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.     

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.     

Ganglioside GD3 and GD3-lactone mediated regulation of the intermolecular organization in mixed monolayers with dipalmitoylphosphatidylcholine

The interactions of dpPC with ganglioside GD3 and two lactones, GD3LacI or GD3LacII, in lipid monolayers occur with reduced, unaltered, or increased molecular area and surface potential/molecule, respectively. dpPC is fully miscible with GD3 and GD3LacI but films with GD3LacII show immiscibility above 75 mol% lactone. At low proportions of GD3 in mixtures with dpPC, GD3 undergoes condensation and depolarization; dpPC is depolarized and its molecular area is reduced above 50 mol% GD3. GD3LacI forms ideally mixed films with dpPC. Mixtures of dpPC with GD3LacII at mole fractions below 0.3 show increased mean molecular area and surface potential/molecule mostly due to lactone alterations. Between mole fractions of 0.3 and 0.75 the surface parameters of dpPC are altered, and above these proportions both lipids are immiscible. Defined variations of molecular properties induced by ganglioside lactonization are selectively transduced to changes of the intermolecular organization and surface electrostatics in mixed interfaces with dpPC. Thus, changes in the relative proportions of a ganglioside and its lactone forms may act as sensitive biotransducers for membrane-mediated cellular functions, without the need for metabolically altering the concentration of gangliosides.     

黑木耳漆酶纯化的研究

目的:研究黑木耳(Auricularia auricula)“黑29”的漆酶纯化,为进一步酶学性质的开展、酶应用和酶基因克隆提供理论基础。方法:采用硫酸铵分级沉淀和柱层析技术分离蛋白,通过PAGE和SDS-PAGE电泳检测蛋白。结果:SDS-PAGE电泳检测发现粗酶液含三种漆酶,分子量大小分别为LacA(60kD)、LacB(34kD)、LacC(19kD);纯化后获得纯化的单一漆酶LacA、LacC组分。结论:得漆酶两个单一组分,为进一步漆酶研究奠定基础。     

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。     

黑木耳漆酶纯化及部分漆酶特性的研究

黑木耳漆酶研究可为漆酶的进一步分离纯化、基因克隆表达和大规模生产应用奠定基础。对黑木耳"黑29"菌株漆酶粗酶液进行硫酸铵分级沉淀后,通过Native SDS-PAGE电泳检测,存在3种漆酶LacA、LacB、LacC,分子量分别为60,34,19 kD。经硫酸铵分级沉淀和DEAE-Sephacel柱层析技术分离得一纯化成分LacC,纯化倍数7.60,酶活性回收4.28%。对LacC的pH、温度、金属离子和Km值等部分酶学性质进行研究发现,该酶氧化ABTS的Km值为1.18×10-6mol/L,催化氧化底物ABTS的最适pH为3.8,在pH 3.0~4.6表现出较强的稳定性;最适反应温度为55℃,低于50℃时有较好的热稳定性;金属离子Ag+对漆酶有激活作用,而Fe3+、Mn2+、Co2+则有抑制作用。     

Fungal laccases: versatile tools for lignocellulose transformation

Conversion of lignocellulosic materials to useful, high value products normally requires a pre-treatment step to transform or deconstruct the recalcitrant and heterogeneous lignin fraction. The development of "green tools" for the transformation of lignocellulosic feedstocks is in high demand for a sustainable exploitation of such resources. This multi-faceted challenge is being addressed by an ever-increasing suite of ligninolytic enzymes isolated from various sources. Among these, fungal laccases are known to play an important role in lignin degradation/modification processes. The white-rot fungus Pleurotus ostreatus expresses multiple laccase genes encoding isoenzymes with different properties. The availability of established recombinant expression systems for P.?ostreatus laccase isoenzymes has allowed to further enrich the panel of P.?ostreatus laccases by the construction of mutated, "better performing" enzymes through molecular evolution techniques. New oxidative catalysts with improved activity and stability either at high temperature and at acidic and alkaline pH have been isolated and characterized.     

Grape seeds: the best lignocellulosic waste to produce laccase by solid state cultures of Trametes hirsuta

Grape seeds were used by Trametes hirsuta as a substrate for laccase production giving 23 kU l^–1, which was 10-fold the value attained in the cultures with no lignocellulosic waste addition. The dyes, Indigo Carmine and Bromophenol Blue, were easily decolourised (100% in 24 h) by the extracellular liquid obtained in such cultures, whereas Methyl Orange (65% in 24 h) and Phenol Red (36% in 24 h) were more resistant to degradation. This shows the specificity of laccase towards different dye structures.     

