Abstract
A novel ‘white’ laccase was purified from the deuteromycete fungus, Myrothecium verrucaria NF-05, which was a high laccase-producing strain (40.2 U·ml−1 on the thirteenth day during fermentation). SDS-PAGE and native-PAGE revealed a single band with laccase activity corresponding to a molecular weight of approximately 66 kDa. The enzyme had three copper and one iron atoms per protein molecule determined by ICP-AES. Furthermore, both UV/visible and EPR spectroscopy remained silence, indicating the enzyme a novel laccase with new metal compositions of active centre and spectral properties. The N-terminal amino acid sequence of the purified protein was APQISPQYPM. Together with MALDI-TOF analysis, the protein revealed a high homology of the protein with that from reported M. verrucaria. The highest activity was detected at pH 4.0 and at 30°C. The enzyme activity was significantly enhanced by Na+, Mn2+, Cu2+ and Zn2+ while inhibited by DTT, NaN3 and halogen anions. The kinetic constant (Km) showed the enzyme was more affinitive to ABTS than other tested aromatic substrates. Twelve structurally different dyes could be effectively decolourised by the laccase within 10 min. The high production of the strain and novel properties of the laccase suggested its potential for biotechnological applications.
Highlights
Laccase (EC 1.10.3.2, benzenediol: oxygen oxidoreductase) is a multicopper oxidase that has become an important industrially relevant enzymes that is used in paper-pulp bleaching [1], synthetic dyes decolourisation [2], bioremediation [3], chemical analysis and bioelectronics [4]
Production and purification of extracellular laccase The production of laccase by M.verrucaria NF-05 was performed in shaking flask cultures at 140 rpm, 30uC that were induced with 1 mM copper for 15 days
No activity of bilirubin oxidase was detected in the fermentation liquid
Summary
Laccase (EC 1.10.3.2, benzenediol: oxygen oxidoreductase) is a multicopper oxidase that has become an important industrially relevant enzymes that is used in paper-pulp bleaching [1], synthetic dyes decolourisation [2], bioremediation [3], chemical analysis and bioelectronics [4]. Laccases are widely distributed among in plants [5], bacteria [6] and especially fungi [7]. Laccases are produced by many ascomycetes [2,8], basidiomycetes [9] and some deuteromycetes [10,11,12] which are involved in plant pathogenesis, pigmentation, detoxification and lignin degradation [7]. Normal copper-containing laccases contain three types of copper that can be distinguished using UV/visible and EPR spectra. T2 copper confers no color, but is EPR detectable. Laccases with a differently structured active site are described in literatures [15,16]. The present study described the purification and characterization of a novel ‘white’ laccase from the strain. The applications of the purified laccase in the decoloursation of various dyes were discussed
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