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1.
Morphological and biochemical analysis of the newly isolated white rot fungal (WRF-1) strain has ability to secrete laccase in the economical medium consisted of synthetic dyes, groundnut shell (GNS) and cyanobacterial biomass (algal bloom) under submerged shaking condition at pH 5.0 and 30 °C ± 2 °C temperature. WRF-1 strain was found to decolorize synthetic dyes efficiently at pH 5.0 and 30 °C ± 2 °C temperature. The laccase activity of strain was purified to homogeneity by chromatography with yield up to 70%. The molecular mass of laccase was found to be 70 kDa by SDS-PAGE and isoelectric point was 4.8. Biotransformation of the dyes was followed spectrophotometrically and dyes were found to decolorize completely after 6 days of fermentation. LC-MS studies were used to decipher the degradation profile of synthetic dyes by WRF-1. Indigo carmine gets degraded to isatin sulfonic acid and 4-amino-3-methylbenzenesulphonic acid whereas methyl orange degraded metabolites were identified as p- N, N′-dimethylamine phenyldiazine and p-hydroxybenzene sulfonic acid. Thus the study would give a road map for the production and application of laccase enzyme on a larger scale using low cost substrate. 相似文献
2.
The use of synthetic dyes for laccase induction in vivo has been scarcely explored. We characterized the effect of adding different synthetic dyes to liquid cultures of Pycnoporus sanguineus on laccase production. We found that carminic acid (CA) can induce 722 % and alizarin yellow 317 % more laccase than control does, and they promoted better fungal biomass development in liquid cultures. Aniline blue and crystal violet did not show such positive effect. CA and alizarin yellow were degraded up to 95 % during P. sanguineus culturing (12 days). With this basis, CA was selected as the best inducer and used to evaluate the induction of laccase on solid-state fermentation (SSF), using sugarcane bagasse (SCB) as substrate, in an attempt to reach selective delignification. We found that laccase induction occurred in SSF, and a slight inhibition of cellulase production was observed when CA was added to the substrate; also, a transformation of SCB under SSF was followed by the 13C cross polarization magic angle spinning (CPMAS) solid-state nuclear magnetic resonance (NMR). Results showed that P. sanguineus can selectively delignify SCB, decreasing aromatic C compounds by 32.67 % in 16 days; O-alkyl C region (polysaccharides) was degraded less than 2 %; delignification values were not correlated with laccase activities. Cellulose-crystallinity index was increased by 27.24 % in absence of CA and 15.94 % when 0.01 mM of CA was added to SCB; this dye also inhibits the production of fungal biomass in SSF (measured as alkyl C gain). We conclude that CA is a good inducer of laccase in liquid media, and that P. sanguineus is a fungus with high potential for biomass delignification. 相似文献
3.
Reactive dyes are one of the major sources of waste-water pollution. Efficient degradation of these dyes with enzymes produced
from agricultural waste has attracted tremendous recent interests in both the scientific community and the general public.
In this study, we took advantage of solid-state fermentation of four agro-byproducts (rape stem, wheat bran, peanut shell
and rice hull) for producing laccase from the fungus Trametes sp. AH28-2. Higher laccase activities were obtained in multiple-substrates media than in single substrate media. The maximum
yield of laccase (2.10 × 10 6 U/kg) was obtained in the medium containing 60% rape stem, 20% peanut shell and 20% wheat bran, without the supplement of
any toxic inducers. Our results further demonstrated that the textile reactive dyes Levafix Blue CA and Cibacron Blue FN-R
(1.0 g/l) were completely decolorized by the crude laccase (5.0 U/l) obtained within 15 h in the absence of any mediator.
Therefore, the agro-byproducts could be re-utilized to produce laccase for the decolorization of textile reactive dyes.
Q.Y. Sun and Y.Z. Hong contributed equally to the study. 相似文献
4.
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. 相似文献
5.
