Comparison of Two Laccases from Trametes versicolor for Application in the Decolorization of Dyes
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It has been previously demonstrated that laccases exhibit great potential for use in severalindustrial and environmental applications. In this paper, two laccase isoenzyme genes, lccBand lccC, were cloned and expressed in Pichia pastoris GS115. The sequence analysis indicatedthat the lccB and lccC genes consisted of 1,563 and 1,584 bp, and their open reading framesencoded 520 and 527 amino acids, respectively. They had 72.7% degree of identity innucleotides and 86.7% in amino acids. The expression levels of LccB and LccC were up to32,479 and 34,231 U/l, respectively. The recombinant laccases were purified by ultrafiltrationand (NH4)2SO4 precipitation, showing a single band on SDS-PAGE, which had a molecularmass of 58 kDa. The optimal pH and temperature for LccB were 2.0 and 55oC with 2,2'-azinobis-[3-ethylbenzthiazolinesulfonic acid (ABTS) as a substrate, whereas LccC exhibited optimalpH and temperature at 3.0 and 60oC. The apparent kinetic parameters of LccB were 0.43 mMfor ABTS with a Vmax value of 51.28 U/mg, and the Km and Vmax values for LccC were 0.29 mMand 62.89 U/mg. The recombinant laccases were able to decolorize five types of dyes. AcidViolet 43 (100 g/ml) was completely decolorized by LccB or LccC (2 U/ml), and thedecolorization of Reactive Blue KN-R (100 g/ml) was 91.6% by LccC (2 U/ml). Thus, the studycharacterizes useful laccase isoenzymes from T. versicolor that have the capability of beingincorporated into the treatment of similar azo and anthraquinone dyes from dyeing industries.
Abstract
It has been previously demonstrated that laccases exhibit great potential for use in severalindustrial and environmental applications. In this paper, two laccase isoenzyme genes, lccBand lccC, were cloned and expressed in Pichia pastoris GS115. The sequence analysis indicatedthat the lccB and lccC genes consisted of 1,563 and 1,584 bp, and their open reading framesencoded 520 and 527 amino acids, respectively. They had 72.7% degree of identity innucleotides and 86.7% in amino acids. The expression levels of LccB and LccC were up to32,479 and 34,231 U/l, respectively. The recombinant laccases were purified by ultrafiltrationand (NH4)2SO4 precipitation, showing a single band on SDS-PAGE, which had a molecularmass of 58 kDa. The optimal pH and temperature for LccB were 2.0 and 55oC with 2,2'-azinobis-[3-ethylbenzthiazolinesulfonic acid (ABTS) as a substrate, whereas LccC exhibited optimalpH and temperature at 3.0 and 60oC. The apparent kinetic parameters of LccB were 0.43 mMfor ABTS with a Vmax value of 51.28 U/mg, and the Km and Vmax values for LccC were 0.29 mMand 62.89 U/mg. The recombinant laccases were able to decolorize five types of dyes. AcidViolet 43 (100 g/ml) was completely decolorized by LccB or LccC (2 U/ml), and thedecolorization of Reactive Blue KN-R (100 g/ml) was 91.6% by LccC (2 U/ml). Thus, the studycharacterizes useful laccase isoenzymes from T. versicolor that have the capability of beingincorporated into the treatment of similar azo and anthraquinone dyes from dyeing industries.
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