Production, Gene Cloning, and Overexpression of a Laccase in the Marine-Derived Yeast Aureobasidium melanogenum Strain 11-1 and Characterization of the Recombinant Laccase.

Abstract

Aureobasidium melanogenum strain 11-1 with a high laccase activity was isolated from a mangrove ecosystem. Under the optimal conditions, the 11-1 strain yielded the highest laccase activity up to 3120.0 ± 170mU/ml (1.2U/mg protein) within 5days. A laccase gene (LAC1) of the yeast strain 11-1 contained two introns and encoded a protein with 570 amino acids and four conserved copper-binding domains typical of the fungal laccase. Expression of the LAC1 gene in the yeast strain 11-1 made a recombinant yeast strain produce the laccase activity of 6005 ± 140mU/ml. The molecular weight of the recombinant laccase after removing the sugar was about 62.5kDa. The optimal temperature and pH of the recombinant laccase were 40°C and 3.2, respectively, and it was stable at a temperature less than 25°C. The laccase was inhibited in the presence of sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), phenylmethanesulfonyl fluoride (PMSF), and DL-dithiothreitol (DTT). The Km and Vmax values of the laccase for 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was 6.3 × 10-2mM and 177.4M/min, respectively. Many synthetic dyes were greatly decolored by the laccase.