Background Laccases have huge potential for biotechnological applications due to their broad substrate spectrum and wide range of reactions
they are able to catalyze. These include, for example, the formation and degradation of dimers, oligomers, polymers, and ring
cleavage as well as oxidation of aromatic compounds. Potential applications of laccases include detoxification of industrial
effluents, decolorization of textile dyes and the synthesis of natural products by, for instance, dimerization of phenolic
acids. We have recently published a report on the cloning and characterization of a CotA Bacillus licheniformis laccase, an enzyme that catalyzes dimerization of phenolic acids. However, the broad application of this laccase is limited
by its low expression level of 26 mg l -1 that was achieved in Escherichia coli. To counteract this shortcoming, random and site-directed mutagenesis have been combined in order to improve functional expression
and activity of CotA. 相似文献
6.
Textile effluent is generally complicated to manage because of its extremely noxious and recalcitrant coloured compositions. Mycoremediation is an extensively used strategy for the competent degradation of hazardous pollutants present in textile effluent. Fungus could be immobilized in synthetic or natural matrices. The current study shows the decolourization of the textile effluent by 85·5 and 98·5% within 6 h using suspended and immobilized fungus, Geotrichum candidum with optimized parameters like inoculum size (5%), pH (4·5), and temperature (30°C). To maintain a high biomass of fungal population and enhance the retention of fungal strain in the contaminated sites, the fungi need to be immobilized. Hence, the fungus was immobilized naturally onto the selected inert support that is, coconut fibres by the means of adsorption, where they grew as active films on the fibres after being grown in the culture broth. The optimized process parameters of inoculum size, fibre quantity and agitation speed for immobilized G. candidum were 5%, 2·2 g l −1 of effluent and 100 rev min −1 respectively. High level of laccase (22 and 25 U l −1 in suspended and immobilized fungal cells treatment respectively) was observed during the process of decolourization and it was found that decolourization was directly proportional to the laccase activity. The UV–vis, FTIR, 1H NMR and GC-MS analyses of treated textile industrial wastewater revealed the degradation of toxic pollutants in the textile effluent and formation of lower molecular weight intermediates. The study revealed a higher efficacy of immobilized G. candidum in comparison to suspended fungal culture, employing ligninolytic enzyme laccase, which catalyzes the degradation/transformation of aromatic dyes in the textile effluent thus decolourizing it. 相似文献
7.
A novel laccase was purified and characterized from an edible mushroom Russula virescens by using a protocol that comprised ammonium sulfate saturation, ion-exchange chromatography on diethylaminoethyl-cellulose, carboxymethyl-cellulose and quaternary amine-Sepharose, and finally gel filtration by fast protein liquid chromatography on Superdex 75. The laccase was a monomeric protein with a molecular mass of 69 kDa. Its N-terminal amino acid sequence was AIGPTAELVV which demonstrated partial sequence homology to those of previously published laccases. Six peptide sequences of the purified laccase were determined by liquid chromatography and linear ion trap quadrupole mass spectrometry. Its optimum pH and temperature were 2.2 and 60 °C, respectively. The laccase was inhibited by inhibitors and several metal ions including Cu 2+ ions. The laccase degraded various phenolic compounds and the Km toward both 2,7-azinobis (3-ethylbenzothia-zolone-6-sulfonic acid) diammonium salt and dimethylphthalate was 0.1 mM. Moreover, the purified laccase decolorizes a large variety of dyes comprising laboratory dyes such as Bromothymol Blue, Eriochrome black T and Malachite Green and textile dyes such as Reactive Brilliant Blue and Reactive Blue R. 相似文献
8.
The decontamination of effluents from textile industries is problematic due to the fact that textile dyes are resistant to degradation in the environment. Enzymes from white rot fungi, especially laccase, are able to degrade various complex aromatic structures, and are therefore able to decolorize textile dyes. The white‐rot fungi Trametes versicolor and Phanerochaete chrysosporium were immobilized, separately, on both pine wood chips and palm oil fiber, and cultivated in the temporary immersion RITA® (R écipient à I mmersion T emporaire A utomatique) System, which was adapted to serve as a fungal bioreactor in a series of four experiments to determine optimal conditions for decolorizing the textile dyes Levafix Blue and Remazol Brilliant Red. The maximum rate of decolorization of both dyes occurred within 24 h of incubation, and laccase was detected in the system. 相似文献
9.
