Laccase activity and putative laccase genes in marine-derived basidiomycetes

Studies of laccases from marine-derived fungi are limited. In the present work, putative laccase genes from three marine-derived basidiomycetes and their laccase activities were evaluated. High amounts of laccase were produced by the fungal strains Marasmiellus sp. CBMAI 1062 (971.2 U L⁻¹) and Peniophora sp. CBMAI 1063 (709.03 U L⁻¹) when grown for 21 d at 28 °C in MA2ASW medium prepared with artificial seawater. Marine-derived basidiomycetes produced multiple distinct laccase sequences of about 200 bp with 73–90 % similarity to terrestrial basidiomycete laccases. Marasmiellus sp. CBMAI 1062 and Tinctoporellus sp. CBMAI 1061 showed the greatest laccase gene diversity with three and four distinct putative laccase sequences, respectively. This is the first report of laccase genes from marine-derived fungi, and our results revealed new putative laccases produced by three basidiomycetes.     

Purification and biochemical characterization of a laccase from the aquatic fungus <Emphasis Type="Italic">Myrioconium</Emphasis> sp. UHH 1-13-18-4 and molecular analysis of the laccase-encoding gene

Myrioconium sp. strain UHH 1-13-18-4 is an ascomycete anamorph isolated from the river Saale, Central Germany. An extracellular, monomeric, and glycosylated laccase with a molecular mass of 72.7 kDa as determined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and an isoelectric point below 2.8 was purified from CuSO₄ and vanillic acid amended liquid fungal cultures grown in malt extract medium. The catalytic efficiencies (k _cat/K _m) for the oxidation of syringaldazine, 2,6-dimethoxyphenol, and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate) were 67.3, 46.9, and 28.2 s⁻¹ mM⁻¹, respectively, with K _m values of 4.2, 67.8, and 104.9 μM. After pre-incubation at different pH values and temperatures for 1 h, more than 80% of the initial laccase activity was retained between pH 4 to 6 and 15°C. The laccase-encoding gene was identified and sequenced at both the genomic and complementary DNA (cDNA) level, and corresponding structural characteristics and putative regulatory elements of the promoter region are reported. The identification of two tryptic peptides of the purified enzyme by mass spectrometry confirmed the identity of the functional laccase protein with the translated genomic sequence of the Myrioconium sp. laccase. Myrioconium sp. laccase shows the highest degree of identity with laccases from ascomycetes belonging to the family Sclerotiniaceae, order Helotiales.     

Characterization of an extracellular laccase, PbLac1, purified from Polyporus brumalis

Polyporus brumalis (strain ibrc05015) secreted high amounts of laccases (Lacs) in liquid medium. With 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) as a substrate, Lac activity was 7.72 U ml⁻¹ and this strain secreted a maximum 0.23 mg ml⁻¹ of total protein. The enzyme, PbLac1 was purified to homogeneity using hydrophobic and anion-exchange chromatography. The purified PbLac1 had a molecular mass of 63.4 kDa as determined by polyacrylamide-gel electrophoresis. PbLac1 oxidized a wide range of substrates such as 3,4-dihydroxy l-phenylalanine (l-DOPA) and catechol, but not tysorine. The activity of PbLac1 was increased by addition of 10.0 mM Cu²⁺. PbLac1 could decolorize several industrial dyes, such as Remazol Brilliant Blue R known as model dyes of environmental xenobiotics. In addition, PbLac1 decolorized a wide range of substrates, such as the carcinogen, Poly R-478, in the presence of violuric acid as mediator. The E^° value of PbLac1 was 0.80 V ± 0.01 versus normal hydrogen electrode, which is a very high redox potential compared to those of other basidiomycetous Lacs. These results suggest the potential utility of PbLac1 for industrial applications.     

