Heterologous expression and characterization of a novel laccase isoenzyme with dyes decolorization potential from Coprinus comatus.
Two new laccase genes, named lac1 and lac2, were cloned from the edible basidiomycete Coprinus comatus. Comparison of the deduced amino acid sequences revealed two laccases showed 66.12 % identity and clustered with lac2 and lac3 from Coprinopsis cinerea in same phylogenetic group. Lac1 and lac2 encode proteins of 517 and 523 amino acids preceded by 18 and 21-residue signal peptides, respectively. Lac1 was functionally expressed in Pichia pastoris. The optimum pHs of recombinant Lac1 were 3.0, 6.0, 5.5 and 6.0 and the optimum temperatures were 65, 55, 70 and 50 °C for ABTS, guaiacol, 2,6-dimethylphenol and syringaldazine, respectively. The Km values of Lac1 were 34, 4,317, 7,611 and 14 μM, and the corresponding kcat values were 465.79, 7.67, 1.15 and 0.60 (s(-1) mM), for ABTS, guaiacol, 2,6-dimethylphenol and syringaldazine, respectively. The enzyme activity was completely inhibited by sodium azide (NaN(3)) and 1,4-dithiothreitol (DTT) at the concentration of 5 mM. Laccase activity was also inhibited by several metal ions, especially Fe(2+), while K(+) and NH(4)                           (+) slightly enhanced laccase activity. Twelve synthetic dyes belonging to anthraquinone, azo and triphenylmethane dyes were decolorized by the recombinant Lac1 at different extents. The recombinant Lac1 decolorized azo dye Reactive Dark Blue KR up to 90 % without any mediator and increasing to 96 % with mediator, indicating its potential in the treatment of industrial effluent containing some recalcitrant synthetic dyes.
Department of Biological Engineering, Nanjing Forestry University, Nanjing, 210037, Jiangsu, People's Republic of China.
Songyuan
S
Bao
Zhen
Z
Teng
Shaojun
S
Ding
D000595:Amino Acid Sequence; D000880:Anthraquinones / Q000737:chemistry; D001391:Azo Compounds / Q000737:chemistry; D001978:Bromphenol Blue / Q000737:chemistry; D003001:Cloning, Molecular; D004396:Coloring Agents / Q000737:chemistry*; D003224:Congo Red / Q000737:chemistry; D003302:Coprinus / Q000201:enzymology*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D005840:Gentian Violet / Q000737:chemistry; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D042845:Laccase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010843:Pichia; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012394:Rosaniline Dyes / Q000737:chemistry
D000880:Anthraquinones; D001391:Azo Compounds; D004396:Coloring Agents; D005656:Fungal Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D012394:Rosaniline Dyes; D001978:Bromphenol Blue; D003224:Congo Red; D042845:Laccase; D005840:Gentian Violet