. The catalytic efficiencies k _cat/K _m determined for ABTS and syringaldazine were 9.2×10⁵ and 8.7×10⁵, respectively. The optimum pH and temperature for the purified enzyme was 5.0 and 40 °C, respectively. Sodium azide completely inhibited the laccase activity. The absorption spectrum revealed type 1 and type 3 copper signals. The purified enzyme decolorized azo dyes such as acid red 18, acid Black 1, and direct blue 71 up to 90, 87, and 72%, respectively. Decolorization ability of P. sajor-caju laccase suggests that this enzyme could be used for decolorization of industrial effluents.     

A surfactant tolerant laccase of <Emphasis Type="Italic">Meripilus giganteus</Emphasis>

A laccase (Lcc1) from the white-rot fungus Meripilus giganteus was purified with superior yields of 34% and 90% by conventional chromatography or by foam separation, respectively. Size exclusion chromatography (SEC) and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) yielded a molecular mass of 55 kDa. The enzyme possessed an isoelectric point of 3.1 and was able to oxidize the common laccase substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at a pH of 2.0, whereas the enzyme was still able to oxidize ABTS and 2,6-dimethoxyphenol (DMP) at pH 6.0. Lcc1 exhibited low K _m values of 8 μM (ABTS) and 80 μM (DMP) and remarkable catalytic efficiency towards the non-phenolic substrate ABTS of 37,437 k _cat/k _m (s⁻¹ mM⁻¹). The laccase showed a high stability towards high concentrations of various metal ions, EDTA and surfactants indicating a considerable biotechnological potential. Furthermore, Lcc1 exhibited an increased activity as well as a striking boost of stability in the presence of surfactants. Degenerated primers were deduced from peptide fragments. The complete coding sequence of lcc1 was determined to 1,551 bp and confirmed via amplification of the 2,214 bp genomic sequence which included 12 introns. The deduced 516 amino acid (aa) sequence of the lcc1 gene shared 82% identity and 90% similarity with a laccase from Rigidoporus microporus. The sequence data may aid theoretical studies and enzyme engineering efforts to create laccases with an improved stability towards metal ions and bipolar compounds.     

灵芝EIM-40漆酶的分离纯化及酶学性质

采用冻干浓缩、（NH4）2S04盐析、HiTrapphenyl（FF）疏水层析和QSepharose FastFlow离子交换层析对灵芝EIM-40发酵液进行分离纯化,获得纯化漆酶,纯化倍数为14．6,回收率为5．3％。SDS-PAGE银染的结果为单一条带,相对分子质量约为6．53×104。以愈创木酚和2,2-联氮-二（3-乙基-苯并噻唑-6-磺酸）二铵盐（ABTS）为催化底物进行酶学性质研究,最适pH分别为4．8和4．5,最适温度分别为55和50℃,2种底物在pH4．0。5．0范围内,温度低于50℃时,酶的稳定性都很好。以愈创木酚为底物,Km=645．0umol／L;以ABTS为底物,Km=22．2txmol／L。Cu2＋对该酶起激活作用,Fe2＋、Ca2＋、Ba2＋则完全抑制酶的活性。     

漆酶高产菌株的诱变选育及其产酶条件

以粗毛栓菌Trametesgallica为出发菌,通过紫外诱变处理其担孢子、PDA-RBBR平板变色法初筛、ABTS法测定培养液漆酶酶活力复筛,获得1株漆酶高产诱变菌株SAH-12。用高氮低碳无机盐培养液(LM3)培养时,其峰值酶活力比出发菌株高出4倍,达到5002.6U/L,且产酶稳定。对SAH-12液体培养产酶条件的研究表明:以纤维二糖和蔗糖为碳源明显优于麦麸、淀粉和葡萄糖,其最高酶活分别达18526U/L和13436U/L;有机氮源较无机氮源更有利于SAH-12漆酶的分泌,以蛋白胨、大豆粕和胰化蛋白胨为氮源时其峰值酶活分别达到20544U/L、19671U/L和16180U/L;适宜初始培养pH为4.0;ABTS、单宁酸、没食子酸对产酶均有明显的诱导作用,其中ABTS和单宁酸的诱导效果相对更好,愈创木酚和吐温80对产酶有一定的抑制作用。     

An alkalophilic laccase from Rheinheimera species isolate: Production and biobleaching of kraft pulp

