MOLECULAR CLONING AND BIOCHEMICAL CHARACTERIZATION OF RECOMBINANT LACCASE FROM RIGIDOPORUS VINCTUS
Laccases are multi-copper oxidases that widely distributed in plants and fungi. These enzymes catalyze oxidation of various compounds including phenolics and non-phenolics, and have been used in many industrial processes such as paper industry, biobleaching and bioremediation. A full length cDNA (1767 bp) encoding a putative laccase (Lac) from Rigidoporus vinctus was cloned by polymerase chain reaction (PCR). The coding region of RvLac encodes 517 amino acids which contained four conserved putative copper-binding regions. To further characterize the RvLac, the coding region was subcloned into an expression vector pET-20b(+) and transformed into E. coli BL21 (DE3) pLysS. Expression of the Lac was induced by IPTG and the recombinant His-tagged Lac was purified by Ni2+ -nitrilotriacetic acid Sepharose superflow column. The purified enzyme was revealed as a single band on SDS-PAGE with molecular mass of ~57 kDa. Furthermore, the enzyme activity and kinetics were determined by ABTS assay. The Michaelis constant value for ABTS is 0.07 mM. The enzyme has a half-life of 4.6 min at 75°C. The enzyme is active under a pH 2 treatment for 30 min.
Kuo, Chia-Yu; Huang, Jenq-Kuen; Wen, Lisa; Pang, Ka-Lai; and Lin, Chi-Tsai
"MOLECULAR CLONING AND BIOCHEMICAL CHARACTERIZATION OF RECOMBINANT LACCASE FROM RIGIDOPORUS VINCTUS,"
Journal of Marine Science and Technology: Vol. 23:
5, Article 28.
Available at: https://jmstt.ntou.edu.tw/journal/vol23/iss5/28