A laccase was purified from Trametes hirsuta. This laccase was classified as a “white” or “yellow” laccase. pH 2.4 was optimal for the oxidation of ABTS and pH 2.5 for
DMP. DMP oxidation was optimal at 85°C. The half-life of this laccase was 70 min at 75°C, and 5 h at 65°C. Non-phenolic dyes,
such as Methyl Red, were oxidized by purified laccase without mediators. The enzyme was not inhibited by Cu 2+, Mn 2+, or EDTA. These are atypical laccase characteristics that make it a good candidate for theoretical and applied research. 相似文献
10.
Cross-linked magnetic chitosan beads were prepared by phase-inversion technique in the presence of epichlorohydrin under alkaline
condition, and used for covalent immobilization of laccase. The activity of the immobilized laccase on the magnetic chitosan
was about 260 U (g/dry beads) with an enzyme loading of about 16.33 ± 0.39 mg [(g/dry beads) mg/g]. Kinetic parameters, V
max and K
m values were determined as 21.7 U/mg protein and 9.4 μM for free enzyme, and 15.6 U/mg protein and 19.7 μM for the immobilized
laccase, respectively. The operational and thermal stabilities of the immobilized laccase were improved compared to free counterpart.
The immobilized laccase was operated in a batch reactor for the decolorization of reactive dyes from aqueous solution. The
laccase immobilized on magnetic chitosan beads was very effective for removal of textile dyes from aqueous solution which
creates an important environmental problem in the discharged textile dying solutions. 相似文献
11.
Background A tannic acid-inducible and mycoviral-regulated laccase3 ( lac 3) from the chestnut blight fungus Cryphonectria parasitica has recently been identified, but further characterization was hampered because of the precipitation of protein products
by tannic acid supplementation. The present study investigated the heterologous expression of the functional laccase3 using
a yeast Saccharomyces cerevisiae. 相似文献
12.
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. 相似文献
13.
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. 相似文献
14.
Biological processes for the degradation of intractable materials are still not considered to be practical due to the slow rates of enzymatic degradation. Cellulosomes are complexed enzyme systems with great degradative potential and one of the strategies for overcoming this problem. In this study, the laccase CueO from Escherichia coli was fused to the dockerin domain of a cellulosome system and further assembled with the scaffoldin miniCbpA, forming a laccase–miniCbpA complex. Compared to the individual subunits, laccase–miniCbpA complex caused a noticeable 2.1-fold increase in enzyme activity levels and enhanced degradation of various synthetic dyes, showing an increase of approximately 1.6-fold. Also, pretreated barley straw by laccase complexes was efficiently converted to bioethanol using a cellulase producing Saccharomyces cerevisiae strain. The laccase complexes caused a 2.6-fold increase in the amount of reduced sugar with an insoluble substrate in conditions with an identical amount of enzymes. The cellulolytic yeast with the aid of laccase complexes produced 2.34 g/L ethanol after 72 h, indicating an increase of approximately 2.1-fold compared to fermentation without the laccase complexes. This demonstrates the feasibility of developing an efficient laccase complex based on the cellulosome and this strategy may be used to degrade recalcitrant materials. 相似文献
15.
[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. 相似文献
16.
Laccase belongs to a family of multi-copper oxidases which is especially useful for biotechnological and industrial applications. A laccase-producing white-rot fungi strain designated as Trametes sp. 5930 was nearly isolated from Shennongjia Nature Reserve in China. Trametes sp. 5930 had the high yield of laccase and was capable of decolorizing different dyes efficiently. Laccase played a very important role in the decolorization of different dyes by this fungus. The laccase gene lac5930-1 and its corresponding full-length cDNA were then cloned and characterized from Trametes sp. 5930. The 1563 bp full-length cDNA of lac5930-1 encoded a mature laccase protein consisting of 499 amino acids preceded by a signal peptide of 21 amino acids. lac5930-1 gene was successfully expressed in Pichia pastoris, which verified the function of lac5930-1 encoding active laccase by means of gene expression. The recombinant laccase produced by the yeast transformant in which lac5930-1 was efficiently expressed, conferred the ability to decolorize different dyes. The capability of decolorizing different dyes was positively related to the laccase activity, which provided strong evidence for the important function of laccase used in decolorizing industrial dyes. 相似文献
17.
