Production, purification, characterization and gene cloning of an esterase produced by Aureobasidium melanogenum HN6.2

Abstract

Abstract Aureobasidium melanogenum HN6.2 was found to able to produce an esterase of 208.1 ± 2.7 units per ml within 72 h. A molecular weight of the purified esterase was 60.2 kDa and its PI was 4.86. The optimal pH and temperature of the purified esterase were 8.0 and 40 °C, respectively. The purified esterase was stable at the temperature less than 40 °C and in the pH range of 7.5–8.0. The esterase activity was greatly inhibited in the presence of Zn 2+ , Hg 2+ , Fe 2+ , Ni 2+ and SDS. K m and V max values of the enzyme for ρ-nitrophenyl butyrate were 68.6 μM and 251.4 μM per min, respectively. Mass analysis showed that it belonged to a carboxylesterase. After an esterase gene ( Car-Est ) cloned from the genomic DNA of the marine yeast strain HN6.2 was disrupted, the disruptant Y44 obtained exhibited a significant decrease in esterase activity. Meanwhile, a transcriptional level of the Car-Est gene of the disruptant Y44 was only 5.18% that of the Car-Est gene of its wild type strain HN6.2, confirming that the cloned esterase gene was indeed closely related to the esterase activity of the yeast HN6.2 strain.