Engineering the Expression and Characterization of Two Novel Laccase Isoenzymes from <i>Coprinus comatus</i> in <i>Pichia pastori</i>s by Fusing an Additional Ten Amino Acids Tag at N-Terminus

<div><p>The detail understanding of physiological/biochemical characteristics of individual laccase isoenzymes in fungi is necessary for fundamental and application purposes, but our knowledge is still limited for most of fungi due to difficult to express laccases heterologously. In this study, two novel laccase genes, named <i>lac3</i> and <i>lac4</i>, encoding proteins of 547 and 532-amino acids preceded by 28 and 16-residue signal peptides, respectively, were cloned from the edible basidiomycete <i>Coprinus comatus</i>. They showed 70% identity but much lower homology with other fungal laccases at protein level (less than 58%). Two novel laccase isoenzymes were successfully expressed in <i>Pichia pastori</i>s by fusing an additional 10 amino acids (Thr-Pro-Phe-Pro-Pro-Phe-Asn-Thr-Asn-Ser) tag at N-terminus, and the volumetric activities could be dramatically enhanced from undetectable level to 689 and 1465 IU/l for Lac3 and Lac4, respectively. Both laccases possessed the lowest <i>K</i>_m and highest <i>k</i>_cat/<i>K</i>_m value towards syringaldazine, followed by ABTS, guaiacol and 2,6-dimethylphenol similar as the low redox potential laccases from other microorganisms. Lac3 and Lac4 showed resistant to SDS, and retained 31.86% and 43.08% activity in the presence of 100 mM SDS, respectively. Lac3 exhibited higher decolorization efficiency than Lac4 for eleven out of thirteen different dyes, which may attribute to the relatively higher catalytic efficiency of Lac3 than Lac4 (in terms of <i>k</i>_cat/<i>K</i>_m) towards syringaldazine and ABTS. The mild synergistic decolorization by two laccases was observed for triphenylmethane dyes but not for anthraquinone and azo dyes.</p></div

Effects of metal ions and inhibitors on Lac3 and Lac4 activity.

<p>Values are the mean of triplicate determinations and standard deviation is less 5%.</p

Schematic representation of constructs for engineering the expression of <i>lac3</i> and <i>lac4</i> (A) and strategy for the construction of expression plasmids pPICZαB-10AALac3, pPICZαB-10AALac4, pPICZαB-10AALac3-ΔMyc and pPICZαB-10AALac4-ΔMyc (B).

<p>M: C-terminal myc epitope; H: polyhistidine tag</p

Effects of pH on the activity of Lac3 (A) and Lac4 (B), effects of temperature on the activity of Lac3 (C) and Lac4 (D) and effects of temperature on the stability of Lac3 (E) and Lac4 (F).

<p>Filled diamond, ABTS; filled square, guaicol; filled triangle, 2,6- Dimethylphenol; and filled circle, Syringaldazine. The values are the mean of triplicate experiments ± SD.</p

Kinetic parameters for recombinant Lac3 and Lac4.

<p>Kinetic parameters for recombinant Lac3 and Lac4.</p

SDS-PAGE of recombinant Lac3 (A) and Lac4 (B).

<p>A: M, Protein markers, lane 1, purified Lac3, lane 2, endoglycosidase-H-treated Lac3; B: M, Protein markers, lane 1, purified Lac4, lane 2, endoglycosidase-H-treated Lac4.</p

Alignment of deduced amino acid sequences of <i>lac3</i> and <i>lac4</i>.

<p>Alignment of deduced amino acid sequences of <i>lac3</i> and <i>lac4</i>.</p

Decolorization of synthetic dyes by Lac3 or Lac4 only (A) and in the presence of HBT (B).

<p>1) Remazol brilliant blue R, 2) Reactive brilliant blue X-BR, 3) Reactive brilliant blue K-GR, 4) Reactive brilliant blue K-3R, 5) Reactive orange 1, 6) Reactive red X-3B, 7) Congo red, 8) Reactive dark blue KR, 9) Coomassie G-250, 10) Malachite green, 11) Bromophenol blue, 12) Methyl violet,13) Victoria blue B. The values are the mean of triplicate experiments ± SD.</p

Detection of laccase activity on BMMY agar plates containing CuSO₄ and ABTS for transformants with <i>lac3</i> gene (A) and <i>lac4</i> gene (B) and the time course of extracellular laccase production in BMMY liquid medium (C).

<p>A: transformants containing 1) pPICZαB-Lac3, 2) pPICZαB-10AALac3, 3) pPICZαB-10AALac3-ΔMyc; B: transformants containing 1) pPICZαB-Lac4, 2) pPICZαB-10AALac4, 3) pPICZαB-10AALac4-ΔMyc; C: transformants containing pPICZαB-10AALac3 (filled diamond), pPICZαB-10AALac4 (filled circle), pPICZαB-Lac3, pPICZαB-Lac4, pPICZαB-10AALac3-ΔMyc and pPICZαB-10AALac4-ΔMyc (filled square).</p

Phenotypic correlations among traits in the RIL population (below diagonal) and the IF₂ population (above diagonal).

<p>Phenotypic correlations among traits in the RIL population (below diagonal) and the IF₂ population (above diagonal).</p