Production of polygalacturonase from <Emphasis Type="Italic">Coriolus versicolor</Emphasis> grown on tomato pomace and its chromatographic behaviour on immobilized metal chelates

Tomato pomace and pectin were used as the sole carbon sources for the production of polygalacturonase from a strain of Coriolus versicolor in submerged culture. The culture of C. versicolor grown on tomato pomace exhibited a peak of polygalacturonase activity (1,427 U/l) on the third day of culture with a specific activity of 14.5 U/mg protein. The production of polygalacturonase by C. versicolor grown on pectin as a sole carbon source increased with the time of cultivation, reaching a maximum activity of 3,207 U/l of fermentation broth with a specific activity of 248 U/mg protein. The levels of different isoenzymes of polygalacturonase produced during the culture growth were analysed by native PAGE. Differential chromatographic behaviour of lignocellulosic enzymes produced by C. versicolor (i.e. polygalacturonase, xylanase and laccase) was studied on immobilized metal chelates. The effect of ligand concentration, pH, the length of spacer arm and the nature of metal ion were studied for enzyme adsorption on immobilized metal affinity chromatography (IMAC). The adsorption of these lignocellulosic enzymes onto immobilized metal chelates was pH-dependent since an increase in protein adsorption was observed as the pH was increased from 6.0 to 8.0. The adsorption of polygalacturonase as well as other enzymes to immobilized metal chelates was due to coordination of histidine residues which are available at the protein surface since the presence of imidazole in the equilibration buffer abolished the adsorption of the enzyme to immobilized metal chelates. A one-step purification of polygalacturonase from C. versicolor was devised by using a column of Sepharose 6B-EPI 30-IDA-Cu(II) and purified enzyme exhibited a specific activity of about 150 U/mg protein, final recovery of enzyme activity of 100% and a purification factor of about 10. The use of short spacer arm and the presence of imidazole in equilibration buffer exhibited a higher selectivity for purification of polygalacturonase on this column with a high purification factor. The purified enzyme preparation was analysed by SDS-PAGE as well as by "in situ" detection of enzyme activity.     

芳香族化合物对斑玉蕈菌丝生物量、漆酶活性及其转录水平的影响

芳香族化合物适当时间适当浓度添加到培养基中,可提高真菌漆酶活性,有助于增强其对木质纤维素的利用效率。为了增强斑玉蕈漆酶活性,本文研究了8种芳香族化合物对其酶活的影响及其与菌丝生物量的相关性。研究发现在无诱导物条件下,斑玉蕈漆酶活性和菌丝生物量相关系数r为0.9956,说明它们呈正相关,但是整个培养过程漆酶活性相对较低;供试的芳香族化合物对漆酶活性都有不同程度的诱导作用,其中添加0.1mmol/L的愈创木酚对斑玉蕈漆酶活性诱导作用最大,达到3倍以上,同时提高了斑玉蕈菌丝生长速度和菌丝生物量;而随着添加时间的延长,部分化合物对漆酶活性和菌丝生物量都产生不同程度的抑制作用,这可能因为化合物对菌丝毒性的延长导致菌丝生长变慢或死亡;进一步研究发现,斑玉蕈3个漆酶同工酶基因lcc2、lcc3和lcc4在诱导剂愈创木酚的影响下转录水平都不同程度地上调。研究结果表明诱导漆酶活性可以提高斑玉蕈菌丝生长速度和生物量,暗示可能通过提高漆酶活性的方法,提高斑玉蕈的培养基利用效率。     

Production and characterisation of lignocellulases of Panus tigrinus and their application

This study on the lignocellulases in broth cultures of the basidiomycete Panus tigrinus indicates that laccase and xylanase enzymes are constitutive and cellulase is inducible. In stationary culture at 28°C, the greatest laccase and xylanase activity was observed after growth for approximately nine days. Laccase production was dependent on the presence, and the particular brand, of malt extract in the growth medium. While production of laccase was enhanced by growth at 37°C and 42°C, xylanase was not. Raising the pH of the growth medium from pH 5.6 to pH 7.0 did not affect xylanase production, but laccase production was reduced at the higher pH. In shake culture, growth was pelleted and biomass lower than in stationary culture, and synthesis of both enzymes was strongly inhibited. Cultures of P. tigrinus decolourised Poly R-478 and the toxic triphenyl methane dye, crystal violet. It was also shown to degrade a natural lignocellulosic waste, sawdust.     

