Characterization of a recombinant laccase from Fusarium oxysporum HUIB02 for biochemical application on dyes removal

A putative laccase was cloned from cDNA of Fusarium oxysporum HUIB02, which resulted in a highly specific amplicon of 1.8 kb. Nucleotide sequence consisting of 1875 nucleotides, encoding for a polypeptide of 623 amino acids and shared 99% similarity to other F. oxysporum putative laccase gene. Hence, the gene was named as FoLacc5. FoLacc5 was expressed in P. pastoris and qualified the expression as extracellular protein. SDS-PAGE and western blot analysis indicated rFoLacc5 has a molecular weight above 100 kDa in glycosylated form. Optimal culture conditions enhanced the enzyme production level to 21,966 U/L. The optimal culture conditions was YPM medium, initial pH of 6.5, shaking speed of 210 rpm, inoculation temperature of 30 °C, and Cu²⁺ concentration of 0.08 mM. rFoLacc5 exhibited maximal activity at pH of 3.5 and temperature of 40 °C. Fe³⁺ and Cu²⁺ showed different effect on rFoLacc5 which Fe³⁺ ion completely inhibited, whereas Cu²⁺ ion increased enzyme activity. rFoLacc5 removed various synthetic dyes including brommothymol blue, methylene blue, methyl orange, remazol brilliant blue R, aniline blue, evans blue, indigo carmine, and orange II. This is the first study conducted on characterization of recombinant laccase rFoLacc5 and its application on synthetic dyes removal. rFoLacc5 shows great potential for biotechnology application on dyes removal.