Medium optimization was carried out to enhance laccase production from a novel Rheinheimera species, isolated from industrial effluent. Out of the 15 variables tested by Placket–Burman design (PBD)—yeast extract, soyabean meal, and peptone were the positively significant ones, enhancing laccase production. Both simple and complex sugars showed a negative effect on laccase production. Central composite design (CCD) of experiments, using the three positively significant variables in combinations, showed that laccase production was not affected by molar carbon, molar nitrogen levels or molar C/N ratio. Maximum laccase yield of 2.5 × 10⁵ nkat L^?1, 31 fold enhancement over the unoptimized medium, was achieved when soyabean meal (0.6%) was used alone as medium showing that laccase production was substrate dependent. Laccase was used, in the presence of 2 mM ABTS, for the biobleaching of eucalyptus kraft pulp resulting in kappa number reduction by 20% and brightness increase by 2.9%. Biobleaching improved further by sequential application of an alkalophilic xylanase (X) and laccase‐ABTS system (LAS) that decreased kappa number by 10, 15, and 35%, increased brightness by 2.7, 3.2, and 5.9% as compared to X treated, LAS treated and untreated control, respectively. XLAS treatment resulted in 15, 13, 10.9% increase in burst factor, tear factor, and viscosity with a 20% reduced consumption of elemental chlorine and hypochlorite. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Laccase activity tests and laccase inhibitors

Sulfhydryl organic compounds described as laccase inhibitors: dithiothreitol, thioglycolic acid, cysteine, diethyldithiocarbamic acid, and sodium azide were tested for their activity toward laccase of Trametes versicolor in different test systems utilising 2, 2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2, 6-dimethoxyphenol as enzyme substrates. Only sodium azide acted as a true laccase inhibitor and showed no significant interference with the enzyme tests. All other substances did not significantly inhibit the laccase activity and the previously reported inhibitory effects result from the reductions of the reaction products such as ABTS radical cation and diquinone or subsequent non-enzymatic interactions during substrate oxidation. The latter apparently forms a complex with unreacted ABTS displaying varied spectral characteristics and resulting in an underestimation of enzyme activity.     

Influence of structure on dye degradation with laccase mediator systems

A new laccase was purified from Trametes hirsuta IMA2002. The laccase had a molecular mass of 62 kDa and an isoelectric point of pH 7. It had an optimum pH of 3.0 and an optimum temperature of 55°C. The laccase was quite stable at 30°C and pH 4.0 with a half-life of more than 100 hours. On ABTS, syringaldazide, and DMP the laccase showed K_M and K_cat values of 75, 12 and 37 μM and 64, 83 and 54 s^?1, respectively. The structurally diverse commercial dyes Indigo Carmine, Lanaset Blue 2R, Diamond Black PV 200 and Diamond Fast Brown were oxidized by the laccase. While the rate and extent of decolorization of the latter dye was significantly enhanced by the presence of different types of mediators, the structurally similar azo-dye Tartrazine was not oxidized. Lanaset Blue 2R, a commercial textile dye containing an anthrachinoid structural fragment acted similarly to anthrachinone sulfonic acid by strongly enhancing the rate of the decolorization reaction. Twenty two model azo-dyes based on the molecular framework of 2,7-dihydroxy-1-phenylazonaphtalene-3,6-disulfonic acid were synthesized and the kinetics of their laccase-catalyzed decolorization was studied. Hydroxy-substituted dyes were the most susceptible to enzyme/mediator action. All reactions were well described by Michaelis–Menten-like kinetics and the Hammett free energy linear relationship could be successfully applied to describe the influence of dye structure (substituents on the aromatic ring) on decolorization. Strongly electron withdrawing substituents such as a nitro-group in the meta-position (+0.7) resulted in positive σ-constants whereas electron donating groups such as para-methyl (?0.3) resulted in negative values for σ-constants.     

Optimization of conditions for submerged cultivation of the basidiomyceteCoriolus hirsutus, a producer of extracellular laccase

The effects of various factors on the biosynthesis of extracellular laccase (EC 1.14.18.1) by the basidiomyceteCoriolus hirsutus (Wulf.:Fr.) Quel. no. 072 during submerged cultivation were examined. Optimal parameters for cultivation in a fermenter of 101 were determined: temperature, 28°C; stirrer rotation speed, 160 rpm; and the inoculum volume, 15% of the working volume of the fermenter. The filtrate contained peroxidase, laccase, and phenol oxidase activities and displayed a high thermal stability.