Abstract
A laccase-producing ascomycete fungus was isolated from soil collected around the premises of a textile dye factory and identified as Nectriella pironii. Efficient laccase production was achieved via the synergistic action of 1 mM copper sulfate and ferulic acid. Extracts of rapeseed oil cake, grass hay, and leaf litter collected in a pocket urban park were used for enzyme production. The highest laccase activity (3,330 U/L) was observed in the culture grown on the leaf litter extract. This is the first report on biosynthesis of laccase by N. pironii. This is also the first study on utilization of naturally fallen park leaves as a substrate for fungal laccase production. The extracellular enzyme possessing laccase activity was purified to homogeneity by ion-exchange and gel filtration chromatographic techniques. The amino acid sequence of the protein revealed highest similarity to the laccase enzyme produced by Stachybotrys chartarum—and considerable homology to those produced by other fungal species. The purified laccase possessed a molecular mass of 50 kDa. The enzyme had an optimum pH of 2.0 or 6.0 and retained more than 50% of residual activity after 3 hours of incubation at pH 3.0–10.6 or 4.0–9.0 when 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid or 2,6-dimethoxyphenol, respectively, were used. Dithiothreitol, β-mercaptoethanol, and sodium azide at 1 mM concentration strongly inhibited the laccase activity, while in the presence of 50 mM urea, the enzyme was found to retain 25% of its activity. The laccase was able to decolorize more than 80% of Indigo Carmine, Remazol Brilliant Blue R, Reactive Orange 16, and Acid Red 27 dyes within 1 h. The possibility of leaf litter use for the production of the laccase enzyme from N. pironii (IM 6443), exhibiting high pH stability and degradative potential, makes it a promising tool for use in different environmental and industrial operations.
Highlights
Laccases (EC 1.10.3.2) are a group of copper-containing enzymes commonly found in plants, bacteria, fungi, and insects
Efficient laccase production was achieved via the synergistic action of 1 mM copper sulfate and ferulic acid
Fungal cultures were checked for zone formation on agar plates containing ABTS or DMP, which are considered as standard substrates for laccase [2]
Summary
Laccases (EC 1.10.3.2) are a group of copper-containing enzymes (multicopper oxidases, MCOs) commonly found in plants, bacteria, fungi, and insects. Fungal laccases are involved in sporulation, pigment production, fruiting body formation, and plant pathogenesis These copper-containing enzymes catalyze the oxidation of a variety of phenolic and nonphenolic substrates with a simultaneous reduction of molecular oxygen to water [1, 2]. Due to their ability to oxidize a wide range of substrates without the requirement of any coenzyme factors and high efficiency, laccases have found application in many industries, medicine, environment protection, and various biotechnological processes [1, 3]. Due to the fact that traditional processes do not remove all dyes and are expensive, laccases provide a safe and efficient alternative for decolorizing and detoxification of dyes before their discharge into the environment [7, 8]
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