Purification and properties of laccase from Botrytis cinerea

The partial purification of an extracellular laccase from Botrytis cinerea is described. Specificity of the enzyme, its K_m for a number of substrates and sensitivity to some inhibitors are described. The enzyme is a typical laccase but has an exceptionally low pI and great stability to acid pH. On gel electrophoresis two isoenzymes could be detected.     

毛木耳漆酶纯化及其部分漆酶特性的研究

对毛木耳AuriculariapolytrichaAP4的粗酶液进行PAGE电泳后发现含有三种漆酶同工酶,并且通过运用NativeSDS-PAGE获得三种漆酶的分子量大小分别约为:LacA(110kD);LacB(84kD);LacC(65kD)。对漆酶粗酶液通过硫酸铵分级沉淀和离子交换柱层析进行纯化,用SDS-PAGE证明获得纯化的单一漆酶LacB。LacB漆酶的反应的最适温度为30℃,最适pH为3.0。此酶氧化ABTS的Km值为6.64×10-mmol/L,金属离子对酶活的影响很大,其中5Ca2+,Mg2+,Zn2+,Na2+,Ag2+对漆酶LacB有明显的激活作用;Co2+,Hg2+,Fe3+,Fe2+,Ba2+等对酶活有明显的抑制作用。LacB和其它真菌漆酶一样具有底物专一性不强的特点,并且LacB对RB亮兰染料有很好的脱色作用。     

刺芹侧耳产漆酶条件优化及对偶氮染料甲基橙的脱色

漆酶是一种含铜的单电子多酚氧化酶,能够催化氧化各种酚类及多种染料,在处理染料废水方面具有巨大的潜力。刺芹侧耳Pleurotus eryngii具有较强的产漆酶能力,但漆酶产量在较大程度上受环境条件限制。本文研究了氮源含量、pH、温度、金属离子等环境条件对刺芹侧耳产漆酶能力的影响,优化了其产漆酶条件,并用其粗酶液对典型偶氮类染料甲基橙进行脱色,结果表明,在氮源0.5%（W/W）、pH 5.5、温度28℃、添加5.0mmol/L Mg ²⁺的培养条件下,刺芹侧耳产漆酶能力最强,培养6d时,漆酶酶活可达78.0U/L。用优化培养的刺芹侧耳粗酶液对偶氮染料甲基橙进行脱色,28h后脱色率可达90%,脱色反应为准一级动力学反应,甲基橙并未完全矿化,而是生成小分子中间产物。     

Inhibition of cellulose enzymatic hydrolysis by laccase‐derived compounds from phenols

The presence of inhibitors compounds after pretreatment of lignocellulosic materials affects the saccharification and fermentation steps in bioethanol production processes. Even though, external addition of laccases selectively removes the phenolic compounds from lignocellulosic prehydrolysates, when it is coupled to saccharification step, lower hydrolysis yields are attained. Vanillin, syringaldehyde and ferulic acid are phenolic compounds commonly found in wheat‐straw prehydrolysate after steam‐explosion pretreatment. These three phenolic compounds were used in this study to elucidate the inhibitory mechanisms of laccase‐derived compounds after laccase treatment. Reaction products derived from laccase oxidation of vanillin and syringaldehyde showed to be the strongest inhibitors. The presence of these products causes a decrement on enzymatic hydrolysis yield of a model cellulosic substrate (Sigmacell) of 46.6 and 32.6%, respectively at 24 h. Moreover, a decrease in more than 50% of cellulase and β‐glucosidase activities was observed in presence of laccase and vanillin. This effect was attributed to coupling reactions between phenoxyl radicals and enzymes. On the other hand, when the hydrolysis of Sigmacell was performed in presence of prehydrolysate from steam‐exploded wheat straw a significant inhibition on enzymatic hydrolysis was observed independently of laccase treatment. This result pointed out that the other components of wheat‐straw prehydrolysate are affecting the enzymatic hydrolysis to a higher extent than the possible laccase‐derived products. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:700–706, 2015     

黄孢原毛平革菌产漆酶优化培养及其对刚果红的脱色降解

以白腐真菌模式菌株黄孢原毛平革菌Phanerochaete chrysosporium为研究对象,探讨培养条件、重金属和芳香族化合物对产漆酶的影响,并进一步研究漆酶对刚果红的降解效果.结果 表明,P.chrysosporium产漆酶最适培养条件:葡萄糖为碳源,蛋白胨为氮源,碳氮比为90.培养8d后,漆酶酶活为911.1...     