Potential of Trametes trogii culture fluids and its purified laccase for the decolorization of different types of recalcitrant dyes without the addition of redox mediators

Trametes trogii BAFC 463 culture fluids (containing 110 U ml⁻¹ laccase; 0.94 U ml⁻¹ manganese peroxidase), as well as its purified laccase were capable of decolorizing azoic, indigoid, triphenylmethane, anthraquinonic and heterocyclic dyes, in the absence of redox mediators. Six dyes: RBBR, Indigo Carmine, Xylidine, Malachite Green, Gentian Violet and Bromophenol Blue were almost completely degraded (more than 85% decolorization after 1 d) by either laccase or T. trogii itself in culture, proving the role of the enzyme in dye decolorization. The purified laccase also decolorized 65% of Fast Blue RR and 30% of Azure B and Methylene Blue after 24 h. The use of redox mediators significantly increased the decolorization rates (90% decolorization of Azure B after 1 h). 1-hydroxybenzotriazole resulted the best redox mediator, but the natural mediator p-hydroxybenzoic acid also demonstrated its efficiency for dye decolorization. Due to their ability to decolorize recalcitrant dyes without addition of redox mediators, high laccase activities, high thermostability and efficient decolorization at 70 °C and pH 7.0, even in the presence of high concentrations of heavy metals (100 mM Cu⁺², Pb⁺² or Cd⁺²) or in a synthetic dyebath, T. trogii culture fluids could be effectively used to decolorize synthetic dyes from effluents.     

Production of laccase, manganese peroxidase and lignin peroxidase by Brazilian marine-derived fungi

Marine-derived fungi are a potential for the search of new compounds with relevant features. Among these, the ligninolytic enzymes have potential applications in a large number of fields, including the environmental and industrial sectors. This is the work aimed to evaluate the enzymatic activities of three marine-derived fungi (Aspergillus sclerotiorum CBMAI 849, Cladosporium cladosporioides CBMAI 857 and Mucor racemosus CBMAI 847) under different carbon sources and salinity conditions by using statistical experimental design. MnP, LiP and laccase were detected when these fungi were cultured in malt extract, however when grown on basal medium containing glucose and wheat bran LiP was not detected and yet an increase in MnP and laccase was observed. Statistical analysis through surface responses was performed and results showed high values of MnP and laccase activities under 12.5% and 23% (w/v) salinity, highlighting the potential use of these fungi for industrial applications and in bioremediation of contaminated sites having high salt concentrations. The highest values for LiP (75376.34 UI L⁻¹), MnP (4484.30 IU L⁻¹) and laccase (898.15 UI L⁻¹) were obtained with the fungus M. racemosus CBMAI 847 and it is the first report concerning ligninolytic enzymes production by a zygomycete from this genus.     

Biochemical and molecular characterization of a novel laccase from selective lignin-degrading white-rot fungus Echinodontium taxodii 2538

A novel laccase was isolated and characterized from a new selective lignin-degrading white-rot fungus Echinodontium taxodii 2538, in which a high yield of laccase was obtained. No laccase isoenzyme was detected in the synthetic liquid media. The purified laccase (designated as EtL2538) had an apparent molecular mass of 56 kDa, pI value of 3.1, and N-terminal amino acid sequence of GIGPVTDLHIVNAAV. EtL2538 showed optimum pH at 3.0 and optimum temperature at 60 °C using ABTS as the substrate. EtL2538 revealed superior thermostability, and retained over 80% of its original activity after incubation for 2 h at 50 °C. The laccase gene, etl2538, was also cloned and sequenced. This gene encoded a mature laccase protein containing 499 amino acids (aa) preceded by a signal peptide of 21 aa, and the deduced protein sequence contained four copper-binding conserved domains of typical laccase protein. EtL2538 was further used in lignin oxidation and dye decolorization. Even without the existence of redox mediators, EtL2538 could cleave the methoxyl groups and β-O-4 ether linkages in lignin from bamboo, and significantly decolorize malachite green and RBBR. These novel properties of EtL2538 may render it as a potential biocatalyst for biotechnological and environmental applications.     

