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

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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°C and 3.75 (2,6-dimethoxyphenol as substrate). At 50 and 60°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 (5.99 × 106 and 3.07 × 106 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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Abbreviations

ABTS:

2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic) acid

BCR:

bubble column reactor

DMP:

2,6-dimethoxyphenol

E m :

relative electrophoretic mobility

HBT:

1-hydroxybenzotriazole

HPI:

N-hydroxyphtalimide

IEF-PAGE:

isoelectric focusing on polyacrylamide gel

K cat :

catalytic rate constant

K m :

Michaelis Menten constant

LME:

lignin-modifying enzyme

OMW:

olive-mill wastewater

PAGE:

polyacrylamide gel electrophoresis

SDS-PAGE:

sodium dodecyl sulfate

TEMPO:

2,2,6,6-tetramethylpiperidin-N-oxide radical

VLA:

violuric acid

XYL:

2,5-xylidine

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Authors and Affiliations

  1. Dipartimento di Agrobiologia & Agrochimica, Università degli Studi della Tuscia, Via San Camillo de Lellis snc, I-01100, Viterbo, Italy

    Daniele Quaratino, Federico Federici, Maurizio Petruccioli, Massimiliano Fenice & Alessandro D’Annibale

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  1. Daniele Quaratino
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  2. Federico Federici
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  3. Maurizio Petruccioli
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  4. Massimiliano Fenice
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  5. Alessandro D’Annibale
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Correspondence to Alessandro D’Annibale.

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Quaratino, D., Federici, F., Petruccioli, M. et al. Production, purification and partial characterisation of a novel laccase from the white-rot fungus Panus tigrinus CBS 577.79. Antonie van Leeuwenhoek 91, 57–69 (2007). https://doi.org/10.1007/s10482-006-9096-4

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