Purification and biochemical characterization of extracellular laccase from the ascomycete Paraconiothyrium variabile

An extracellular laccase-producing ascomycete was isolated from soil and identified as Paraconiothyrium variabile using rDNA sequence analysis. Typical laccase substrates including 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), 2,6-dimethoxyphenol (DMP), and guaiacol were oxidized by the purified enzyme (designated as PvL). The molecular mass of PvL was 84 kDa and it showed a pI value of 4.2. The enzyme acted optimally at pH 4.8 and exhibited an optimum temperature of 50 °C. Using ABTS, PvL represented Km and Vmax of 203 μM and 40 μmol min(-1) mg(-1), respectively. After 24 h incubation at pH 4.8 and 4 °C, 80% of the initial activity of PvL remained. The enzyme was inhibited by Fe2+, Hg2+, and Mn2+, but induced by Cu2+. EDTA (10 mM), 1,4-dithiothreitol (DTT) (0.1 mM), and NaN3 (10 mM) were found to completely inhibit PvL. Sixty-eight percent of Malachite green was decolorized by 4 U/mL of PvL after 15 min incubation at 30 °C.     

Engineering the Expression and Characterization of Two Novel Laccase Isoenzymes from Coprinus comatus in Pichia pastoris by Fusing an Additional Ten Amino Acids Tag at N-Terminus

The detail understanding of physiological/biochemical characteristics of individual laccase isoenzymes in fungi is necessary for fundamental and application purposes, but our knowledge is still limited for most of fungi due to difficult to express laccases heterologously. In this study, two novel laccase genes, named lac3 and lac4, encoding proteins of 547 and 532-amino acids preceded by 28 and 16-residue signal peptides, respectively, were cloned from the edible basidiomycete Coprinus comatus. They showed 70% identity but much lower homology with other fungal laccases at protein level (less than 58%). Two novel laccase isoenzymes were successfully expressed in Pichia pastoris by fusing an additional 10 amino acids (Thr-Pro-Phe-Pro-Pro-Phe-Asn-Thr-Asn-Ser) tag at N-terminus, and the volumetric activities could be dramatically enhanced from undetectable level to 689 and 1465 IU/l for Lac3 and Lac4, respectively. Both laccases possessed the lowest K _m and highest k _cat/K _m value towards syringaldazine, followed by ABTS, guaiacol and 2,6-dimethylphenol similar as the low redox potential laccases from other microorganisms. Lac3 and Lac4 showed resistant to SDS, and retained 31.86% and 43.08% activity in the presence of 100 mM SDS, respectively. Lac3 exhibited higher decolorization efficiency than Lac4 for eleven out of thirteen different dyes, which may attribute to the relatively higher catalytic efficiency of Lac3 than Lac4 (in terms of k _cat/K _m) towards syringaldazine and ABTS. The mild synergistic decolorization by two laccases was observed for triphenylmethane dyes but not for anthraquinone and azo dyes.     

烟管菌漆酶的分离纯化及部分酶学性质研究

烟管菌Bjerkandera adustaWZFF.W-Y11漆酶粗酶液经过丙酮分级沉淀、DEAE-Cellulose离子交换层析、Sephadex G-100凝胶过滤,得到了三种电泳纯的漆酶同工酶,总酶活回收率达到65.5%,其中LacA平均纯化了29.2倍,LacB纯化了5.1倍,LacC纯化了18.5倍;三种同工酶的分子量分别为LacA:68.7kDa、LacB:80.2kDa、LacC:77.2kDa。LacA、LacC氧化愈创木酚的Km大于氧化ABTS的Km,最适作用温度在45-70℃,最适反应pH3.0-5.5,65℃时LacC比LacA稳定,LacC在pH3.5-6.0稳定,LacA在pH5.0-9.0稳定,Al3+对LacA、LacC的酶活有促进作用,Cl-、Fe3+、Hg2+抑制LacA、LacC的酶活,Cu2+抑制LacC的酶活,而对LacA的活性没有明显影响。     