Purification and characterization of laccase II of Aspergillus nidulans

Sexual development in Aspergillus nidulans is a morphogenetic differentiation process triggered by internal and environmental signals. As a first step in analyzing the developmental pathway at the molecular level, laccase II (EC 1.10.3.2), which is specifically expressed in early stages of fruitbodies, was isolated. The enzyme was purified to apparent homogeneity from a mutant strain (SMS1) in which the sexual cycle dominates and the number of cleistothecia is increased tenfold. Laccase II was enriched 560-fold to a specific activity of 892 U (mg protein)^–1. The apparent molecular mass was determined to be 80 kDa under denaturing conditions and to be 100–120 kDa under native conditions. The internal peptide sequences gained from the protein will allow the isolation of the corresponding gene as a first step in determining the key regulators of sexual development. Received: 8 January 1998 / Accepted: 14 April 1998     

Role of laccase in lignin degradation by white-rot fungi

Abstract Laccase is commonly found in white-rot fungi and catalyses the abstraction of one electron from the phenolic hydroxyl group to polymerize or depolymerize lignin model compounds. Laccase degrades both β-1 and β-O-4 dimers via C _α - C _β cleavage, C _α oxidation and alkyl-aryl cleavage. Also, aromatic ring cleavage may be detected following the action of laccase. Laccase can also oxidize non-phenolic compounds when primary mediators, such as 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate), are co-present. Laccase produces Mn(III) chelates which allow wood-decaying enzymes to penetrate wood cell walls. Laccase is considered to be capable of degrading lignin together with lignin peroxidase and manganese peroxidase.     

Heterologous production of a temperature and pH-stable laccase from Bacillus vallismortis fmb-103 in Escherichia coli and its application

To enhance laccase yield, the laccase gene from Bacillus vallismortis fmb-103 was cloned and heterologously expressed in Escherichia coli BL21 (DE3) cells. The auto-induction strategy was applied during fermentation, and the process was controlled, as follows: Cu²⁺ was added when the optical density at 600 nm (OD₆₀₀) was 0.3, the fermentation temperature was adjusted to 16 °C when the OD₆₀₀ was 0.9, and fermentation was stopped after 50 h. The yield of recombinant laccase was up to 3420 U/L, as assayed by 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). Recombinant laccase was purified 4.47-fold by heating for 10 min at 70 °C and dialyzing against 50–60% ammonium sulfate, retained more than 50% activity after 10 h at 70 °C, and demonstrated broad pH stability. Malachite green was efficiently degraded by recombinant laccase, especially in combination with mediators. These results provided a basis for the future application of recombinant laccase to malachite green degradation.     

Purification and partial characterization of a thermostable laccase from an unidentified basidiomycete

A thermostable laccase was isolated from an unidentified fungal isolate [Enz. Microb. Technol. 33 (2003) 212], and tentatively named UD4. This work indicates that the enzyme has unique properties other than its thermostability. Investigation into the kinetic parameters of the thermostable laccase yielded an unusually high affinity for ABTS as a substrate (low K_m) when compared with available published data for other laccase isozymes. The specificity constant (k_cat/K_m) was found to be considerably higher than laccase from other sources and is comparable to “white” laccase from Pleurotus ostreatus (POXA1). However, POXA1 isozyme exhibits a large turnover number (k_cat) that contributes to its high specificity constant whereas the high specificity constant for UD4 laccase is achieved by having a high substrate affinity. The UD4 thermostable laccase, like most other laccases, is able to utilize guaiacol as a substrate, whereas POXA1 is unable to oxidize guaiacol, indicating a broader substrate range for the thermostable laccase from UD4. The thermostable laccase is inhibited by sodium azide through non-competitive inhibition, and by thioglycolic acid and hydroxylamine through competitive inhibition. The high specificity constant, substrate affinity and broader substrate range of the thermostable laccase from UD4 indicates that it is a highly favourable candidate enzyme for industrial application.     

