Production, purification and partial characterisation of a novel laccase from the white-rot fungus Panus tigrinus CBS 577.79. Antonie Van Leeuwenhoek

Dipartimento di Agrobiologia & Agrochimica, Università degli Studi della Tuscia, Via San Camillo de Lellis snc, I-01100, Viterbo, Italy.
Antonie van Leeuwenhoek (Impact Factor: 1.81). 02/2007; 91(1):57-69. DOI: 10.1007/s10482-006-9096-4
Source: PubMed

ABSTRACT Extracellular laccase from Panus tigrinus CBS 577.79 was produced in a bubble-column reactor using glucose-containing medium supplemented with 2,5-xylidine under conditions of nitrogen sufficiency. The main laccase isoenzyme was purified to apparent homogeneity by ultra-filtration, anion-exchange chromatography and gel filtration that led to a purified enzyme with a specific activity of 317 IU (mg protein)-1 and a final yield of 66%. Laccase was found to be a monomeric protein with a molecular mass of 69.1 kDa, pI of 3.15 and 6.9% N-glycosylation of the high mannose type. Temperature and pH optima were 55 degrees C and 3.75 (2,6-dimethoxyphenol as substrate). At 50 and 60 degrees C, the enzyme half-lives were 281 and 25 min, respectively. The P. tigrinus laccase oxidized a wide range of both naturally occurring and synthetic aromatic compounds: the highest catalytic efficiencies were for 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic) acid and 2,6-dimethoxyphenol (5.99x10(6) and 3.07x10(6) M-1 s-1, respectively). Catalytic rate constants for typical N-OH redox mediators, such as 1-hydroxybenzotriazole (2.6 s-1), violuric acid (8.4 s-1) and 2,2,6,6-tetramethylpiperidin-N-oxide radical (7.8 s-1), were found to be higher than those reported for other high redox potential fungal laccases.

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Available from: Alessandro D'Annibale, Feb 17, 2014
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    • "Laccases are important in several industrial and environmental applications (Thurston 1994). Studies geared towards understanding their molecular mechanism therefore shall provide a scientific basis for the employment of these enzymes in biotechnological processes and have been a focus of recent interest (Quaratino et al. 2007; "
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    ABSTRACT: Biophysical characterization of laccase enzyme isoforms from two different xerophytic plants Cereus pterogonus and Opuntia vulgaris was carried out using EPR, fluorescence and circular dichroism (CD) spectroscopy while their thermal denaturation profiles were investigated employing differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). EPR analysis revealed the presence of endogenous copper. The hyperfine splitting of EPR spectrum reduced with increase in the complexity of enzyme protein. Raise in temperature did not alter the protein fluorescence emission suggestive of high temperature stability of the enzyme, causing the tryptophan and tyrosine residues to remain buried within the protein structure. Far-UV CD spectrum revealed existence of 60 % random coils in enzyme structure even at elevated temperatures and in presence of metal ions and protein denaturants. DSC analysis provided a Tm in the range 95–121 °C for the native and 158–199 °C for the metal associated laccase isoforms. Loss in weight of the enzyme protein by 10–18 % was noted up to 100 °C when determined through TGA. The thermostable plant xerophytic laccase enzyme isoforms will be of potential use in textile, dyeing, pulping and biotechnology applications.
    Cellulose 02/2013; 20(1):115. DOI:10.1007/s10570-012-9811-4 · 3.57 Impact Factor
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    • "Another important aspect on enzyme recovery from SMS is the optimisation of the extraction methods. Certain methods for extraction and purification of enzymes, for example, laccase have been reported, including dialysis, ultrafiltration , anion-exchange chromatography and gel filtration (Chen et al. 2004; Nagai et al. 2002; Quaratino et al. 2007; Ullrich et al. 2005), However, most of the investigations were carried out using fruiting body or mycelium of mushroom , not the SMS. Potential use of aqueous two-phase systems (Benavides and Rito-Palomares 2008) is also gaining interest and this method resulted in " one-single primary recovery stage " that gave a 95 % yield of laccase recovery (Mayolo-Deloisa et al. 2009). "
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    ABSTRACT: Mushroom industries generate a virtually in-exhaustible supply of a co-product called spent mushroom substrate (SMS). This is the unutilised substrate and the mushroom mycelium left after harvesting of mushrooms. As the mushroom industry is steadily growing, the volume of SMS generated annually is increasing. In recent years, the mushroom industry has faced challenges in storing and disposing the SMS. The obvious solution is to explore new applications of SMS. There has been considerable discussion recently about the potentials of using SMS for production of value-added products. One of them is production of lignocellulosic enzymes such as laccase, xylanase, lignin peroxidase, cellulase and hemicellulase. This paper reviews scientific research and practical applications of SMS as a readily available and cheap source of enzymes for bioremediation, animal feed and energy feedstock.
    Applied Microbiology and Biotechnology 09/2012; 96(4):863-73. DOI:10.1007/s00253-012-4446-9 · 3.34 Impact Factor
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    • "References: Panus tigrinus CBS 577.79 [29], Panus tigrinus BKM F3616D [30], Panus tigrinus 8/18 [31–34], Tricholoma mongolicum [35], Lentinus edodes [36], Lentinus edodes (lacI) [37], Lentinus edodes (lacII) [38], Pleurotus eryngii [39], Hericium erinaceus [19], Tricholoma giganteum [18]. "
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    ABSTRACT: A 59 kDa laccase with inhibitory activity against HIV-1 reverse transcriptase (IC(50) = 2.4 μM) was isolated from the broth of mycelial culture of the mushroom Lentinus tigrinus. The isolation procedure involved ion exchange chromatography on DEAE-cellulose and CM-cellulose, and gel filtration by fast protein liquid chromatography on Superdex 75. The laccase was adsorbed on both types of ion exchangers. About 95-fold purification was achieved with a 25.9% yield of the enzyme. The procedure resulted in a specific enzyme activity of 76.6 U/mg. Its N-terminal amino acid sequence was GIPDLHDLTV, which showed little similarity to other mushroom laccase and other Lentinus tigrinus strain laccase. Its characteristics were different from previously reported laccase of other Lentinus tigrinus strain. Maximal laccase activity was observed at a pH of 4 and at a temperature of 60°C, respectively. This study yielded the information about the potentially exploitable activities of Lentinus tigrinus laccase.
    BioMed Research International 03/2012; 2012:536725. DOI:10.1155/2012/536725 · 2.71 Impact Factor
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