One-copper laccase-related enzyme from Marasmius sp.: purification, characterization and bleaching of textile dyes

In the culture filtrate of a Marasmius sp. strain isolated in Indonesia during a screening for fungi with the ability to decolorize textile dyes, two laccase-related enzymes (laccase-related enzyme I and II) were detected. Laccase-related enzyme I was purified to homogeneity by ion exchange and hydrophobic interaction chromatography. The native enzyme was shown to have a molecular mass of 53 kDa, an N-terminal amino acid sequence characteristically seen in laccases and an isoelectric point of pH 3.8. The enzyme accepts typical laccase substrates including 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), syringaldazine and guaiacol, but has no tyrosinase activity. The pH optimum is at pH 3.0 for ABTS and at 6.0 for syringaldazine and the enzyme is stable up to pH 10. The UV/vis spectrum of the laccase-related enzyme is non-typical for laccases and metal content analysis revealed that the enzyme contains only a single copper atom per enzyme molecule. This suggests that this enzyme could be related to the group of the so-called "white" laccases, however, no zinc or any other metal ion could be detected in this enzyme, suggesting that the enzyme is a unique laccase-related enzyme. Comparison of the bleaching activity of the whole fungus with that of the isolated laccase-related enzyme showed that this enzyme is the major bleaching enzyme produced by this Marasmius sp. strain and was able to bleach violet, red, orange and yellow dyes in addition to a number of blue dyes.     

Extracellular laccase produced by an edible basidiomycetous mushroom, Grifola frondosa: purification and characterization

A major laccase isozyme (Lac 1) was isolated from the culture fluid of an edible basidiomycetous mushroom, Grifola frondosa. Lac 1 was revealed to be a monomeric protein with a molecular mass of 71 kDa. The N-terminal amino acid sequence of Lac 1 was highly similar to those of laccases of some other white-rot basidiomycetes. Lac 1 showed the typical absorption spectrum of a copper-containing enzyme. The enzyme was stable in a wide pH range (4.0 to 10.0), and lost no activity up to 60 °C for 60 min. The optimal pH of the enzyme activity varied among substrates. The K(m) values of Lac 1 toward 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), 2,6-dimethoxyphenol, guaiacol, catechol, and 3,4-dihydroxy-L-phenylalanine were 0.0137 mM, 0.608 mM, 0.531 mM, 2.51 mM, and 0.149 mM respectively. Lac 1 activity was remarkably inhibited by the chloride ion, in a reversible manner. Lac 1 activity was also inhibited by thiol compounds.     

Purification,molecular characterization and reactivity with aromatic compounds of a laccase from basidiomycete <Emphasis Type="Italic">Trametes</Emphasis> sp. strain AH28-2

A recently isolated basidiomycete, Trametes sp. strain AH28-2, can be induced to produce a high level of laccases when grown on a cellobiose-asparagine liquid medium. After induction by kraft lignin, two major isozymes were detected in the fermentation supernatant of the fungus. The principal component laccase A, which accounts for about 85% of the total activity, can be purified to electrophoretic homogeneity by three chromatographic steps: DEAE-Sepharose FF, Superdex-200 and Mono-Q. The solution containing purified laccase is blue in color, and the ratio of absorbance at 280 nm to that at 600 nm is 22. The molecular mass of laccase A is estimated to be 62 kDa by SDS-PAGE, 57 kDa by FPLC, and measured as 58522 Da by MALDI mass spectrum. Laccase A is a monomeric glycoprotein with a carbohydrate content of 11-12% and an isoelectric point of 4.2. The optimum pH and temperature for oxidizing guaiacol are 4.5 and 50 degrees C, respectively. The half-life of the enzyme at 75 degrees C is 27 min. The enzyme shows a good stability from pH 4.2 to pH 8.0. The K(m) values of the enzyme toward substrates 2,2'-azino-bis (3-ethylbenzothazoline-6-sulfonate) (ABTS), guaiacol and 2,6-dimethoxyphenol are 25, 420 and 25.5 microM, respectively, and the corresponding V(max) values are 670, 66.8, and 79 microM min(-1) x mg(-1), respectively. Laccase A activity is strongly inhibited by 0.1 mM NaN(3) or 0.1 mM cyanide. Two units of laccase A alone is able to completely oxidize 100 micromol 2,6-chlorophenol in 6 h. In the presence of 1 mM ABTS and 1-hydroxybenzotriazole, 15.0 U laccase A is able to oxidize 45% and 70% of 50 micromol fluorene in 12 and 18 h, respectively. The laccase A gene was cloned by a PCR method, and preliminary analysis of its sequence indicates 87.0% similarity to the corresponding segment in the phenoloxidase gene from Coriolus hirsutus.     