Characterization of a gene encoding Trametes versicolor laccase A and improved heterologous expression in Saccharomyces cerevisiae by decreased cultivation temperature

Laccase can be used for enzymatic detoxification of lignocellulosic hydrolysates. A Saccharomyces cerevisiae strain with enhanced resistance to phenolic inhibitors and thereby improved ability to ferment lignocellulosic hydrolysates would presumably be obtained by heterologous expression of laccase. Sequencing of the cDNA for the novel laccase gene lcc2 from the lignin-degrading basidiomycete Trametes versicolor showed that it encodes an isoenzyme of 499 amino-acid residues preceded by a 21-residue signal peptide. By comparison with Edman degradation data, it was concluded that lcc2 encodes an isoenzyme corresponding to laccase A. The gene product of lcc2 displays 71% identity with the previously characterized T. versicolor lcc1 gene product. An alignment of laccase sequences revealed that the T. versicolor isoenzymes in general are more closely related to corresponding isoenzymes from other white-rot fungi than to the other T. versicolor isoenzymes. The multiplicity of laccase is thus a conserved feature of T. versicolor and related species of white-rot fungi. When the T. versicolor lcc2 cDNA was expressed in S. cerevisiae, the production of active enzyme was strongly dependent on the temperature. After 3 days of incubation, a 16-fold higher laccase activity was found when a positive transformant was kept at 19 °C instead of 28 °C. Similar experiments with Pichia pastoris expressing the T. versicolor laccase gene lcc1 also showed that the expression level was favoured considerably by lower cultivation temperature, indicating that the observation made for the S. cerevisiae expression system is of general significance. Received: 8 December 1998 / Received revision: 9 April 1999 / Accepted: 16 April 1999     

Production of Laccase and Optimization of Its Production by Bacillus sp. Using Distillery Spent Wash as Inducer

A bacillus sp. isolated from the sediments of a distillery mill was used for laccase production under optimized culture conditions. The distillery effluent was used as an inducer for overproduction of laccase by employing the Taguchi approach. Screening of different medium components and their effect on laccase production was studied using an M-16 orthogonal array. The formation of laccase was considerably increased by addition of 1 mM copper sulfate (51.95 U/ml), which was further enhanced by the use of different inducers. The usefulness of the Taguchi method for optimization of culture conditions was investigated with five selected factors at four levels, and it was observed that the optimized medium resulted in a 9-fold increase in extracellular laccase production compared with the control. The optimized medium composition for laccase production was dextrose (1%), tryptone (0.1%), CuSO₄ (1 mM), and an inducer (distillery effluent 10% [v/v]) at pH 7, which altogether resulted in 107.32 U/ml extracellular laccase activity. Hence, the Taguchi approach proved to be a reliable tool in optimizing culture conditions and achieving the best possible combination for enhanced laccase production.     

Overproduction of laccase by a monokaryotic strain of Pycnoporus cinnabarinus using ethanol as inducer

AIMS: Laccase production by the monokaryotic strain Pycnoporus cinnabarinus ss3 was studied using ethanol as inducer in the culture medium. METHODS AND RESULTS: The effect of ethanol was tested at 10, 20, 30, 35 and 45 g l-1 and compared with that of ferulic acid, known until now as the most efficient inducer for laccase expression by P. cinnabarinus ss3. In the presence of 35 g l-1 ethanol, laccase activity (266 600 U l-1) and productivity (19 000 U l-1 day-1) were nine and fivefold higher compared with ferulic acid-induced cultures, and 155- and 65-fold higher compared with non-induced cultures, respectively. In vivo, ethanol added to the culture medium of P. cinnabarinus ss3 favoured a continuous and high expression of laccase gene. Under these conditions, P. cinnabarinus ss3 produced preferentially the isoenzyme LAC I. Ethanol added in vitro to the purified P. cinnabarinus ss3 laccase typically inhibited the enzymatic activity. CONCLUSIONS: In spite of an initial inhibitory effect on mycelial growth, ethanol was shown to be a very strong inducer for laccase expression by P. cinnabarinus ss3 allowing an average yield of 1-1.5 g l-1 laccase. SIGNIFICANCE AND IMPACT OF THE STUDY: This study identified P. cinnabarinus ss3 as an outstanding producer of laccase in the presence of ethanol as inducer. Ethanol is an inexpensive agricultural by-product and the process is simple to scale-up for industrial production.