Characterization of Trametes versicolor laccase for the transformation of aqueous phenol

Laccase (oxygen oxidoreductase, EC 1.10.3.2) from Trametes versicolor was thoroughly characterized in terms of its catalytic stability and its effectiveness as a biocatalyst under various reaction conditions when using phenol as a model substrate. This enzyme demonstrated high or moderate degrees of stability at pHs from 5 to 8 at 25 degrees C and at temperatures from 10 to 30 degrees C at pH 6. Exponential decay expressions were successfully used to model laccase inactivation when incubated under various conditions of pH and temperature. Phenol transformation was optimum at pH 6, but significant transformation was observed over a pH range of 4-7, provided that sufficient laccase was present in the reacting solution. Partial inactivation of laccase was observed during the oxidation of phenol, even under conditions of optimal stability (pH 6 and 25 degrees C).     

平菇漆酶基因在毕赤酵母中的分泌表达及酶学性质研究

采用RTPCR技术克隆到一个平菇(Pleurotusostreatus)漆酶基因的全长cDNA,命名为lccPo1,其序列提交GenBank,登录号为AY450404。将其ORF克隆到毕赤酵母表达载体pHBM906,转化3株毕赤酵母GS115、KM71和SMD1168,该漆酶基因在3种毕赤酵母菌株中均实现了分泌表达。3种摇瓶培养条件①25℃,1.0%(VV)甲醇;②20℃,1.0%(VV)甲醇;③20℃,0.5%(VV)甲醇,进行比较研究后发现适当提高甲醇浓度有利于漆酶在低温条件下表达,而降低培养温度到20℃则可以提高漆酶的产量2～6倍。3株重组毕赤酵母在其最适反应条件下测得三者粗酶液最高漆酶酶活分别为3.19UmL[GS115(pHBM565)]、2.56UmL[KM71(pHBM565)]和2.49UmL[SMD1168(pHBM565)]。对重组酶进行相关的酶学性质分析表明,三者的最适反应pH值约为4.2,最适反应温度约为60℃。重组毕赤酵母GS115(pHBM565)所产酶的热稳定性稍好,在pH稳定性方面三者没有太大差异。     

Purification and characterization of laccase from <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> and decolorization of an anthraquinone dye by the enzyme

The white rot fungus Pycnoporus sanguineus produced high amount of laccase in the basal liquid medium without induction. Laccase was purified using ultrafiltration, anion-exchange chromatography, and gel filtration. The molecular weight of the purified laccase was estimated as 61.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme oxidized typical substrates of laccases including 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate), 2,6-dimethoxyphenol, and syringaldazine. The optimum pH and temperature for the purified laccase were 3.0 and 65°C, respectively. The enzyme was stable up to 40°C, and high laccase activity was maintained at pH 2.0–5.0. Sodium azide, l-cysteine, and dithiothreitol strongly inhibited the laccase activity. The purified enzyme efficiently decolorized Remazol Brilliant Blue R in the absence of added redox mediators. The high production of P. sanguineus laccase as well as its decolorization ability demonstrated its potential applications in dye decolorization.     

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.     

天然介体在产漆酶真菌氧化蒽和芘中的重要作用（英文稿）

【目的】研究了氧化还原介体在产漆酶真菌氧化蒽和芘的作用。【方法】通过非变性电泳和酶活力分析。【结果】发现血红密孔菌Z-1和木蹄层孔菌Z-5只产漆酶,其最大酶产量分别为11.90 U/mL和4.83 U/mL,不产木质素过氧化酶和锰过氧化物酶。木蹄层孔菌Z-5的胞外液尽管具有较低的漆酶活性,但是氧化了74.3%的蒽和12.4%的芘,高于血红密孔菌Z-1对蒽和芘的氧化率,提示天然介体可能存在于真菌胞外液中并且影响了漆酶对多环芳烃的氧化。实验进一步表明,木蹄层孔菌Z-5灭活和不灭活的超滤液以及灭活的胞外液对纯漆酶氧化多环芳烃的促进作用均大于血红密孔菌Z-1,说明木蹄层孔菌Z-5的天然介体比血红密孔菌Z-1能够更为有效地促进多环芳烃氧化。【结论】氧化还原结体在产漆酶真菌降解底物过程中发挥了重要作用,这也解释了木蹄层孔菌Z-5胞外液尽管漆酶活性不高,但是具有较大多环芳烃氧化率的原因。     