Purification and characterization of a novel laccase from Coprinus cinereus and decolorization of different chemically dyes

Laccase is a blue copper oxidase with multiple copper ions and widely distributed in higher plant and fungi. To date, numerous fungal laccases have been reported by many researchers. In present work, a new laccase gene, named CcLCC5I, from Coprinus cinereus was synthesized chemically according to the yeast bias codon and integrated into Pichia pastoris GS115 genome by electroporation. SDS-PAGE analysis showed that the recombinant laccase has a molecular mass of approximately 56.8 kDa. Its biochemical properties was carried out using substrate 2-2^′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). It was showed that the optimum pH and temperature of the laccase is 3.0 and 55 °C, respectively. Except for copper ions, most metal ions inhibited the laccase activity at a high concentration about 10 mM. Sodium sulfite can also highly inhibit laccase activity whereas EDTA had no inhibitory effect on the laccase activity. The CcLCC5I have high ability to decolor not only azo but also aryl methane dyes. The recombinant laccase decolored 44.6 % orange G, 54.8 % Crystal Violet, and 87.2 % Malachite green at about 2.6 h. The novel laccase may be a good candidate for breeding engineering strains used in the treatment of industrial effluent containing azo and aryl methane dyes.     

The effects of aeration and veratryl alcohol on the production of two laccases by the ascomycete Botryosphaeria sp

The ascomycete, Botryosphaeria sp, produced two extracellular constitutive laccases (PPO-I and PPO-II) active toward the substrates: 2, 2(1)-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) [ABTS], and 2,6-dimethoxyphenol (DMP), respectively. The production of both laccases increased when the fungal isolate was grown in the presence of veratryl alcohol, and resulted in optimal laccase production (100- and 25- fold, respectively) at 40 mM. The effect of aeration on growth and laccase production was studied in baffled flasks, and showed that aeration of the cultures increased the production of both enzymes 4-5 fold in the presence of veratryl alcohol. Both laccases were susceptible to inhibition by azide, acetate and chloride anions. Veratryl alcohol inhibited the laccase-catalyzed polymerization of DMP. Growing cultures of Botryosphaeria sp. produced an exopolysaccharide of the beta-glucan type whose synthesis was depressed when grown in the presence of veratryl alcohol.     

Heterologous expression and characterization of a novel laccase isoenzyme with dyes decolorization potential from Coprinus comatus