The aim of this review is to determine the trends of state-of-art of laccase sources, properties, structure and recent application of fungal laccase in various fields. Laccases are biotechnologically important multi copper proteins that have broad substrate specificity towards aromatic and non-aromatic compounds. Fungi are the major laccase producers especially ascomycetes, deuteromycetes and basidiomycetes, and laccases have an average molecular weight between 50 and 130 kDa. Fungal laccases are used in biotechnological applications for preparation of anticancerous and anti-oxidant hormonal drugs, stabilization of food products, and laccase application is also extended to preparation of biosensors, DNA labeling, immunochemical assay, bioorganic compound synthesis etc. The environmental application of laccase is for biodegradation of dyes, phenols and pesticides, and the mechanism of degradation has been briefly explained. Analysis of the biodegraded dye sample by FT-IR and Mass (ESI)-spectrum has been discussed in a detailed manner. Modeling kinetics has been discussed with respect to degradation of wastes in order to understand the factors involved in the degradation process. 相似文献
18.
Myrothecium verrucaria NF-05 is a deuteromycete fungus capable of producing a white laccase. The optimal concentration of Cu 2+ for laccase production by this strain is 0.2 mM (43.23 ± 1.16 U mL ? 1). A comprehensive investigation of the induction demonstrated that NF-05 laccase production could be synergistically enhanced by various inducers, including aromatic phenols, amines and recalcitrant dyes, in the presence of 0.2 mM Cu 2+. Sixteen phenols, fourteen amines and four dyes exhibited significant inductive effects on laccase production. The best inducer was 3, 3’-dimethylbenzidine, which increased laccase production to 258.1 ± 11.1 U mL ? 1. These results suggest that M. verrucaria NF-05 is a promising industrial laccase producer. Based on the increased production, purified NF-05 laccase was used to decolorize dyes of various structural types in the presence of six redox mediators. Among the 26 tested dyes, the decolorization rate of six azo dyes, chromotrope 2R, orange G6, Congo red, Ponceau S, amaranth and reactive yellow 135 and two arylmethane dyes, fast green 3 and neutral red, were significantly increased by each of the six mediators. These results demonstrate the potential use of the NF-05 laccase for the decolorization of recalcitrant dyes in dye bleaching and effluent detoxification. 相似文献
19.
White-rot fungi were studied for the decolorization of 23 industrial dyes. Laccase, manganese peroxidase, lignin peroxidase,
and aryl alcohol oxidase activities were determined in crude extracts from solid-state cultures of 16 different fungal strains
grown on whole oats. All Pleurotus ostreatus strains exhibited high laccase and manganese peroxidase activity, but highest laccase volumetric activity was found in Trametes hispida. Solid-state culture on whole oats showed higher laccase and manganese peroxidase activities compared with growth in a complex
liquid medium. Only laccase activity correlated with the decolorization activity of the crude extracts. Two laccase isoenzymes
from Trametes hispida were purified, and their decolorization activity was characterized.
Received: 26 May 1998 / Accepted: 7 August 1998 相似文献
20.
During dye decoloration by Trametes versicolor ATCC 20869 in modified Kirk’s medium, manganese peroxidase (MnP) and laccase were produced, but not lignin peroxidase, cellobiose dehydrogenase or manganese-independent peroxidase. Purified MnP decolorized azo dyes [amaranth, reactive black 5 (RB5) and Cibacron brilliant yellow] in Mn 2+-dependent reactions but did not decolorize an anthraquinone dye [Remazol brilliant blue R (RBBR)]. However, the purified laccase decolorized RBBR five to ten times faster than the azo dyes and the addition of a redox mediator, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), did not alter decoloration rates. Amaranth and RB5 were decolorized the most rapidly by MnP since they have a hydroxyl group in an ortho position and a sulfonate group in the meta position relative to the azo bond. During a typical batch decoloration with the fungal culture, the ratio of laccase:MnP was 10:1 to 20:1 (based on enzyme activity) and increased to greater than 30:1 after decoloration was complete. Since MnP decolorized amaranth about 30 times more rapidly than laccase per unit of enzyme activity, MnP should have contributed more to decoloration than laccase in batch cultures. 相似文献
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