Heterologous expression of Stlac2, a laccase isozyme of Setosphearia turcica, and the ability of decolorization of malachite green

Abstract

The active laccases of ascomycetous fungus Setosphaeria turcica were identified by Native-PAGE and ESI-MS/MS, and one of these isozymes Stlac2 was heterologous expressed to investigate the decolorization of malachite green. Setosphaeria turcica produced three active laccase isozymes: Stlac1, Stlac2, and Stlac6. Stlac2 was heterologously expressed in both eukaryotic and prokaryotic expression systems. The eukaryotic recombinant Stlac2 expressed in Pichia pastoris was inactive, and also showed a higher molecular weight than predicted because of glycosylation. The depression of laccase activity was attributable to the incorrect glycosylation at Asn97. Stlac2 expressed in Escherichia coli and the recombinant Stlac2 exhibited activity of 28.23 U/mg with 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. The highest activity was observed at pH of 4.5 and the temperature of 60 °C. The activity of recombinant Stlac2 was inhibited by 10 mM Na⁺, Mg²⁺, Ca²⁺, Mn²⁺, and increased by 10 mM of Fe³⁺ with a relatively activity of 315% compared with no addition. Cu²⁺ did not affect enzyme activity. Recombinant Stlac2 was capable of decolorizing 67.08% of 20 mg/L malachite green in 15 min without any mediators. CONCLUSIONS: Generally, recombinant protein of fungal laccase Stlac2 was active without glycosylation and decolorize malachite green efficiently, which has potential industrial applications.

Keywords: Decolorization; Laccase isozyme; Setosphearia turcica.