Two new laccase genes, named lac1 and lac2, were cloned from the edible basidiomycete Coprinus comatus. Comparison of the deduced amino acid sequences revealed two laccases showed 66.12 % identity and clustered with lac2 and lac3 from Coprinopsis cinerea in same phylogenetic group. Lac1 and lac2 encode proteins of 517 and 523 amino acids preceded by 18 and 21-residue signal peptides, respectively. Lac1 was functionally expressed in Pichia pastoris. The optimum pHs of recombinant Lac1 were 3.0, 6.0, 5.5 and 6.0 and the optimum temperatures were 65, 55, 70 and 50 °C for ABTS, guaiacol, 2,6-dimethylphenol and syringaldazine, respectively. The Km values of Lac1 were 34, 4,317, 7,611 and 14 μM, and the corresponding kcat values were 465.79, 7.67, 1.15 and 0.60 (s^?1 mM), for ABTS, guaiacol, 2,6-dimethylphenol and syringaldazine, respectively. The enzyme activity was completely inhibited by sodium azide (NaN₃) and 1,4-dithiothreitol (DTT) at the concentration of 5 mM. Laccase activity was also inhibited by several metal ions, especially Fe²⁺, while K⁺ and NH₄ ⁺ slightly enhanced laccase activity. Twelve synthetic dyes belonging to anthraquinone, azo and triphenylmethane dyes were decolorized by the recombinant Lac1 at different extents. The recombinant Lac1 decolorized azo dye Reactive Dark Blue KR up to 90 % without any mediator and increasing to 96 % with mediator, indicating its potential in the treatment of industrial effluent containing some recalcitrant synthetic dyes.     

Purification and characterization of two thermostable laccases with high cold adapted characteristics from Pycnoporus sp. SYBC-L1

The white-rot fungus Pycnoporus sp. SYBC-L1 produced large amount of laccase in submerged fermentation. Two laccase isozymes (LacI and LacII) were purified using (NH₄)₂SO₄ fractionation, DEAE-cellulose and Sephadex G-100 column chromatography. The molecular masses of LacI and Lac II were 55.89 and 63.07 kDa, respectively by SDS-PAGE. Both the laccases showed acidic pH optima and high catalytic activities at low temperature for oxidations of 2,6-dimethoxyphenol (DMP), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonate acid) (ABTS), syringaldazine and guaiacol. LacI and LacII were not only with high cold adaptation, but also fairly stable at high temperature. The half-lives of LacI at 50, 60 and 70 °C were 69.31, 2.58 and 0.13 h, respectively, whereas LacII was more stable with half-lives of 256.72, 21.00 and 2.06 h respectively. The best substrates for the enzymes were both found to be ABTS, in which the K_m values of LacI and LacII were 0.0166 and 0.0435 mM and the catalytic efficiencies were 19640.36 and 31172.64 S⁻¹ mM⁻¹, respectively. EDTA and low concentration of Cu²⁺ and Mn²⁺ almost had non-inhibitions on their activities. LacII with syringaldehyde efficiently decolorized Remazol Brilliant Blue R. The high thermostabilities as well as cold adapted properties made Pycnoporus sp. SYBC-L1 laccases to be excellent candidates in harsh industry.     

Purification and characterisation of a novel laccase from the ascomycete Melanocarpus albomyces

A novel laccase from the ascomycete Melanocarpus albomyces was purified and characterised. The enzyme was purified using anion exchange chromatography, hydrophobic interaction chromatography and gel filtration, and the purified laccase was biochemically characterised. It had activity towards typical substrates of laccases including 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate), dimethoxyphenol, guaiacol, and syringaldazine. The laccase showed good thermostability and it had a pH optimum at neutral pH, both unusual properties for most known fungal laccases. The activity of the laccase from M. albomyces was highest at 60-70 degrees C. With guaiacol and syringaldazine the pH optima were rather broad: 5-7.5 and 6-7, respectively. It retained 50% of its activity after 5 h incubation at 60 degrees C. The molecular weight of the laccase was about 80 kDa and the isoelectric point 4.0. The ultraviolet-visible absorption and electron paramagnetic resonance spectra of the purified laccase indicated that the typical three types of copper were present.     

