Orkun PİNAR, Candan TAMERLER, Ayten KARATAŞ YAZGAN

Heterologous expression and characterization of a high redox potential laccase from Coriolopsis polyzona MUCL 38443

In this study, a novel laccase gene, named as Cplcc1, and its corresponding cDNA were isolated and characterized from the Coriolopsis polyzona MUCL 38443 strain. The Cplcc1 gene consists of a 1563-bp open reading frame encoding a protein of 520 amino acids with a 20-residue putative signal peptide. The size of the Cplcc1 gene is 2106 bp and it contains ten introns and five potential N-glycosylation sites. Additionally, the isolated full-length Cplcc1 cDNA was successfully expressed in Pichia pastoris. The heterologous expression conditions were also optimized and the highest activity value increased to 800 U L 1 with 1.5% methanol, 0.8 mM CuSO4 , and 0.6% L-alanine supplementation. The recombinant laccase was partially purified and the molecular weight was found as approximately 54 kDa. The maximum oxidation activity was observed for 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at pH 3.0. The optimal temperature was found as 70 °C. On the other hand, at 30 °C, the enzyme was stable for more than a week and its half-life was longer than 8 h. The Km, Vmax, kcat, and kcat Km1 values of the recombinant laccase were identified as 0.137 mM, 288.6 µmol min 1 L 1, 5.73 × 105 min 1, and 4.18 × 106 min 1 mM 1,respectively. Sodium azide, L-cysteine, and SDS were found as usual inhibitors.

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