Phenolic removal of olive-mill dry residues by laccase activity of white-rot fungi and its impact on tomato plant growth

We studied the influence of the laccase activity of two white-rot fungi on the toxic effect of water-soluble substances from dry residues of olives (ADOR) on tomato plants. Pycnoporus cinnabarinus and Coriolopsis rigida decreased the phenol content of ADOR to 73% after 15 days. P. cinnabarinus and C. rigida produced laccase activity after 5 and 15 days, respectively, and the highest activity in both fungi was detected at 20 days. The treatment of ADOR with these white-rot fungi decreased the phytotoxicity of this residue on tomato plants. A close relationship was found between the amount of laccase produced, the decrease in phenol content of ADOR by the saprobic fungi, decrease of phytotoxicity of ADOR, and the increase in dry weight of tomato plants. These results show that phenol removal by the laccase activity of white-rot fungi can be important in the elimination of phytotoxic substances present in olive-mill dry residues.     

Amelioration of the ability to decolorize dyes by laccase: relationship between redox mediators and laccase isoenzymes in Trametes versicolor

The effect of redox mediators in the dye decolorization by two laccase isoenzymes from Trametes versicolor cultures supplemented with barley bran has been investigated. All the redox mediators tested, 1-hydroxybenzotriazole (HBT), promazine (PZ), para-hydroxybenzoic acid (pHBA) and 1-nitroso-2-naphthol-3,6-disulfonic acid (NNDS), led to higher dye decolorization than those obtained without mediator addition. Among the different tested mediators, PZ was the most effective one at a low range of concentration (0.5–50 μM) and the natural mediator employed, pHBA did not improve significantly the degree of decolorization, and was slightly inhibitory.The two laccase isoenzymes, LacI and LacII, showed different decolorization capability depending on the mediator used. No significant differences were detected for NNDS, however LacII was more effective than LacI in the presence of PZ, while in the presence of HBT LacI was the fastest and the most effective isoenzyme.     

漆酶在磁性壳聚糖微球上的固定及其酶学性质研究

以磁性壳聚糖微球为载体,戊二醛为交联剂,共价结合制备固定化漆酶。探讨了漆酶固定化的影响因素,并对固定化漆酶的性质进行了研究。确定漆酶固定化适宜条件为：50 mg磁性壳聚糖微球,加入10mL 0.8mg/mL 漆酶磷酸盐缓冲液（0.1mol/L,pH 7.0）,在4℃固定2h。固定化酶最适pH为3.0, 最适温度分别为10℃和55℃,均比游离酶降低5℃。在pH 3.0,温度37℃时,固定化酶对ABTS的表观米氏常数为171.1μmol/L。与游离酶相比,该固定化漆酶热稳定性明显提高,并具有良好的操作和存储稳定性。     

一株产漆酶细菌的分离鉴定及酶学性质研究

[目的]分离获得产漆酶的细菌菌株,研究漆酶的酶学性质并应用于染料脱色.[方法]利用含铜的富集培养基筛选产漆酶细菌;通过形态特征、生理生化试验及16SrDNA序列分析等方法进行鉴定;以丁香醛连氮为底物测定漆酶的酶学性质;通过测定染料在最大吸收波长下吸光值的变化评价漆酶对染料的脱色效果.[结果]从森林土壤中筛选到一株漆酶高产菌株LS05,初步鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens);菌株LS05的芽孢漆酶以丁香醛连氮为底物的最适pH为6.6,最适温度为70℃;该酶具有较好的稳定性,经70℃处理10h或在pH 9.0条件下放置10d后可保留活性.对抑制剂SDS和EDTA具有一定的抗性,在碱性条件下可有效脱色不同的工业染料,RB亮蓝、活性黑和靛红1h内的脱色率达93％以上.[结论]Bacillus amyloliquefaciens LS05的芽孢漆酶在高温和碱性条件下稳定性强,相对于真菌漆酶具有更好的工业应用特性,可有效用于工业染料废水的处理.