Novel thermotolerant laccases produced by the white-rot fungus <Emphasis Type="Italic">Physisporinus rivulosus</Emphasis>

The white-rot basidiomycete Physisporinus rivulosus strain T241i is highly selective for degradation of softwood lignin, which makes this fungus suitable for biopulping. In order to promote laccase production, P. rivulosus was cultivated in nutrient-nitrogen sufficient liquid media containing either charcoal or spruce sawdust as supplements. Two laccases with distinct pI values, Lac-3.5 and Lac-4.8, were purified from peptone-spruce sawdust-charcoal cultures of P. rivulosus. Both laccases showed thermal stability at up to 60°C. Lac-4.8 was thermally activated at 50°C. Surprisingly, both laccases displayed atypically low pH optima (pH 3.0–3.5) in oxidation of the commonly used laccase substrates syringaldazine (4-hydroxy-3,5-dimethoxybenzaldehyde azine), 2,6-dimethoxyphenol and guaiacol (2-methoxyphenol). Steady-state kinetic measurements pointed to unusually low affinity to guaiacol at low pH, whereas the kinetic constants for the methoxyphenols and ABTS were within the ranges reported for other fungal laccases. The combination of thermotolerance with low pH optima for methoxylated phenol substrates suggests that the two P. rivulosus T241i laccases possess potential for use in biotechnological applications.     

Characterization of the Alkaline Laccase Ssl1 from Streptomyces sviceus with Unusual Properties Discovered by Genome Mining

Fungal laccases are well investigated enzymes with high potential in diverse applications like bleaching of waste waters and textiles, cellulose delignification, and organic synthesis. However, they are limited to acidic reaction conditions and require eukaryotic expression systems. This raises a demand for novel laccases without these constraints. We have taken advantage of the laccase engineering database LccED derived from genome mining to identify and clone the laccase Ssl1 from Streptomyces sviceus which can circumvent the limitations of fungal laccases. Ssl1 belongs to the family of small laccases that contains only few characterized enzymes. After removal of the twin-arginine signal peptide Ssl1 was readily expressed in E. coli. Ssl1 is a small laccase with 32.5 kDa, consists of only two cupredoxin-like domains, and forms trimers in solution. Ssl1 oxidizes 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and phenolic substrates like 2,6-dimethoxy phenol, guaiacol, and syringaldazine. The k_cat value for ABTS oxidation was at least 20 times higher than for other substrates. The optimal pH for oxidation reactions is substrate dependent: for phenolic substrates the highest activities were detected at alkaline conditions (pH 9.0 for 2,6-dimethoxy phenol and guaiacol and pH 8.0 for syringaldazine), while the highest reaction rates with ABTS were observed at pH 4.0. Though originating from a mesophilic organism, Ssl demonstrates remarkable stability at elevated temperatures (T_1/2,60°C = 88 min) and in a wide pH range (pH 5.0 to 11.0). Notably, the enzyme retained 80% residual activity after 5 days of incubation at pH 11. Detergents and organic co-solvents do not affect Ssl1 stability. The described robustness makes Ssl1 a potential candidate for industrial applications, preferably in processes that require alkaline reaction conditions.     

The Determination of Assay for Laccase of Bacillus subtilis WPI with Two Classes of Chemical Compounds as Substrates

Ligninolytic enzyme complexes are involved in lignin degradation. Among them laccases are outstanding because they use molecular oxygen as a co-substrate instead of hydrogen peroxide as used by peroxidases. Bacterial laccase of Bacillus genus was first reported in Claus and Filip (Microbiol Res 152:209–216, 1997), since then more bacterial laccases have been found. In this research, laccase-producing bacteria were screened from pulp and paper industry wastewater, bagass and sugarcane rhizosphere. Nutrient agar medium containing 0.5 mM of guaiacol was used. It was observed that the laccase-producing strains developed brown colour from which 16 strains of Bacillus were identified. One of the isolated strains was identified as Bacillus subtilis WPI based on the results of biochemical tests and 16S rDNA sequence analysis. This strain showed laccase-like activity towards the oxidizing substrates ABTS and guaiacol. In this study guaiacol was used as the substrate of laccase activity assay. For determination of laccase activity of this isolate guaiacol was used as a substrate of assay for the first time in this study. SDS-PAGE and Native-PAGE confirmed the presence of laccase.     

Characterization of peroxidase in buckwheat seed

A peroxidase (POX)-containing fraction was purified from buckwheat seed. The POX consisted of two isozymes, POX I and POX II, that were purified 6.6- and 67.4-fold, respectively. Their molecular weights were estimated to be 46.1 kDa (POX I) and 58.1 kDa (POX II) by gel filtration. While POX I and II each oxidized quercetin, o-dianisidine, ascorbic acid and guaiacol, only POX II oxidized ABTS. Kinetic studies revealed that POX I and II had lower K(m) values for quercetin (0.071 and 0.028 mM), ABTS (0.016 mM for POX II) and ascorbic acid (0.043 and 0.029 mM) than for o-dianisidine (0.229 and 0.137 mM) and guaiacol (0.288 and 0 ). The optimum pHs of POX I and II for various substrates were almost the same, except for quercetin; pH 8.0 for POX I and pH 4.5 for II. Their optimal temperatures were 30 degrees C (POX I) and 10 degrees C (POX II), and POX I was more stable than POX II above 30 degrees C.     

Purification and characterization of a secreted laccase of Gaeumannomyces graminis var. tritici.

We purified a secreted fungal laccase from filtrates of Gaeumannomyces graminis var. tritici cultures induced with copper and xylidine. The active protein had an apparent molecular mass of 190 kDa and yielded subunits with molecular masses of 60 kDa when denatured and deglycosylated. This laccase had a pI of 5.6 and an optimal pH of 4.5 with 2,6-dimethoxyphenol as its substrate. Like other, previously purified laccases, this one contained several copper atoms in each subunit, as determined by inductively coupled plasma spectroscopy. The active enzyme catalyzed the oxidation of 2, 6-dimethoxyphenol (Km = 2.6 x 10(-5) +/- 7 x 10(-6) M), catechol (Km = 2.5 x 10(-4) +/- 1 x 10(-5) M), pyrogallol (Km = 3.1 x 10(-4) +/- 4 x 10(-5) M), and guaiacol (Km = 5.1 x 10(-4) +/- 2 x 10(-5) M). In addition, the laccase catalyzed the polymerization of 1, 8-dihydroxynaphthalene, a natural fungal melanin precursor, into a high-molecular-weight melanin and catalyzed the oxidation, or decolorization, of the dye poly B-411, a lignin-like polymer. These findings indicate that this laccase may be involved in melanin polymerization in this phytopathogen's hyphae and/or in lignin depolymerization in its infected plant host.     

Production of two novel laccase isoforms by a thermotolerant strain of Pycnoporus sanguineus isolated from an oil-polluted tropical habitat

A thermotolerant and halotolerant strain of Pycnoporus sanguineus was isolated from an oil-polluted site in a tropical area located in Veracruz, Mexico. This strain was able to grow at 47 degrees C and in culture medium containing 500 mM NaCl. The strain was also tolerant to the presence of 30,000 ppm of crude Maya oil. A 68-kDa protein purified from submerged cultures exhibited laccase activity towards 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), guaiacol, syringaldazine, and o-dianisidine, for which it presented the highest affinity (Km = 43 microM). Two-dimensional gel electrophoresis analysis showed that, unusual for laccases, the enzyme has two active isoforms, with isoelectric points of 7.00 and 7.08. The purified enzyme showed high thermostability, retaining 40% of its original activity after 3 h at 60 degrees C. This property seems to correlate with a long "shelf-life," given that at 40 degrees C enzyme activity was only gradually lost over a 5-day period incubation. Both the fungus and its laccase are likely to have high potential for biotechnological applications.     

Use of laccase as a novel, versatile reporter system in filamentous fungi

Laccases are copper-containing enzymes which oxidize phenolic substrates and transfer the electrons to oxygen. Many filamentous fungi contain several laccase-encoding genes, but their biological roles are mostly not well understood. The main interest in laccases in biotechnology is their potential to be used to detoxify phenolic substances. We report here on a novel application of laccases as a reporter system in fungi. We purified a laccase enzyme from the ligno-cellulolytic ascomycete Stachybotrys chartarum. It oxidized the artificial substrate 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate) (ABTS). The corresponding gene was isolated and expressed in Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. Heterologously expressed laccase activity was monitored in colorimetric enzyme assays and on agar plates with ABTS as a substrate. The use of laccase as a reporter was shown in a genetic screen for the isolation of improved T. reesei cellulase production strains. In addition to the laccase from S. charatarum, we tested the application of three laccases from A. nidulans (LccB, LccC, and LccD) as reporters. Whereas LccC oxidized ABTS (Km = 0.3 mM), LccD did not react with ABTS but with DMA/ADBP (3,5-dimethylaniline/4-amino-2,6-dibromophenol). LccB reacted with DMA/ADBP and showed weak activity with ABTS. The different catalytic properties of LccC and LccD allow simultaneous use of these two laccases as reporters in one fungal strain.     

粗毛栓菌诱变菌株SAH-12漆酶的分离纯化及酶学性质研究

粗毛栓菌Trametes gallica诱变菌株SAH-12是通过紫外诱变选育所得的漆酶高产菌株,Active-PAGE分析表明SAH-12在高氮低碳无机盐培养液(LM3)中至少分泌3种漆酶同工酶(Lac1、Lac2、Lac3)。采用硫酸铵盐析、透析和Sephadex-G75分子筛层析从其培养液中分离纯化得到电泳纯的Lac1,纯化倍数6.54,酶活性回收59.7%。Lac1经SDS-PAGE验证为一条带,其表观分子量为61.5kDa。Lac1为一种糖蛋白,含糖量11.6%,等电点pI4.40,催化氧化底物ABTS的最适反应温度为60℃,最适pH为2.6,Km值为25μmol/L。Lac1在40℃(pH4.0)以下和pH1.5～5.0(28℃)范围内稳定。金属离子Fe2+、Ag+、Hg2+和Cr3+与抑制剂DTT、SDS、EDTA和DMSO对Lac1有抑制作用,其中Fe2+和DTT完全抑制酶活,而Cu2+对酶有明显激活作用,Mn2+、Zn2+对酶活影响不大。Lac1不仅可使一些合成染料明显脱色,而且对苹果汁多酚祛除也有较好效果。40℃用该酶(1U/mL)处理苹果汁5h,其多酚含量可降低40%。     

Purification,characterization, and gene cloning of two laccase isoenzymes (Lac1 and Lac2) from Trametes hirsuta MX2 and their potential in dye decolorization

In this study, two laccase isoenzymes (Lac1 and Lac2) from the culture supernatant of Trametes hirsuta MX2 were purified, and the genes (Lac1 and Lac2) coding the isoenzymes were cloned. Both Lac1 and Lac2 contained an open reading frame of 1563 bp with an identity of 79%. The two isoenzymes showed significant biochemical differences. The maximal activities of Lac1 and Lac2 were at pH 2.5 with 2-2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS), and the optimal temperatures for the activities of Lac1 and Lac2 were 60 and 50 °C, respectively. Lac1 exhibited excellent resistance to acidic conditions and retained 62.17% of its initial activity at pH 2.5 after a 72-h incubation. Lac2 was more thermostable than Lac1 with half-lives (t_1/2) of 9.58 and 3.12 h at 50 and 60 °C, respectively; the t_1/2 of Lac1 were only 4.19 and 0.88 h, respectively. Both Lac1 and Lac2 isoenzymes have a strong tolerance to Mg²⁺, Mn²⁺, Cu²⁺, and EDTA (50 mM). At a low concentration of 0.05 U mL^?1, the enzymes could decolorize towards Remazol Brilliant Blue R, Acid Red 1, Crystal Violet, and Neutral Red in the presence of ABTS. These unusual properties demonstrated that the two laccases have strong potential for specific industrial applications.